WHO Expert Committee On Biological Standardization, 1011

This report presents the recommendations of a WHO Expert
Committee commissioned to coordinate activities leading to the
1011
adoption of international recommendations for the production W H O Te c h n i c a l R e p o r t S e r i e s
and control of vaccines and other biological substances, and the
establishment of international biological reference materials. 1011
Following a brief introduction, the report summarizes a number
WHO Expert Committee on Biological Standardization

of general issues brought to the attention of the Committee. The
next part of the report, of particular relevance to manufacturers
and national regulatory authorities, outlines the discussions
held on the development and adoption of new and revised
WHO Recommendations, Guidelines and guidance documents.
Following these discussions, WHO Guidelines on the quality,
safety and efficacy of Ebola vaccines, and WHO Guidelines
on procedures and data requirements for changes to approved
biotherapeutic products were adopted on the recommendation
of the Committee. In addition, the following two WHO
WHO Expert Committee

guidance documents on the WHO prequalification of in vitro
diagnostic medical devices were also adopted: (a) Technical
Specifications Series (TSS) for WHO Prequalification –
Diagnostic Assessment: Human immunodeficiency virus
(HIV) rapid diagnostic tests for professional use and/or self-
testing; and (b) Technical Guidance Series (TGS) for WHO
on Biological
Prequalification – Diagnostic Assessment: Establishing stability
of in vitro diagnostic medical devices. Standardization
Subsequent sections of the report provide information on the
current status, proposed development and establishment of
international reference materials in the areas of: antibiotics,
biotherapeutics other than blood products; blood products Sixty-eighth report
and related substances; in vitro diagnostics; and vaccines and
related substances.
A series of annexes are then presented which include an
updated list of all WHO Recommendations, Guidelines and
WHO Technical Report Series

other documents on biological substances used in medicine
(Annex 1). The above four WHO documents adopted on
the advice of the Committee are then published as part
of this report (Annexes 2–5). Finally, all additions and
discontinuations made during the 2017 meeting to the list of
International Standards, Reference Reagents and Reference
Panels for biological substances maintained by WHO are
summarized in Annex 6. The updated full catalogue of
WHO International Reference Preparations is available at:
http://www.who.int/bloodproducts/catalogue/en/.
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Printed in Malta
Contents
Abbreviations xiii
1. Introduction 1
2. General 3
2.1 Current directions 3
2.1.1 Strategic directions in the regulation of medicines and other health
technologies 3
2.1.2 Vaccines and biotherapeutics: recent and planned activities in biological
standardization 5
2.1.3 Blood products and in vitro diagnostics: recent and planned activities in
biological standardization 8
2.2 Reports 10
2.2.1 Report from the WHO Blood Regulators Network 10
2.2.2 Report from the WHO network of collaborating centres on standardization
and regulatory evaluation of vaccines 12
2.2.3 Report from the WHO network of collaborating centres for blood products
and in vitro diagnostics 13
2.3 Feedback from custodian laboratories 13
2.3.1 Developments and scientific issues highlighted by custodians of WHO
biological reference preparations 13
2.4 Cross-cutting activities of other WHO committees and groups 16
2.4.1 Update from the WHO Expert Committee on Specifications for
Pharmaceutical Preparations 16
2.4.2 WHO Global Benchmarking Tool 17
2.4.3 Development of WHO guidelines on good regulatory practices 18
2.4.4 Snake-bite envenoming 19
2.4.5 Update from the WHO Product Development for Vaccines Advisory
Committee 21
2.4.6 Pilot WHO prequalification of biosimilar monoclonal antibodies 22
2.4.7 Model NRA Lot Release Certificate for prequalified vaccines 23
2.4.8 Planned proficiency testing study of a standardized method for
determining total and free saccharide content of Hib liquid combined
vaccines 24
2.4.9 Vaccine prequalification – establishment of the WHO-NNB 24
2.5 Strategic issues 25
2.5.1 Standards for priority pathogens for public health emergencies 25
2.5.2 International standards and reference preparations – revision of TRS 932
Annex 2 27
3. International Recommendations, Guidelines and other matters
related to the manufacture, quality control and evaluation of
biological substances 29
3.1 Biotherapeutics other than blood products 29
3.1.1 Guidelines on procedures and data requirements for changes to approved
biotherapeutic products 29
iii
3.2 Cellular and gene therapies 30
3.2.1 Global activities in cell therapy products 30
3.3 In vitro diagnostics 31
3.3.1 WHO IVD prequalification: update report 31
3.3.2 Human immunodeficiency virus rapid diagnostic tests for professional
use and/or self-testing 33
3.3.3 Establishing stability of in vitro diagnostic medical devices 34
3.3.4 WHO consultation on the First WHO International Standard for anti-rubella
immunoglobulin 35
3.4 Vaccines and related substances 36
3.4.1 Guidelines on the quality, safety and efficacy of Ebola vaccines 36
4. International reference materials – antibiotics 38
4.1 Proposed new projects and updates – antibiotics 38
4.1.1 Proposed Third WHO International Standard for erythromycin 38
5. International reference materials – biotherapeutics other than
blood products 39
5.1 WHO International Standards and Reference Reagents – biotherapeutics other
than blood products 39
5.1.1 Second WHO International Standard for parathyroid hormone 1-34
(recombinant, human) 39
5.1.2 First WHO International Standard for rituximab 40
5.1.3 First WHO International Standard for infliximab 41
6. International reference materials – blood products and related
substances 44
6.1 WHO International Standards and Reference Reagents – blood products and
related substances 44
6.1.1 First WHO Reference Reagent for activated blood coagulation
factor X (human) 44
6.1.2 Second WHO International Standard for activated blood coagulation
factor IX (human) 45
6.1.3 First WHO International Standard for blood coagulation factor XII (plasma,
human) via assignment of additional analytes to the current Second WHO
International Standard for blood coagulation factor XI (plasma, human) 46
7. International reference materials – in vitro diagnostics 48
7.1 WHO International Standards and Reference Reagents – in vitro diagnostics 48
7.1.1 First WHO Reference Reagent for lupus anti-dsDNA serum 48
7.1.2 Third WHO International Standard for hepatitis A virus RNA for
NAT-based assays 49
7.1.3 Fourth WHO International Standard for HIV‑1 RNA for NAT-based assays 50
7.1.4 First WHO International Standard for Ebola virus antibodies (plasma,
human); and First WHO Reference Panel for Ebola virus antibodies
(plasma, human) 51
7.1.5 First WHO Reference Panel for genomic KRAS codons 12 and 13 mutations 52
7.1.6 First WHO International Standard for human herpes virus 6B DNA for
NAT-based assays 54
7.1.7 First WHO International Standard for Plasmodium falciparum antigens 55
iv
7.1.8 First WHO International Standard for anti-cytomegalovirus
immunoglobulin G 56
7.1.9 First WHO International Standard for chikungunya virus RNA for
NAT-based assays 57
7.1.10 First WHO International Standard for Zika virus antibodies
(immunoglobulin G and immunoglobulin M) (human) 58
7.2 Proposed new projects and updates – in vitro diagnostics 59
7.2.1 Proposed First WHO Reference Panel for cancer mutation detection 59
7.2.2 Proposed Third WHO International Standard for prekallikrein activator 60
7.2.3 Proposed First WHO Reference Reagent for anti-human platelet
antigen 15b 60
7.2.4 Proposed First WHO International Standard for anti-cyclic citrullinated
peptide antibodies 61
7.2.5 Proposed Second WHO International Standard for rheumatoid factor 62
7.2.6 Proposed Second WHO reference reagents for dengue virus subtypes 1–4 63
7.2.7 Proposed First WHO International Standard for cutaneous leishmaniasis;
and First WHO Reference Panel for cutaneous leishmaniasis 63
7.2.8 Proposed First WHO International Standard for Plasmodium vivax
antigens; and First WHO Reference Reagent for anti-malaria (Plasmodium
vivax) serum 64
7.2.9 Proposed First WHO International Standard for anti-MERS-CoV serum 65
7.2.10 Proposed First WHO International Standard for MERS-CoV RNA for
NAT-based assays 65
7.2.11 Proposed Sixth WHO International Standard for hepatitis C virus RNA for
NAT-based assays 66
8. International reference materials – vaccines and related substances 68
8.1 WHO International Standards and Reference Reagents – vaccines and related
substances 68
8.1.1 First WHO international standards for oral poliomyelitis vaccines 68
8.1.2 Second WHO International Standard for pertussis toxin 69
8.1.3 First WHO international standards for Citrobacter freundii and Salmonella
Typhi Vi polysaccharides 70
8.1.4 First WHO International Standard for anti-typhoid capsular Vi
polysaccharide immunoglobulin G (human) 72
8.1.5 First WHO International Standard for antiserum to respiratory
syncytial virus 73
8.2 Proposed new projects and updates – vaccines and related substances 74
8.2.1 Proposed First WHO Reference Panel for Vibrio cholera O1 and O139
lipopolysaccharides 74
8.2.2 Proposed First WHO Reference Panel for anti-Vibrio cholera O1 and O139
lipopolysaccharide serums (rabbit) 75
8.2.3 Proposed First WHO International Standard for Vibrio cholera vaccine
(oral, inactivated) 76
8.2.4 Proposed First WHO International Standard for antibody to the influenza
virus haemagglutinin stem domain 76
8.2.5 Proposed First WHO international standards for influenza virus
pathogenicity for safety testing 77
8.2.6 Proposed Third WHO International Standard for anti-rabies
immunoglobulin (human) 78
v
Annex 1
WHO Recommendations, Guidelines and other documents related to the
manufacture, quality control and evaluation of biological substances used in medicine 81
Annex 2
Guidelines on the quality, safety and efficacy of Ebola vaccines 87
Annex 3
Guidelines on procedures and data requirements for changes to approved
biotherapeutic products 181
Annex 4
Technical Specifications Series (TSS) for WHO Prequalification – Diagnostic Assessment 281
Annex 5
Technical Guidance Series (TGS) for WHO Prequalification – Diagnostic Assessment 315
Annex 6
Biological substances: WHO International Standards, Reference Reagents and
Reference Panels 377
vi
17 to 20 October 2017
Committee members1
Professor K. Cichutek, Paul-Ehrlich-Institut, Langen, Germany (Chair)
Dr M. Darko,2 Food and Drugs Authority, Accra, Ghana
Dr J. Epstein, Center for Biologics Evaluation and Research, Food and Drug Administration,
Silver Spring, MD, United States of America (USA) (also Blood Regulators Network
(BRN) representative)
Dr S. Hindawi, Blood Transfusion Services, Jeddah, Saudi Arabia
Mrs T. Jivapaisarnpong, Lad Prao, Bangkok, Thailand (Rapporteur for the plenary sessions
and for the vaccines and biotherapeutics track)
Dr H. Klein, National Institutes of Health, Bethesda, MD, the USA (Vice-Chair)
Dr C. Morris, National Institute for Biological Standards and Control, Potters Bar, the United
Kingdom (Rapporteur for the blood products and in vitro diagnostics track)
Mr V.R. Reddy, South African National Blood Service, Weltevreden Park, South Africa
Dr P. Strengers, Sanquin, Amsterdam, Netherlands
Dr Y. Sohn, Seoul National University, Seoul, Republic of Korea
Dr D. Teo, Health Sciences Authority, Singapore
Dr J. Wang, National Institutes for Food and Drug Control, Beijing, China
Temporary Advisers
Dr E. Griffiths, Kingston-upon-Thames, the United Kingdom (Rapporteur for the plenary
sessions and for the vaccines and biotherapeutics track)
Dr M. Gruber,3 Center for Biologics Evaluation and Research, Food and Drug Administration,
Rockville, MD, the USA
Dr H. Hamel, Biologics and Genetic Therapies Directorate, Health Canada, Ottawa, Canada
1
The decisions of the Committee were taken in closed session with only members of the Committee
present. Each Committee member had completed a Declaration of Interests form prior to the meeting.
These were assessed by the WHO Secretariat and no declared interests were considered to be in conflict
with full meeting participation.
2
Unable to attend.
3
Participated via teleconference.
vii
WHO Expert Committee on Biological Standardization Sixty-eighth report
Dr E. Lacana,4 Center for Drug Evaluation and Research, Food and Drug Administration,
Bethesda, MD, the USA
Dr J. Reinhardt, Paul-Ehrlich-Institut, Langen, Germany (Rapporteur for the blood products
and in vitro diagnostics track)
Dr Y. Sun, Paul-Ehrlich-Institut, Langen, Germany
Dr M. Udell, Medicines and Healthcare Products Regulatory Agency, London, the United
Kingdom
Dr A.M.H.P. van den Besselaar,4 Leiden University Medical Centre, Leiden, Netherlands
Dr A.L. Waddell, Stanley, the United Kingdom (Freelance writer)
Participants
Dr F. Agbanyo, Biologics and Genetic Therapies Directorate, Health Canada, Ottawa,
Canada (also BRN representative)
Dr N. Almond, National Institute for Biological Standards and Control, Potters Bar, the
United Kingdom
Dr P. Bowyer,5 National Institute for Biological Standards and Control, Potters Bar, the
United Kingdom
Dr J. Boyle,5 National Institute for Biological Standards and Control, Potters Bar, the United
Kingdom
Dr C. Burns, National Institute for Biological Standards and Control, Potters Bar, the United
Kingdom
Dr G. Cooper,5 National Institute for Biological Standards and Control, Potters Bar, the
United Kingdom
Dr B. Cowper,5 National Institute for Biological Standards and Control, Potters Bar, the
United Kingdom
WHO Technical Report Series, No. 1011, 2018
Dr L. Elmgren, Biologics and Genetic Therapies Directorate, Health Canada, Ottawa,

Canada (also BRN representative)
Dr O. Engelhardt,5 National Institute for Biological Standards and Control, Potters Bar,
the United Kingdom
Dr B. Fox,5 National Institute for Biological Standards and Control, Potters Bar, the United
Kingdom
4
Unable to attend.
5
viii
Dr S. Govind,6 National Institute for Biological Standards and Control, Potters Bar, the
United Kingdom
Dr E. Gray,6 National Institute for Biological Standards and Control, Potters Bar, the United
Kingdom
Dr I. Hamaguchi, National Institute of Infectious Diseases, Tokyo, Japan (also BRN
representative)
Dr C.C. Ilonze, National Agency for Food and Drug Administration and Control, Lagos,
Nigeria
Dr A. Kato, National Institute of Infectious Diseases, Tokyo, Japan
Dr D. Kaslow, Chair of the Product Development for Vaccines Advisory Committee,
Center for Vaccine Innovation and Access, Seattle, WA, the USA
Dr J. Kress,6 Paul-Ehrlich-Institut, Langen, Germany
Dr F-X. Lery, European Directorate for the Quality of Medicines & Healthcare, Strasbourg,
France
Dr K. Markey,6 National Institute for Biological Standards and Control, Potters Bar, the
United Kingdom
Dr C. Metcalfe,6 National Institute for Biological Standards and Control, Potters Bar, the
United Kingdom
Dr H. Meyer, Paul-Ehrlich-Institut, Langen, Germany
Dr P. Minor, Consultant, St Albans, the United Kingdom
Dr K. Nam, Ministry of Food and Drug Safety, Chungcheongbuk-do, Republic of Korea
Dr M. Ochiai, National Institute of Infectious Diseases, Tokyo, Japan
Dr H. Oh, Ministry of Food and Drug Safety, Chungcheongbuk-do, Republic of Korea
Dr D. Padley,6 National Institute for Biological Standards and Control, Potters Bar, the
United Kingdom
Dr M. Page,6 National Institute for Biological Standards and Control, Potters Bar, the
United Kingdom
Dr S. Prior,6 National Institute for Biological Standards and Control, Potters Bar, the
United Kingdom
Dr S. Rijpkema,6 National Institute for Biological Standards and Control, Potters Bar, the
United Kingdom
Dr S-r. Ryu, Ministry of Food and Drug Safety, Chungcheongbuk-do, Republic of Korea
6
ix
Dr C. Schärer, Swiss Agency for Therapeutic Products, Bern, Switzerland (also BRN
representative)
Dr H. Scheiblauer, Paul-Ehrlich-Institut, Langen, Germany
Dr C. Schneider, National Institute for Biological Standards and Control, Potters Bar, the
United Kingdom
Dr G. Smith, Therapeutic Goods Administration, Woden, ACT, Australia (also BRN
representative)
Dr J. Southern, Representative of South African National Regulatory Authority, Simon’s
Town, South Africa
Dr D. Stahl, Paul-Ehrlich-Institut, Langen, Germany (also BRN representative)
Dr P. Stickings, National Institute for Biological Standards and Control, Potters Bar, the
United Kingdom
Dr L. Studholme,7 National Institute for Biological Standards and Control, Potters Bar, the
United Kingdom
Dr C. Thelwell,7 National Institute for Biological Standards and Control, Potters Bar, the
United Kingdom
Dr R. Thorpe, Consultant, Welwyn, the United Kingdom
Dr A. Vasheghani, Food and Drug Organization, Tehran, the Islamic Republic of Iran
Dr N. Verdun, Center for Biologics Evaluation and Research, Food and Drug Administration,
Rockville, MD, the USA (also BRN representative)
Dr D. Williams, University of Melbourne, Melbourne, Australia
Dr D. Wilkinson, National Institute for Biological Standards and Control, Potters Bar, the
United Kingdom
Dr M. Xu, National Institutes for Food and Drug Control, Beijing, China
Dr S.H. Yoo, Ministry of Food and Drug Safety, Chungcheongbuk-do, Republic of Korea
Dr C. Zhang, National Institutes for Food and Drug Control, Beijing, China
Representation from non-state actors

Chinese Pharmacopoeia Commission
Professor R. Yueming, Beijing, China
7
x
Developing Countries Vaccine Manufacturers Network 8

Dr N. Dellepiane, Serum Institute of India Pvt. Ltd, Nyon, Switzerland
Dr V. Paradkar, Biological E Ltd, Hyderabad, India
Dr Y. Yufeng, Institute of Medical Biological Chinese Academy of Medical Sciences,
Kunming, China
European Directorate for the Quality of Medicines & HealthCare
Dr K-H. Buchheit, Department of Biological Standardisation, OMCL Network & HealthCare,
Strasbourg, France
Dr E. Charton, European Pharmacopoeia Department, Strasbourg, France
Dr M. Wierer, Department of Biological Standardisation, OMCL Network & HealthCare,
Strasbourg, France
International Alliance for Biological Standardization
Dr P. Neels, International Alliance for Biological Standardization, Geneva, Switzerland
International Federation of Pharmaceutical Manufacturers & Associations 8
Dr T. Schreitmueller, Hoffmann-La Roche, Basel, Switzerland
Dr M. van Ooij, Janssen Vaccines & Prevention B.V., Leiden, Netherlands
Dr K. Watson, AbbVie, Maidenhead, the United Kingdom
International Generic and Biosimilar Medicines Association
Dr I. Schwarzenberger, European Generic Medicines Association, Brussels, Belgium
Plasma Protein Therapeutics Association
Dr D. Misztela, Plasma Protein Therapeutics Association, Brussels, Belgium
United States Pharmacopeial Convention
Dr F. Atouf, Global Biologics, Rockville, MD, the USA
WHO Secretariat
Regional Office
AFRO – Dr B.D. Akanmori
Headquarters
Dr S. Hill, Director, EMP
Ms E. Cooke, Head, RHT/EMP
8
A maximum of two representatives of the DCVMN and two representatives of the IFPMA were present in
the meeting room during discussion of any one agenda item.
xi
Dr I. Knezevic, TSN/EMP (Committee Secretary, a.i.; Lead for the vaccines and biotherapeutics
track)
Dr M. Nübling, TSN/EMP (Lead for the blood products and in vitro diagnostics track)
Ms S. Jenner, TSN/EMP
Dr M. Friede, IVB/IVR
Dr H-N Kang, TSN/EMP
Dr A. Khadem, RSS/EMP
Dr S. Kopp, TSN/EMP
Dr D. Lei, TSN/EMP
Dr R. Meurant, PQT/EMP
Dr D. Mubangizi, PQT/EMP
Dr U. Rosskopf, RSS/EMP
Dr I. Shin, TSN/EMP
Dr M. Ward, RSS/EMP
Dr T. Zhou, TSN/EMP
Dr I.A. Cree, IARC/WHO, Lyon
xii
Abbreviations
Ad human adenovirus
Ag antigen
anti-CCP anti-cyclic citrullinated peptide
anti-dsDNA antibody to double-stranded DNA
APTT activated partial thromboplastin time
ASTM ASTM International
BDBV Bundibugyo ebolavirus
BRN WHO Blood Regulators Network
BSE bovine spongiform encephalopathy
CBER Center for Biologics Evaluation and Research
ChAd3 chimpanzee adenovirus type 3
CHO Chinese hamster ovary
CEG Core Expert Group
CHIKV chikungunya virus
CLSI Clinical and Laboratory Standards Institute
CMV cytomegalovirus
CRF circulating recombinant form
CTP cell therapy product
CV coefficient of variation
CVV candidate vaccine virus
DCVMN Developing Countries Vaccine Manufacturers Network
DNA deoxyribonucleic acid
EBOV Ebola virus
ECSPP WHO Expert Committee on Specifications for Pharmaceutical
Preparations
EDQM European Directorate for the Quality of Medicines & HealthCare
EIA enzyme immunoassay
ELISA enzyme-linked immunosorbent assay
xiii
EUAL WHO emergency use assessment and listing (procedure)

EVD Ebola virus disease
FIXa activated blood coagulation factor IX
FXa activated blood coagulation factor X
FXII blood coagulation factor XII
FXII:Ag blood coagulation factor XII (antigen value)
FXII:C blood coagulation factor XII (functional activity)
GCP good clinical practice
GCV geometric coefficient of variation
GLP good laboratory practice(s)
GMP good manufacturing practice(s)
GP glycoprotein
HA haemagglutinin
HAV hepatitis A virus
HBsAg hepatitis B surface antigen
HBV hepatitis B virus
HCV hepatitis C virus
HHV human herpes virus
HIST histamine sensitization test
HIV human immunodeficiency virus
HPA human platelet antigens

HPLC high-performance liquid chromatography
HPV human papillomavirus
HRP2 histidine-rich protein 2
ICDRA International Conference of Drug Regulatory Authorities
ICH International Council for Harmonisation of Technical
Requirements for Pharmaceuticals for Human Use
ICP immune correlate of protection
IFPMA International Federation of Pharmaceutical Manufacturers
& Associations
xiv
IFU instructions for use

IgA immunoglobulin A
IgG immunoglobulin G
IgM immunoglobulin M
ISO International Organization for Standardization
IU International Unit(s)
IVD in vitro diagnostic
KRAS Kirsten rat sarcoma viral oncogene homolog
LMIC low- and middle-income countries
LPS lipopolysaccharide(s)
LVV lentiviral vector
mAb monoclonal antibody
MARV Marburg virus
MCB master cell bank
MERS-CoV Middle East respiratory syndrome coronavirus
MVA modified vaccinia Ankara
NAT nucleic acid amplification technique
NCL national control laboratory
NGS next-generation sequencing
NIBSC National Institute for Biological Standards and Control
NRA national regulatory authority
OCV oral cholera vaccine
OPV oral poliomyelitis vaccine
PAS prior approval supplement
PCR polymerase chain reaction
PEI Paul-Ehrlich-Institut
PKA prekallikrein activator
pLDH plasmodial lactate dehydrogenase
PT prothrombin time
xv
PTx pertussis toxin

QA quality assurance
QC quality control
RDT rapid diagnostic test
RESTV Reston ebolavirus
RF rheumatoid factor
rhPTH1-34 parathyroid hormone 1-34 (recombinant, human)
RNA ribonucleic acid
RSV respiratory syncytial virus
rVSV recombinant vesicular stomatitis virus
SAGE WHO Strategic Advisory Group of Experts
SBP similar biotherapeutic product
SLE systemic lupus erythematosus
SoGAT Standardisation of Genome Amplification Techniques (group)
SUDV Sudan ebolavirus
TAFV Tai Forest ebolavirus
TGT thrombin generation test
TGS WHO Technical Guidance Series
TP Treponema pallidum
TSE transmissible spongiform encephalopathy
TSS WHO Technical Specifications Series

Vi PS Vi polysaccharide
VLP virus-like particle
VSV vesicular stomatitis virus
WCB working cell bank
WHOCC WHO collaborating centre
ZEBOV Zaire ebolavirus
ZIKV Zika virus
xvi
1. Introduction
The WHO Expert Committee on Biological Standardization met in Geneva
from 17 to 20 October 2017. The meeting was opened on behalf of the Director-
General of WHO by Dr Suzanne Hill, Director of Essential Medicines and Health
Products (EMP) and currently Acting Assistant Director-General for the Health
Systems and Innovations Cluster. Dr Hill welcomed the Committee, other meeting
participants and observers, and briefly outlined several key changes in WHO staff
and structures that had occurred since the previous meeting of the Committee.
The election of the new Director-General, Dr Tedros Adhanom Ghebreyesus, in
May 2017 meant that this was a time of transition for the Organization.
Three new clusters had now been formed from the previous Health
Systems and Innovations Cluster, and senior management posts filled. One of
the new clusters – Access to Medicines, Vaccines and Pharmaceuticals – would
be led by a new Assistant Director-General, Dr Mariângela Batista Galvão Simão.
Dr Hill also announced that Dr David Wood, Coordinator, Technologies,
Standards and Norms – and Secretary to the Committee – had retired in April,
and that Dr Francois-Xavier Lery, previously of the European Directorate for the
Quality of Medicines & HealthCare, had been appointed as his successor.
Dr Hill highlighted the commitment of the Director-General to champion
universal health coverage, including through improved access to medicinal
products of assured quality, safety and efficacy. It was envisaged that this would
be achieved through a coherent country ownership approach backed up by
the strong normative efforts of WHO. As the longest-standing WHO Expert
Committee, the WHO Expert Committee on Biological Standardization has long
emphasized the importance of pursuing a coherent approach to improving access
to medicines and strengthening WHO’s normative work in this area. There would
now be an opportunity to review the ways in which the Committee functioned
and to decide how best to prioritize its substantial workload. On behalf of WHO,
Dr Hill expressed her thanks to the Committee, to WHO Collaborating Centres,
and to all the experts, institutions and professional societies working in this
field, whose efforts provided vital support to WHO programmes in global public
health. She concluded by reminding participants that Committee members acted
in their personal capacities as experts and not on behalf of their organizations
or countries.
Dr Ivana Knezevic, Acting Secretary to the Committee, outlined the
working arrangements of the meeting before moving on to the election of
the meeting officials. Professor Klaus Cichutek was elected as Chair and
Dr Elwyn Griffiths as Rapporteurs for the plenary sessions and for the track
considering vaccines and biotherapeutics. Mrs Teeranart Jivapaisarnpong was
also Rapporteur for the vaccines and biotherapeutic track. Dr Harvey Klein
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was elected as Chair and Dr Clare Morris and Dr Jens Reinhardt as Rapporteurs
for the track considering blood products and in vitro diagnostics. Dr Klein was
also elected as Vice-Chair for the plenary sessions of the Committee.
Dr Knezevic then gave a brief overview of WHO Expert Committees
and of their important and greatly valued role in providing assistance to WHO
Member States. She noted that the meeting of the WHO Expert Committee on
Specifications for Pharmaceutical Preparations meant that two WHO Expert
Committees were meeting concurrently. Also meeting during the same week
were the WHO Strategic Advisory Group of Experts (SAGE) on Immunization
and the annual Consultation on International Nonproprietary Names (INN) for
Pharmaceutical Substances. Dr Knezevic introduced the members of the 2017
Expert Committee on Biological Standardization and presented the declarations
of interests made by Committee members, Temporary advisers and participants.
After evaluation, WHO had concluded that none of the declarations made
constituted a significant conflict of interest and that the individuals concerned
would be allowed to participate fully in the meeting.
Following participant introductions, the Committee adopted the
proposed agenda (WHO/BS/2017.2331).
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2.1 Current directions
2.1.1 Strategic directions in the regulation of medicines
and other health technologies
Ms Emer Cooke, newly appointed Head of Regulation of Medicines and other
Health Technologies (RHT), presented an overview of WHO strategic directions
in the regulation of medicines and other health technologies, with a particular
focus placed on biologicals. After outlining the overall vision of the new Director-
General, Ms Cooke provided more details of the new WHO Cluster on Access to
Medicines, Vaccines and Pharmaceuticals. The cluster would comprise EMP and
the two underlying groups, Innovation, Access and Use (IAU) led by Dr Sarah
Garner and RHT. RHT would consist of Technologies, Standards and Norms,
Regulatory System Strengthening, the WHO Prequalification Team and Safety
and Vigilance. Mention was made of the fact that the WHO Prequalification
Team now had responsibility for medical devices and vector-control products in
addition to vaccines, medicines and diagnostics. Ms Cooke then expressed her
thanks to Dr Knezevic, who had been the Acting Secretary to the Committee
since the retirement of Dr Wood in May 2017, for her invaluable support and
that of her team.
Ms Cooke noted that two strategic aspects of EMP activities which
particularly impacted upon biologicals were access to medicines and public
health emergencies. Since the previous meeting of the Committee, an Expert
Consultation on improving access to and use of similar biotherapeutic products
(SBPs) 1 had been held in Geneva, as had a WHO Informal Consultation on
options to improve regulatory preparedness to address public health emergencies.2
In addition, an EMP strategic framework entitled “Towards Access 2030” 3
had been published, with a supporting RHT strategy now under development.
Furthermore, a new Strategic Advisory Group of Experts on In Vitro Diagnostics
(SAGE IVD) had been established. The SAGE IVD would act as an advisory
body to WHO on matters of global policies and strategies related to IVDs.
1
Report on the Expert Consultation on improving access to and use of similar biotherapeutic products.
Geneva, 2–3 May 2017. Geneva: World Health Organization; 2017 (http://www.who.int/medicines/access/
biotherapeutics/FINAL_Report-improving-access-to-and-use-of-biotherapeutics_October2017.pdf,
accessed 2 April 2018).
2
WHO Informal Consultation on options to improve regulatory preparedness to address public health
emergencies. Geneva, 17–19 May 2018. Geneva: World Health Organization; 2017 (http://www.who.int/
medicines/news/2017/PHEmeeting-reportIK-EG16_Nov_2017.pdf, accessed 2 April 2018).
3
Towards Access 2030. WHO medicines and health products programme strategic framework 2016–
2030. Geneva: World Health Organization; 2017 (WHO/EMP/2017.01; http://www.who.int/medicines/
publications/Towards_Access_2030_Final.pdf?ua=1, accessed 2 April 2018).
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A wide range of stakeholders had participated in the Expert Consultation

on improving access to and use of SBPs, including clinicians, regulators,
health economists, experts in public health and from academia, members of
WHO Expert Committees and advisory panels, manufacturers, and patient
organizations and professional societies. Recurrent themes which emerged
included the need to ensure SBP quality, safety and efficacy (notably through
robust regulation and guidance), the need for clarity regarding nomenclature
and other aspects of terminology, and the need for education and effective
communication regarding SBPs. The need for strengthened pharmacovigilance
was also emphasized. Ms Cooke informed the Committee that no consensus
had been reached on whether WHO should continue with plans for a Biological
Qualifier and so WHO had decided not to proceed with this nomenclature.
However, it was agreed that WHO would review and provide clarification
of its 2009 Guidelines on evaluation of SBPs in order to reflect, where
necessary, technological and analytical advances. WHO would also pilot the
prequalification of two SBPs – trastuzumab and rituximab – in an attempt to
extend the considerable experience of stringent regulatory authorities (SRAs) in
this area to national regulatory authorities (NRAs) in low- and middle-income
countries (LMIC). Manufacturers of rituximab and trastuzumab would be
invited to submit expressions of interest to WHO.
The Committee was also informed that the WHO Informal Consultation
on options to improve regulatory preparedness to address public health
emergencies had been well attended by regulators from high-income countries
and from LMIC, as well as by manufacturers, subject matter experts and other
stakeholders. Pertinent issues relating to in vitro diagnostics, therapeutics and
vaccines had been discussed, and available regulatory tools and pathways –
including the WHO Emergency Use Assessment and Listing (EUAL) procedure
– were reviewed, along with regulatory collaboration arrangements between
countries, and capacity-building activities. Preliminary outcomes highlighted
the need to map out current emergency provisions in LMIC and to address
legal or regulatory deficiencies, to clarify and revise the current EUAL procedure
based on experience to date, and to consider a possible “pre-EUAL” submission
process for priority pathogens. Clarification was also needed of what happens
following a EUAL procedure regarding, for example, national licensing, product
procurement, importation and liability issues. It had been proposed that
WHO guidance be developed on procedures and pathways to enable the use of
unlicensed products during a public health emergency, including guidance on
the minimum competencies required by NRAs to deal with such situations. The
possibility of a diagnostics preparedness consortium was raised, as was the use
of “mock-up” exercises in expediting the review of submissions or clinical trials
in emergency contexts. A trial tabletop exercise was scheduled to take place at
the end of November 2017. The Committee was informed that WHO would now
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reflect on all the recommendations made for moving this agenda forward, with
a view to developing an action plan based on priorities and available resources.
The Committee was then reminded of the two main strategic roles of
EMP – namely, to act as a facilitator, supporting innovation and promoting
access to health products, and as a guardian in efforts to strengthen regulatory
capacity and practices and thus ensure the quality, safety and efficacy of products
in order to secure health gains. An integrated approach across all RHT activities
would be needed to achieve these goals. This would include strengthening
the ability of NRAs to effectively regulate medicines, and promoting, where
appropriate, the concept of regulatory reliance. Ms Cooke emphasized that the
work of the Committee was a fundamental enabler of many of the normative
activities of WHO, and noted its strong links with other WHO activities and
entities such as the SAGE on Immunization. In addition, WHO initiatives in the
area of public health emergencies – including the WHO Blueprint for Research
and Development: Responding to Public Health Emergencies of International
Concern (R&D Blueprint) and collaboration with other international initiatives
such as the Coalition for Epidemic Preparedness Innovations – would continue
to depend upon sound regulatory science, standards and norms.
The Committee thanked Ms Cooke for sharing this helpful overview of
current WHO developments in the regulation of medicines and other health
technologies and raised a number of issues for clarification. In particular,
clarification was sought of the remit of the newly established SAGE IVD and
of how its work would complement the longstanding responsibilities of the
Committee in this area. Ms Cooke indicated that this was not yet clear but
agreed that it would be very important to avoid both excessive workload and
the overlapping of responsibilities. It was also noted that the Committee would
normally provide the SAGE on Immunization with a report of its work on
vaccine standardization and related issues. However, this year both groups were
meeting in parallel and so representatives of the Committee were only able to
participate in parts of the SAGE on Immunization meeting. A full report of the
work of the Committee in this regard would be presented at the next SAGE on
Immunization meeting. The Committee requested that consideration be given
by WHO to improving the coordination of meetings at WHO headquarters of
all the various advisory and other groups working in this area.
2.1.2 Vaccines and biotherapeutics: recent and planned

activities in biological standardization
Dr Knezevic reported on recent and planned activities in the area of
standardization and regulatory evaluation of vaccines and biotherapeutics.
Current WHO Recommendations, Guidelines and guidance documents (“written
standards”) in the area of biological standardization are primarily vaccine
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specific (or relevant to all vaccines), a number specifically cover biotherapeutic

products, while others apply to both vaccines and biotherapeutic products.
Two new WHO Guidelines – one on Ebola vaccines and the other
on post-approval changes to biotherapeutic products, including similar
biotherapeutic products (SBPs) – were being considered for adoption at
the present meeting (see sections 3.1.1 and 3.4.1 respectively). Dr Knezevic
reminded the Committee that written standards relating specifically to blood
products are listed on the separate WHO Blood Products website (http://www.
who.int/bloodproducts/en/) and suggested that the coordination between the
two sites could be improved.
Dr Knezevic then outlined the range of recently adopted WHO
measurement standards in this area. Such standards are crucial elements in the
development, licensing and ongoing oversight of biological medicines. Nine new
WHO international standards for vaccines and related substances were being
considered for establishment at the present meeting (see sections 8.1.1–8.1.5).
Ideally, measurement standards and written standards should be developed
simultaneously but this is not always feasible, and in cases where a written
standard is adopted before the establishment of the international measurement
standard there may be a need to update the former. Currently, there are nine
WHOCCs contributing to this work and their evolving role in the development
of measurement standards was under discussion.
Dr Knezevic informed the Committee of the progress being made in
the development of guidelines on the quality, safety and efficacy of respiratory
syncytial virus (RSV) vaccines. A WHO consultation on RSV vaccines held
in September 2017 had highlighted a significant surge in RSV vaccine and
monoclonal antibody (mAb) development, with 42 candidate vaccines and four
mAbs in development, targeting different populations (paediatric, pregnant
women and the elderly). Although a WHO international standard for antiserum
to RSV for standardizing RSV neutralization assays was being submitted for
establishment at the present meeting, its suitability as a standard in the mAb

competition assay was still being assessed and additional international reference
materials for such products may be required. Consideration was also being
given to the development of a manual on the standardization of such assays with
subsequent evaluation of training needs.
The Committee was also informed that WHO Recommendations to
assure the quality, safety and efficacy of hepatitis E vaccines were also under
development. Such vaccines had already been developed, licensed and used
in China but several more were in development. A range of issues had been
discussed at a WHO meeting in May 2017 and a drafting group established.
A first round of public consultation on the draft Recommendations document
was scheduled for late 2017 and a second meeting of the drafting group planned
for April 2018.
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An update was then provided on the revision of the WHO Guidelines

on the safe production and quality control of inactivated poliomyelitis vaccines
manufactured from wild polioviruses in the context of the biocontainment
needs described in the WHO Global Action Plan to minimize poliovirus facility-
associated risk after type-specific eradication of wild polioviruses and sequential
cessation of oral polio vaccine use (GAPIII). The Committee was reminded that
the focus of the WHO Guidelines on production safety and product quality
was complementary to the focus of GAP III on environmental safety and
biocontainment following the eradication of wild-type polioviruses. A first
round of public consultation on the draft Guidelines document was scheduled
for late 2017 and a third meeting of the working group planned for April 2018.
The Committee was also reminded of the crucial importance of
harmonizing international biosafety expectations for both pilot- and large-
scale production of human pandemic influenza vaccines. Such harmonization
was a key element in facilitating influenza vaccine development during the
interpandemic period and thus in ensuring the timely availability of vaccine in
the event of an influenza pandemic. The current WHO document on biosafety
risk assessment and the production and quality control of human influenza
pandemic vaccines had been adopted by the Committee in 2007. Following an
expert group review of the document in the light of existing biosafety guidance,
new knowledge and other inputs, it had been concluded that the text required
updating in a number of key areas and expansion of its scope to cover all
influenza A virus subtypes. A first draft of the revised guidance was expected
to be ready for public consultation in late 2017 with further rounds of revision
and public consultation scheduled for 2018, with the aim of submitting the final
revised document for consideration by the Committee in October 2018.
Dr Knezevic then reported on the outcomes of a 2017 WHO consultation
on improving access to SBPs. One of the proposals made was for WHO to review
and revise its 2009 Guidelines on evaluation of similar biotherapeutic products to
reflect recent technological and analytical advances. However, during a previous
WHO implementation workshop it had been concluded that the principles set
out in the current Guidelines were still relevant and it had instead been suggested
that a Questions & Answers (Q&A) document might better clarify and inform
the current understanding of the principles outlined. An early draft Q&A text
was now undergoing public consultation with the intention of publishing the
resulting document on the WHO website in 2018.
The vital role played by implementation workshops in promoting and
clarifying WHO written standards, and in moving the biologicals field forward,
was highlighted. During 2016, three such WHO workshops had been held – two
(in China and Thailand) on human papillomavirus (HPV) vaccines, and one
(in Indonesia) on typhoid conjugate vaccines. In July 2017, an implementation
workshop had been held on biotherapeutics (including SBPs) for Russian-
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speaking countries, organized by the WHO Regional Office for Europe in

Copenhagen. Further implementation workshops were scheduled for late 2017
on good manufacturing practices (GMP) for biologicals (in Thailand) and on
typhoid conjugate vaccines (in the Republic of Korea).
Dr Knezevic concluded by outlining the ways in which WHO written and
measurement standards contribute to the process of regulatory convergence by
promoting a common understanding in all WHO Member States of the complex
issues involved and by providing international standards for the regulatory
evaluation of medicinal products. Implementation workshops then provide
valuable educational and training tools for improving the expertise of NRAs. After
noting that many challenges remain, Dr Knezevic outlined a number of successful
collaborations that had been undertaken, and the numerous opportunities
that exist for further collaboration with other organizations and networks in
promoting the use of WHO standards and thus regulatory convergence.
The Committee thanked Dr Knezevic for her informative overview and
expressed its support for the proposed initiatives. In particular, the Committee
agreed that a Q&A document for SBPs should be developed. The Committee also
reiterated the view that the conducting of technically detailed implementation
workshops, including through the use of case studies, will continue to be an
important element in promoting regulatory convergence.
2.1.3 Blood products and in vitro diagnostics: recent and

planned activities in biological standardization
Dr Micha Nübling presented an overview of activities in the areas of blood
products and in vitro diagnostics over the past 12 months, highlighting five
main topics: (a) the recommendations of a workshop on blood products held
during the 17th International Conference of Drug Regulatory Authorities
(ICDRA) in South Africa in December 2016; (b) progress in the assessment of
snake antivenoms; (c) blood regulation in Zambia; (d) the impact of emerging
infections on blood supply; and (e) issues related to the First WHO International
Standard for anti-rubella immunoglobulin.
The ICDRA blood products workshop recommendations emphasized
the need for countries to implement blood regulations, to regulate reagents
and devices associated with the use of blood, to model new regulations on
already-existing regulations in other countries and to regulate snake antivenoms.
In support of this, WHO was urged to provide assistance in the assessment of
national blood regulation, provide training for inspectors and assessors with a
focus on regional networks, update the global database on snakes and antivenoms,
develop regional reference standards for venoms, and continue in its assessment
and listing of snake antivenoms.
The Committee was informed that snake-bites lead to more than
100 000 fatalities each year, with women and children in rural areas being the
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most affected. However, at present, antivenoms are still widely unregulated and
often of unknown quality. WHO had therefore implemented its programme
of assessment and listing of snake antivenoms in response to the need for
antivenoms of assured quality. Manufacturers of antivenoms are invited to
submit a dossier for evaluation along with product samples for laboratory testing
of their efficacy and other characteristics. GMP inspections are also carried
out. Following a call to manufacturers of antivenoms specifically intended for
use in sub-Saharan Africa, seven dossiers had been submitted for evaluation
and five product samples for laboratory testing. Antivenoms meeting WHO
requirements and with a favourable risk–benefit ratio will be listed on the WHO
website for easy access by procurement agencies and other relevant parties. Dr
Nübling indicated that a WHO prequalification process for antivenoms may be
developed if the conducting of the risk–benefit assessments indicates that this
would generate significant public health benefits.
The Committee was informed that an invited assessment of the Zambia
Medicines Regulatory Agency (ZAMRA) by staff from the WHO Regional Office
for Africa and WHO headquarters, together with a member of the BRN, had
taken place in 2017. The assessment indicated that although legal preconditions
and medicines regulation is in place, blood regulation in Zambia is still at a very
early stage. The Zambia National Blood Transfusion Service (ZNBTS) was not
overseen by ZAMRA but instead was auto-regulated. It was considered that the
current medicines regulations should be used as a blueprint for blood regulation
in the country. Advancing blood regulation would require both training – which
could be delivered using ZNBTS expertise – and twinning with (or having greater
reliance on) mature NRAs in other countries.
Dr Nübling reported that a WHO Global Technical Expert Consultation
on estimating the impact of emerging infections on the blood supply: requirements
for risk estimation and decision-making support had been held in Geneva on
14–15 June 2017. Consultation participants had noted that regulatory decisions
on the protection of the blood supply are often taken on an ad hoc basis when
confronted with the emergence of a pathogen. Such decisions may include the
introduction of a new test for screening blood donors, deferral of certain donors
from blood donation or quarantining of blood components. In these situations,
promoting the public perception of “safe blood” or political expectations were
sometimes more dominant factors than scientific considerations. There was
thus a recognized need for consistency in estimating threats to the blood supply
and making subsequent regulatory decisions. It was proposed that a prototype
guidance tool could be developed covering different aspects (such as quantitative
risk estimates, potential interventions and the evaluation of risk outcomes
in terms of benefits and costs) in order to guide the potential scientific,
epidemiological and regulatory considerations involved. Such considerations
would include whether or not the pathogen is novel and previously unknown,
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and the extent to which the precautionary principle may therefore prevail. In
cases where an infection is re-emerging there would be far fewer uncertainties in
relation to potential interventions and outcomes. Since considerable differences
exist between low-, middle- and high-income countries not only in the experience
of using decision tools but also in the considerations to be taken into account,
the pilot testing of such a tool should include users in a range of countries.
Dr Nübling then informed the Committee of the outcomes of a WHO
Consultation on the First WHO International Standard for anti-rubella
immunoglobulin held in Geneva in June 2017. The current international
standard was comprised of polyclonal antibodies and was used in standardizing
the different results of assays of different design and purpose. This raises issues
when working with the immunity threshold of 10 IU/ml and questions have been
raised as to the commutability of this International Standard. It was concluded
that the International Standard is well characterized and should continue to be
used but that the lack of commutability must be clearly communicated to users
in the instructions for use (IFU). However, uncertainty remained as to whether
the immunity threshold was still appropriate and whether this standard should
still be used in qualitative assays of high specificity. Further work would be
necessary to resolve the outstanding issues and, depending on the conclusions
reached, the revision of WHO guidelines and regulatory requirements may need
to be considered.
Dr Nübling concluded by outlining the documents and measurement
standards in the areas of blood products and in vitro diagnostics to be considered
for adoption and establishment respectively by the Committee this year. These
consisted of two WHO prequalification guidance documents, one on the
performance evaluation of HIV rapid diagnostic tests (RDTs) and another on
establishing the stability of in vitro diagnostic medical devices (see sections 3.3.2
and 3.3.3 respectively); three measurement standards for blood products and
related substances (see sections 6.1.1–6.1.3) and 10 measurement standards for
in vitro diagnostics (see sections 7.1.1–7.1.10).

The Committee thanked Dr Nübling for his wide-ranging report and
after making a number of observations and comments looked forward to being
updated on the progress made in these important areas of public health at its
next meeting.
2.2 Reports
2.2.1 Report from the WHO Blood Regulators Network
Dr Christian Schärer began by reminding the Committee that the objectives
of the BRN were to identify issues and share expertise and information,
to promote the science-based convergence of regulatory policy (including
through fostering the development of international consensus on regulatory
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approaches) and to propose solutions to specific blood-related issues. After

presenting an overview of the current BRN membership, Dr Schärer informed
the Committee that a new BRN Chair, Dr Anneliese Hilger (PEI, Germany), had
recently been elected.
Following its face-to-face meeting during the previous Committee
meeting in October 2016, four teleconferences and several further meetings had
been held. At these meetings, discussions had been held on a wide range of
topics, including the sharing of information on the hepatitis A and Zika virus
situations, the storage of red blood cells, follow-up of the results of Ebola virus
(EBOV) clinical trials using convalescent plasma in Guinea, national decision-
making on donor deferral for men who have sex with men, and raising awareness
of the need to promote the topic of blood products.
Dr Schärer then outlined a number of BRN work products which
included a BRN Position Statement on Collection of blood for transfusion in
the setting of a vaccination campaign against yellow fever, and a BRN Position
Paper on Use of convalescent plasma, serum or immune globuline concentrates
as an element in response to an emerging virus. The latter paper was a follow-up
to previous BRN position papers on the use of convalescent plasma in specific
situations, and takes a more generic approach. Both papers had been published
on the WHO BRN website. Other work products included efforts made to
strengthen stakeholder involvement in BRN activities.
During this period BRN had also provided support for the integration
of the BRN assessment criteria for a national blood regulatory system into the
WHO global benchmarking tool (GBT) for evaluation of national regulatory
systems (see section 2.4.2). During a workshop held in Geneva in August 2017,
BRN representatives from the Food and Drug Administration (the USA), PEI
(Germany), the Ministry of Health, Labour and Welfare (Japan) and Swissmedic
(Switzerland) and other workshop participants reviewed a large number of
datasets and identified a number of gaps in the current version of the BRN
assessment criteria. The integration of BRN criteria into the WHO GBT is
intended to be completed by the end of 2017. BRN had also organized a workshop
on blood products at the ICDRA meeting held in South Africa in December
2016 and was involved in the WHO assessment of the blood regulatory system in
Zambia using the BRN assessment criteria (see section 2.1.3 above). A number
of additional BRN work products and engagements were then outlined.
Dr Schärer concluded by informing the Committee that this year the
BRN face-to-face meeting had been held immediately prior to the meeting of the
Committee. Among the main outcomes of this meeting had been the election of
the new BRN Chair and development of the 2017–2018 BRN workplan, which
would incorporate the provision of specific BRN support to the WHO Regional
Office for Africa.
The Committee thanked Dr Schärer and noted his report.
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2.2.2 Report from the WHO network of collaborating centres on

standardization and regulatory evaluation of vaccines
Work of the Core Expert Group
Dr Lindsay Elmgren presented a progress report on the work of the network’s
Core Expert Group (CEG). The Committee was reminded that the original
rationale for the establishment of the CEG was to reduce and streamline the
workload of the Committee in the vaccines area. At present, the reviewing of large
numbers of proposals for new or replacement measurement standards consumed
a significant amount of Committee time and it had been suggested that relevant
expertise within the network could be utilized to expedite both the review and
priority-setting processes. At its previous meeting, the Committee had agreed
that as a first step the CEG could pre-review selected measurement standards.
Dr Elmgren reported that six CEG WebEx meetings had been held at
regular intervals between September 2016 and September 2017. The specific
issues discussed included the structure of the CEG, the feasibility of expanding
its scope to include written standards and biotherapeutic products, and the likely
timeframe available for pre-review. During discussions, it became clear that the
proposed timeframe might be problematic and that expanding the scope of
the CEG might conflict with the Terms of Reference of individual WHOCCs.
Dr Elmgren concluded by outlining some of the options for the next steps, along
with the potential advantages and disadvantages of each option, and closed by
presenting an overview schematic of the envisaged CEG process.
The Committee thanked Dr Elmgren for his report and provided
a number of inputs in relation to the role of the CEG process in facilitating
its work.
Proposal to simplify the structure of type-specific vaccine written standards

Dr Elmgren reported on discussions which had been undertaken by the network
regarding the most practical way of developing and structuring new type-
specific vaccine guidelines. It had been concluded that a case could be made
for simplifying guidelines on the quality, safety and efficacy of specific vaccine
types by focussing only on the major points to be considered for such vaccines.
A proposal was therefore made to change and simplify the present structure of
product-specific vaccine guidelines – a structure which could be considered
duplicative and which had now been in use for many years. It was proposed that
a pilot guidelines document be developed for group B meningococcal vaccines
focussing only on the key points to consider, and possibly taking the form of a
discussion paper.
Although the Committee was sympathetic to this suggestion, and
considered that drafting groups might indeed welcome a reduction in workload
when preparing such guidelines, little support was expressed for this proposal.
Although the idea might seem to have merit in principle, there were concerns
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that in practice such a document would not meet the needs of users of WHO
guidelines, who often required a comprehensive document – and one which
could potentially be adopted as national guidelines. In addition, WHO guidelines
were also used to inform decisions on the WHO prequalification of vaccines
and thus on United Nations procurement.
2.2.3 Report from the WHO network of collaborating centres

for blood products and in vitro diagnostics
Dr Clare Morris reported on a number of recent activities of the network. Three
WebEx meetings on blood products and IVDs had been held to address issues
prior to the 2017 meeting of the Committee, help set the meeting agenda and
share practices between network member WHOCCs. Several of the issues
covered would be discussed in detail during the upcoming Blood Products and
IVD track sessions. However, Dr Morris raised the concern that although some
standards (such as anti-rubella serum and new diagnostic reagents for HPV)
had been established in the Vaccines track, the use of these materials also had
implications for the diagnosis of infectious disease (see section 3.3.4 below).
There was therefore a need to ensure awareness of such potential overlaps
between the diagnostics and vaccines fields, which are currently dealt with
independently in the different tracks of Committee meetings.
Dr Morris then outlined a number of issues discussed at the 2017
meeting of the Standardisation of Genome Amplification Techniques (SoGAT)
group with particular relevance to the current meeting.
The Committee thanked Dr Morris for her report and requested
clarification on one specific point raised at the SoGAT group meeting in relation to
the need for standardization in the expanding area of next generation sequencing.
Discussion also took place on the need for improved interaction between the
currently separate tracks of Committee meetings and on the network’s role in
assisting the work of the Committee by means of the preparatory WebEx meetings.
2.3 Feedback from custodian laboratories

2.3.1 Developments and scientific issues highlighted by
custodians of WHO biological reference preparations
The Committee was informed of recent developments and issues identified by the
following custodians of WHO biological reference preparations.
National Institute for Biological Standards and Control

(NIBSC), Potters Bar, the United Kingdom
Dr Christian Schneider informed the Committee that NIBSC currently holds
more than 800 000 ampoules/vials of 354 WHO international standards.
During the period October 2016 to October 2017, 351 different standards were
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distributed to 75 different countries. Of the standards distributed, approximately

66% went to commercial organizations and the rest to other organizations,
including national control authorities and universities. On the assumption that
the projects to be submitted to the Committee at this meeting are endorsed
NIBSC will have 83 active projects under way for new or replacement WHO
international standards. Twelve of these projects involve biotherapeutic
products, eight involve blood products, 20 involve IVDs and 22 involve vaccines
– with 21 projects awaiting confirmation and assignment.
Among the issues raised by Dr Schneider was the difficulty sometimes
experienced in meeting the current deadlines for establishment by the
Committee of WHO standards for emerging/priority pathogens. In the past, the
Committee had demonstrated flexibility and had accepted the late submission
of study reports (for example, in establishing interim EBOV standards).
Dr Schneider suggested that, going forward, consideration should be given to
developing alternatives to the current fixed cycle of submission/establishment
of WHO international standards.
Dr Schneider also highlighted the increasing difficulty of sourcing
biological materials for the development of candidate standards and/or for
inclusion as samples in collaborative studies. Such difficulties can delay the
development and availability of both new and replacement standards, which in
turn can negatively impact upon public health worldwide. Despite a number of
concerted efforts made by NIBSC, accessing suitable biological materials would
remain an ongoing issue and the support of the Committee, regional centres and
other WHOCCs would be vital. Dr Schneider also pointed out that publishing
the collaborative study reports in high-impact scientific journals targeted at a
relevant audience was one of the best ways to promote the availability and use
of WHO international standards. Dr Schneider enquired about the feasibility of
WHO waiving copyright to allow for such publication.
The Committee thanked Dr Schneider and noted his report.
European Directorate for the Quality of Medicines &

HealthCare (EDQM), Strasbourg, France
Dr Karl-Heinz Buchheit outlined a number of recent EDQM activity areas in
biological standardization, including the European Pharmacopoeia standards,
international standards for antibiotics and the biological standardization
programme, in which WHO has Observer status. The Committee was reminded
that EDQM is the custodian centre for international standards for antibiotics – a
responsibility it took over from NIBSC in 2006. At present, 23 such standards are
available for “old” antibiotics, with several of these antibiotics being on the WHO
Essential Medicines List. Since 2006, eight replacement standards had been
established, and in 2017 the Committee was being asked to endorse a proposal
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to establish the Third WHO International Standard for erythromycin due to low
stocks of the existing standard.
Dr Buchheit then discussed a number of recent activities of the EDQM
biological standardization programme – the goal of which is to establish
European Pharmacopoeia biological reference preparations and to standardize
methods. The programme of work is established by a Steering Committee and,
whenever possible, collaboration and common projects were undertaken with
WHO and non-European partners. Current EDQM projects of potential interest
to the Committee included the ongoing development of an ELISA to replace the
current in vivo potency test for human rabies vaccines.
Dr Buchheit reminded the Committee that the development of
alternatives to animal experiments remained a major EDQM commitment in
line with European Union directives. WHO was once again strongly urged to
consider the incorporation of the 3Rs principles (Replacement, Reduction,
Refinement) into its written standards and other guidance where appropriate.
Such a step would be key to the global acceptance of these principles. The
Committee was also reminded that one of the main outcomes of a 2015
International Alliance for Biological Standardization meeting on the 3Rs
concept was a formal request to WHO to initiate steps to delete the abnormal
toxicity test from all its written standards.
Dr Buchheit concluded by proposing that the Committee evaluate the
possibility of its more active involvement in the validation of alternative quality
control assays aligned with the 3Rs principles. In addition, there was need for the
Committee to reflect upon the consequences of replacing in vivo potency assays
with in vitro assays.
The Committee thanked Dr Buchheit and noted his report.
Paul-Ehrlich-Institut (PEI), Langen, Germany

Dr Heidi Meyer provided the Committee with an update on the activities of PEI
in the development of WHO international standards. This year, two candidate
standards would be proposed for establishment by the Committee. One of these
was a proposed First WHO International Standard for anti-cytomegalovirus
immunoglobulin G for serological assays, which had been developed to improve
the comparability of the divergent results generated by current assays. PEI
was also proposing the establishment of a First WHO International Standard
for chikungunya virus RNA for NAT-based assays. Dr Meyer outlined to the
Committee the main steps and timeframe involved for each of these standards.
In addition, a number of other standards projects were ongoing at PEI.
These included the development of a WHO reference panel for hepatitis E
virus (HEV) antibodies, a WHO reference reagent for anti-chikungunya virus
(CHIKV) immunoglobulin M (IgM) and immunoglobulin G (IgG), and the
15
further extension of the First WHO Repository of platelet transfusion relevant

bacterial strains.
The Committee thanked Dr Meyer and noted her report.
Center for Biologics Evaluation and Research (CBER), Silver Spring, MD, the USA
Dr Jay Epstein informed the Committee that CBER was currently involved in
several international projects evaluating new vaccine-related standards. These
included a study into the potential use of a pneumococcal reference serum as a
standard in the pneumococcal opsonophagocytosis assay (OPA), the international
collaborative study to establish the First WHO International Standard for Zika
virus antibodies (see section 7.1.10) and the international collaborative study
to establish the First WHO International Standard for antiserum to respiratory
syncytial virus (see section 8.1.5). CBER had also provided support for the
development of new vaccine technologies, including the development of a
sequence database to facilitate detection of novel adventitious viruses using next
generation sequencing (NGS). CBER had also participated in a series of projects
on the standardization of influenza vaccines.
In the area of blood products, CBER had distributed a number of both
European and WHO international standards. The limited supply and restricted
distribution of one such standard – the Second WHO International Standard
for thrombin – was highlighted and a recommendation made to develop a
replacement standard. CBER also recommended developing an international
standard for human activated factor X (FXa) to support the development of
genetically modified FXa therapies.
CBER was also actively involved in developing reference preparations for a
range of different IVDs. FDA support had also been provided in the development
of technical guidance documents on the WHO prequalification of IVDs, including
one on HIV rapid diagnostic tests (RDTs) scheduled for consideration by the
Committee this year (see section 3.3.2). In addition, funding provided through
a CBER-WHO Cooperative Agreement was being used to support the revision

of the current WHO EUAL procedures introduced as part of strengthening
regulatory preparedness for public health emergencies of international concern.
The Committee thanked Dr Epstein for his report and noted the potential
importance of NGS as a tool for regulatory evaluation in the future.
2.4 Cross-cutting activities of other WHO committees and groups

2.4.1 Update from the WHO Expert Committee on
Specifications for Pharmaceutical Preparations
Dr Sabine Kopp presented an update of the work of the WHO Expert
Committee on Specifications for Pharmaceutical Preparations (ECSPP). The
scope of ECSPP activities covers the life-cycle of medicines from development
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to delivery to the patient. Within this overall context, the ECSPP has to date
actively overseen the development and publication of 85 official WHO guidance
texts and guidelines on medicines, quality assurance and related regulatory
standards. A new CD‑ROM including all guidelines and the International
Pharmacopoeia had been produced. In 2017, seven new or revised annexes
were to be proposed for adoption by the ECSPP, which meets each year during
the same week as the Committee.
Dr Kopp outlined a number of cross-cutting issues across the two Expert
Committees, including: (a) good regulatory practices (GRP); (b) the definition of
a stringent regulatory authority (SRA); (c) the use of a collaborative procedure
for medical products; (d) aspects of GMP; (e) issues related to the transition from
microbiological to physicochemical assays in monographs on capreomycin API
and products; (f) the need for additional data for products already on the market;
and (g) comparison between microbiological and chemical methods of analysis.
Dr Kopp indicated that some of these issues could appear on the agenda of the
Expert Committee on Biological Standardization in future and that a working
group might be established to deal with some of them.
Dr Kopp then informed the Committee that the ECSPP also oversees
the revision of the International Pharmacopoeia and the establishment
of International Chemical Reference Substances (ICRS). In 2017, 17 new
specifications and general texts were adopted for inclusion in the International
Pharmacopoeia and four new ICRS established.
The Committee thanked Dr Kopp for the update on the activities of the
ECSPP. During subsequent discussion it was noted that several of the above
issues of common interest to the two Expert Committees will require proactive
coordination, and the example of the WHO Global Benchmarking Tool was
given as an issue that was to be discussed this year at both Expert Committees.
2.4.2 WHO Global Benchmarking Tool

Dr Alireza Khadem informed the Committee of the progress made in the
development of the unified WHO Global Benchmarking Tool (GBT) for assessing
national regulatory systems. The aim of the project was to align all of the various
benchmarking tools developed by different WHO programmes and by other
agencies. It is anticipated that this will allow for greater alignment of policy
and scope, and greater consistency in standards and approach. This will lead to
improved outcomes and impact, and to a reduced burden of assessments, costs
and duplication for WHO Member States, as well as for WHO and its partners
working in regulatory systems strengthening.
The project started in 2013 with the unification of the WHO vaccines and
medicines benchmarking tools and is now progressing towards the integration
of the Pan American Health Organization (PAHO) assessment tools, medical
devices assessment tool and blood products assessment criteria. The Committee
17
heard that there had been several revisions of the proposed tool, extensive global
discussion and a number of pilot studies, with work still ongoing. The integration
of the blood products assessment criteria into the WHO GBT had involved
support from the WHO BRN.
The Committee thanked Dr Khadem for his report and raised a number
of points. The value of a harmonized tool was acknowledged and it was suggested
that linking NRA assessment to the WHO prequalification scheme may provide
an additional incentive for countries to undertake an NRA assessment. The
Committee was informed that internal WHO discussions along these lines had
already been scheduled. It was further suggested that reliance between regulatory
agencies might become less of an issue once the results of assessment are widely
accepted. A number of other important points were then raised in relation to
the use of the unified tool. Topics discussed included the need for transparency
in the criteria used to define a stringent NRA, whether the definition of blood
included plasma and plasma-derived products, and the selecting of countries for
piloting of the tool. The need to ensure precision in the questions asked was also
highlighted, for example to ascertain whether all relevant guidance from WHO
and ICH was being implemented rather than simply being in place.
2.4.3 Development of WHO guidelines on good regulatory practices

Dr Mike Ward reported on the progress being made in the development of
WHO GRP guidelines for NRAs. This initiative had been undertaken in response
to requests from WHO Member States through ICDRA and at various WHO
consultations for guidance on best practices for collaboration and cooperation
between NRAs – in areas such as information exchange, joint assessments and
inspections, and activities aimed at reducing duplication. This foundational
document would apply internationally accepted GRP principles to the regulation
of all medical products, and was intended for a range of audiences, including
senior policy-makers responsible for the formulation of health policies, laws
and regulations, NRAs and other interested parties. Dr Ward then outlined the
concept of GRP as:
Internationally recognised processes, systems, tools and methods for
improving the quality of regulations. GRP systematically implements
public consultation and stakeholder engagement as well as impact
analysis of government proposals, before they are implemented to
make sure they are fit for purpose and will deliver what they are set
out to achieve.4
4
See: http://www.oecd.org/gov/regulatory-policy/asean-oecd-good-regulatory-practice-
conference-2015.htm
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The Committee was informed that a draft document had been developed
and subjected to public consultation. The document included sections on
background and scope, on the principles of GRP and on the implementation of
regulations. Three appendices cover the details of regulatory impact assessment,
legal instruments and international regulatory cooperation. Although the
draft document was well received, and considered to be helpful for regulatory
convergence, public consultation indicated a need for streamlining and other
refinements of the content. Further inputs from a consultation held in July 2017
included requests for the text to be made more understandable and usable by its
intended readers, and to become more guidance-like in nature, while ensuring
that key messages were both clear and relevant to LMIC. Other suggestions
included the addition of practical tools such as examples and checklists to assist
in the implementation of the guidance provided. Revision was now underway
and it was expected that a final draft would be available for presentation to the
ECSPP for endorsement in 2018. It had been suggested that the path forward
should also include exploration of a pilot phase that would serve to validate the
relevance and usability of the guideline in LMIC.
The Committee thanked Dr Ward for his report and raised a number
of points in relation to the language used in the document, its content and
the applicability of some of the specific guidance given, especially in LMIC.
A number of potential challenges in implementation were also raised, including
the need to avoid damaging existing national systems, and the need for high-
level advocacy and political engagement in this area.
2.4.4 Snake-bite envenoming

Dr David Williams reminded the Committee that antivenoms are immunoglobulin
preparations manufactured from equine (or ovine) plasma and can be either
monospecific to individual snake species or polyspecific. Currently, antivenoms
are the only effective therapy against snake-bite envenoming but because
of regulatory deficiencies in affected regions, falling numbers of antivenom
producers and the fragility of current production systems, envenoming had
become a crucial global health issue. Following international advocacy efforts,
snake-bite envenoming became a category one neglected tropical disease (NTD)
in May 2017.
There was now an ongoing initiative to address the situation and the
Committee was provided with an update of current WHO activities in this area.
The period 2016–2017 had been one of rapid progress, new opportunities and
key challenges. The Committee was reminded of its adoption of the updated
WHO Guidelines for the production, control and regulation of snake antivenom
immunoglobulins at its meeting in 2016. Other activities had included the
19
updating of the WHO antivenoms website 5 and the revision and expansion of
antivenom manufacturer data, which now included package inserts and published
literature citations for each product. In addition, the names and photographs of
snake species are being updated along with information on their geographical
distribution. Links to clinical treatment information resources are also now being
incorporated into the WHO website.
A WHO assessment of antivenoms intended for use in sub-Saharan Africa
had also been initiated. A call for applications for the evaluation of products
suitable for use in this region resulted in the submission of nine applications.
Following an initial assessment and selection process, five products are now
undergoing laboratory evaluation, including physicochemical characterization,
specific venom immune-recognition and potency testing. Corresponding GMP
inspections of production facilities in Costa Rica, India, Mexico, South Africa
and the United Kingdom are also ongoing. Once these studies and inspections
are completed, WHO will be in a position to recommend suitable products to
procurement agencies.
Where GMP deficiencies have been found, manufacturers had
demonstrated a willingness to respond but resources are very limited and further
support from WHO and other stakeholders is needed. Dr Williams indicated that
a roadmap for addressing snake-bite envenoming issues is being developed and
will be supported by a WHO technical working group. A stakeholders meeting
is planned for 2018 prior to the publication of the roadmap but resources and
funding will be required if the plan is to be implemented. There is however
growing political will to address this issue and a draft World Health Assembly
resolution has been developed, led by Costa Rica and supported by 30 other
WHO Member States. It is expected that the resolution will go to the WHO
Executive Board in January 2018 and to the World Health Assembly in May 2018.
The Committee thanked Dr Williams for his report and discussed
some of the issues raised. In response to one query, Dr Williams pointed out
that although there are around 260 medically relevant snake species there was
also some degree of commonality of toxins. The production of antisera against
the venoms of around 100 different snake species might allow for the treatment
of most snake-bites. It should also be possible to prepare polyvalent sera that
can neutralize several different snake venoms. However, the characterization of
antivenoms based on ED 50 was challenging. It was further pointed out that the
standardization of venom would be crucial, with the importance of reference
venoms having been identified at the previous ICDRA meeting and efforts
already under way in some countries to produce reference standards. Both
the improved characterization of venoms to help assure the quality of the raw
5
See: http://www.who.int/bloodproducts/snake_antivenoms/en/
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material used to raise animal antisera and the development of in vitro assays
for potency testing to replace ED 50 testing would likely result in significant
improvements in future. One other issue of concern was that animal welfare
standards in some countries were not always sufficiently addressed which had
led to legitimate concerns by animal-rights campaigners and which potentially
jeopardized antivenom manufacture.
2.4.5 Update from the WHO Product Development

for Vaccines Advisory Committee
Dr David Kaslow provided the Committee with a brief overview of the outcomes
of the 2017 WHO Product Development for Vaccines Advisory Committee
(PDVAC). A range of vaccine development and related issues across a wide range
of pathogen areas had been addressed. This had involved discussion of current
developments in the areas of HIV, tuberculosis, malaria, influenza, gonorrhoea,
RSV, Group B streptococci, enteropathogenic Escherichia coli, shigella, herpes
simplex virus (HSV) and CMV. In addition, following the publication by WHO
in 2017 of the first-ever list of antibiotic-resistant pathogens that pose the greatest
threat to human health, Dr Kaslow reported that PDVAC had recommended the
development of a quantitative framework through which the public health impact
of vaccines in combating antimicrobial resistance (AMR) could be evaluated.
A number of cross-cutting issues were then identified by Dr Kaslow with
particular relevance to the activities of the Committee. For example, PDVAC had
expressed an interest in the development of several candidate vaccines designed
to elicit antibodies to conserved epitopes on the haemagglutinin head or stem
of influenza viruses. If successful, these would become the next generation of
influenza vaccines. It was also noted that the concept of heterologous prime-
boost was under consideration for a number of candidate vaccines, including HIV
vaccines. The question had therefore been raised as to whether the Committee
might need to provide guidance on the testing or licensing strategies for vaccines
using such regimes. PDVAC had also discussed the progress made in developing
the new generation of DNA and RNA-based vaccines, a number of which are
now in pre-clinical and early clinical development, including candidate vaccines
for Zika and influenza viruses. In relation to this, PDVAC had asked if the
Committee needed to update current guidelines on nucleic acid based vaccines
to include RNA and considerations for maternal immunization. Among the other
main topics discussed were developments in passive immunization using mAbs.
It was noted that a number of such products were in development against an
increasing number of pathogens, including HIV, RSV, Staphylococcus aureus and
rabies virus. As PDVAC had recommended evaluation of the technical, regulatory
and commercial barriers to the development, licensure and availability of mAbs
specifically for use in LMIC, the need for guidance from the Committee on the
testing and licensing of mAbs for use in neonates was also raised.
21
The Committee thanked Dr Kaslow for his helpful update and noted the
specific points raised for its consideration. In particular, the interest expressed
by PDVAC in the new projects outlined complemented that of the Committee.
An international standard for RSV antiserum along with proposed new projects
on international standards for candidate influenza vaccines based on conserved
antigens were scheduled for consideration by the Committee this year (see
sections 8.1.5 and 8.2.4/5). It was also pointed out that WHO guidance is already
available on the clinical evaluation of vaccines to be used in heterologous prime-
boost regimens.6 The issue of recent developments in nucleic acid based vaccines
was another topic of common interest to both PDVAC and the Committee, with
a WHO consultation on this subject planned for 2018.
2.4.6 Pilot WHO prequalification of biosimilar monoclonal antibodies

Dr Deus Mubangizi reminded the Committee that, as outlined in an earlier
presentation (see section 2.1.1 above), WHO was intending to pilot the
prequalification of two SBPs as part of efforts to improve access to biotherapeutics
by extending the considerable experience of SRAs to NRAs in LMIC. The two
SBPs selected for the pilot project – trastuzumab and rituximab – were now
included in the WHO Model List of Essential Medicines and manufacturers
would be invited to send expressions of interest.
Dr Mubangizi went on to provide the Committee with further details
of the project. The pilot prequalification would assess SRA-approved originator
products, SRA-approved SBPs and SBPs approved by non SRAs, using one SRA-
approved reference biotherapeutic product as a comparator. Project procedures
had been developed and were now undergoing public review and comment. For
WHO prequalification purposes, candidate products to be provided through the
United Nations for use in different countries must meet the quality, safety and
efficacy criteria set out in relevant WHO guidelines,7, 8 including compliance
with GMP, GCP and good distribution practices.
6
Guidelines on clinical evaluation of vaccines: regulatory expectations. In: WHO Expert Committee on
Biological Standardization: sixty-seventh report. Geneva: World Health Organization; 2017: Annex 9
(WHO Technical Report Series, No. 1004; http://www.who.int/entity/biologicals/expert_committee/WHO_
TRS_1004_web_Annex_9.pdf?ua=1, accessed 6 April 2018).
7
Guidelines on evaluation of similar biotherapeutic products (SBPs). In: WHO Expert Committee on
Biological Standardization: sixtieth report. Geneva: World Health Organization; 2013: Annex 2 (WHO
Technical Report Series, No. 977; http://who.int/biologicals/publications/trs/areas/biological_therapeutics/
TRS_977_Annex_2.pdf, accessed 6 April 2018).
8
Guidelines on evaluation of monoclonal antibodies as similar biotherapeutic products (SBPs). In: WHO
Expert Committee on Biological Standardization: sixty-seventh report. Geneva: World Health Organization;
2017: Annex 2 (WHO Technical Report Series, No. 1004; http://who.int/biologicals/biotherapeutics/WHO_
TRS_1004_web_Annex_2.pdf?ua=1, accessed 6 April 2018).
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General
Two pathways had been proposed for the pilot project – an abridged
assessment of SRA-approved innovator products or SBPs (which may lead to
waivers for requirements) and full assessment of SBPs already registered by non-
SRAs using the SRA-approved reference biotherapeutic product as a comparator
and marketed in the authorized country. Dr Mubangizi provided details of the
proposed process, which would involve the concept of reliance and the exchange
of relevant information between WHO and the SRA or applicant. One major issue
arising from the public consultation process was the need to better distinguish
between the two assessment pathways – that is, for applicants with products
approved by an SRA and those with products approved by other NRAs.
The Committee thanked Dr Mubangizi for his presentation and looked
forward to being updated on the outcome of this project.
2.4.7 Model NRA Lot Release Certificate for prequalified vaccines

Dr Ute Rosskopf informed the Committee of a proposal, presently under
discussion, to develop a unified certificate for the lot release by NRAs of WHO
prequalified vaccines. At present, NRA lot release certificates vary in their content
depending on the vaccine, and different certificates may be in use for vaccine
released onto domestic or export markets. The objective of developing a common
lot release certificate would be to harmonize release practices and to increase
acceptance of vaccine lot release certificates by recipient countries.
Dr Rosskopf highlighted that vaccine-specific guidance and model
lot release certificates were typically provided by WHO written standards for
vaccines, with specific advice on certificate issuance also available.9 In addition,
the WHO National Control Laboratory (NCL) Network for Biologicals (WHO-
NNB) had been established in 2016 (see section 2.4.9 below), the objectives of
which included harmonizing lot release standards and practices, and fostering
reliance on member NCL lot release to reduce redundant testing in recipient
countries. The Committee was informed that in collaboration with a WHO-NNB
working group, WHO had now developed a proposed template for a model lot
release certificate for prequalified vaccines. Dr Rosskopf outlined the format and
content of the template certificate which had been provided to the Committee for
its consideration.
The Committee thanked Dr Rosskopf for drawing its attention to this
development. The view was expressed that numerous issues remained to be
resolved in moving towards a harmonized model lot release certificate for
9
Guidelines for independent lot release of vaccines by regulatory authorities. In: WHO Expert Committee
on Biological Standardization: sixty-first report. Geneva: World Health Organization; 2013: Annex 2 (WHO
Technical Report Series, No. 978; http://www.who.int/biologicals/TRS_978_Annex_2.pdf?ua=1, accessed
8 April 2018).
23
prequalified vaccines. The Committee also noted that lot release certificates
needed to convey relevant information to the recipient on the vaccine lot
released which may not be covered in the general format presented. One point
to consider would be whether lot release was on the basis of independent testing
by the releasing NCL or on the basis of testing by the manufacturer followed by
review of the lot summary protocol by the releasing NRA. The latter procedure
is considered to be the minimum basis for vaccine lot release and this may be
an important consideration for some NRAs. There was also the question of the
impact of using a harmonized model lot release certificate on the product-specific
model lot release certificates usually provided in WHO Recommendations and
Guidelines for vaccines.
2.4.8 Planned proficiency testing study of a standardized

method for determining total and free saccharide
content of Hib liquid combined vaccines
Dr Rosskopf reminded the Committee that a key test in the quality control of
Haemophilus influenzae type b (Hib) conjugate vaccines is measurement of the
quantities of conjugated and unconjugated polyribosyl-ribitol-phosphate (PRP)
present to ensure that these are within the approved specifications. Only PRP
that is covalently bound to the carrier protein – that is conjugated PRP – is
immunologically important for clinical protection. However, testing liquid-
formulated combination vaccines containing Hib conjugate is challenging and
different protocols are used by different manufacturers and different control
laboratories.
The Committee was informed of plans by WHO to undertake a
proficiency testing study on the use of High-Performance Anion-Exchange
Chromatography with Pulsed Amperometric Detection (HPAEC-PAD) for the
determination of total and free saccharide content of Hib vaccines. This study
will aim to evaluate the performance of laboratories in applying a previously
identified standardized HPAEC-PAD test protocol that is applicable to all eight

WHO prequalified vaccine combinations containing the whole cell pertussis
component. The collaborative study is planned to start in the first quarter of 2018
and will involve around 25–30 participating laboratories.
The Committee thanked Dr Rosskopf for her presentation and looked
forward to being updated on the outcome of the study in due course.
2.4.9 Vaccine prequalification – establishment of the WHO-NNB

Dr Rosskopf provided the Committee with an update on recent developments
in the area of WHO vaccine prequalification activities. To date, the independent
laboratory testing of vaccines both pre- and post-prequalification had been
undertaken by a limited number of WHO-contracted NCLs. However, the present
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system faced a number of challenges, including: (a) the increasing number of

sophisticated and complex vaccines; (b) the globalization of the vaccine industry
(with an increasing number of production sites); (c) the limited capacities of
regulatory authorities in both developed and developing countries; and (d)
redundant testing by multiple countries leading to delays in vaccine supply and
subsequent shortages. In addition, there number of applications for vaccine
prequalification is increasing and pre- and post-prequalification quality control
testing is both costly and demanding.
In response to these challenges, and in an effort to improve the efficiency
of the WHO prequalification process and utilization of resources, the WHO
Vaccine Prequalification Team was now working on a number of initiatives.
These included efforts to: (a) harmonize test methods; (b) provide hands-on
training in quality control methods; (c) reach agreements with manufacturers
of prequalified vaccines to enable confidential reporting of lot release data by
NCLs to WHO; (d) use the NCL of the country of production for quality control
testing; and (e) shortening lead times for vaccine shipments.
Against this backdrop, the need for a WHO network of NCLs involved
in prequalification testing had been raised by WHO laboratories and other key
stakeholders. At a 2016 meeting, attended by representatives from 21 NCLs,
manufacturers’ associations and EDQM, it was agreed that a WHO National
Control Laboratory Network for Biologicals (WHO-NNB) should be established.
This proposal then received subsequent support in the form of a recommendation
adopted by the 17th ICDRA held in Cape Town, South Africa in late 2016.
Dr Rosskopf reported that the WHO-NNB terms of reference had now been
developed and information-sharing agreements were being worked out. The first
meeting of the new network was scheduled to take place in India in late 2017.
The Committee thanked Dr Rosskopf for her presentation and noted
these developments.
2.5 Strategic issues

2.5.1 Standards for priority pathogens for public health emergencies
Dr Martin Friede reminded the Committee of the new WHO initiative –
the Blueprint for Research and Development: Responding to Public Health
Emergencies of International Concern (R&D Blueprint) – which had been
brought to the attention of the Committee at its previous meeting. The R&D
Blueprint had been developed in the light of previous epidemics, particularly
the 2014–2016 Ebola epidemic, in order to accelerate R&D preparedness
and effective collaboration in advance of any new epidemic. A list of priority
pathogens had been developed and roadmaps constructed, with target product
profiles for vaccines and diagnostics now under development.
25
A number of gaps in regulatory preparedness had also been identified

at a recent WHO Informal Consultation.10 These included a lack of coordinated
emergency regulatory processes, weaknesses and lack of capacity in drug regulatory
systems, limited capacity and experience in stakeholder communication, poor
engagement of product developers with affected regulators and weakness in the
regulation of supply chains. Meeting participants had also reviewed the WHO
EUAL procedure developed to provide regulatory decision-making support to
impacted countries and United Nations procurement. Details of the range of
outcomes and proposed actions resulting from the review process can be found
in section 2.1.1 above.
The Committee was informed that the R&D Blueprint also covers the
development of tools used to evaluate vaccines, as well as tools for collaboration,
data exchange and sample sharing. Of particular relevance to the work of the
Committee was the development of reference reagents for priority pathogens and
the potential need for new norms and standards tailored to the epidemic context.
The Committee thanked Dr Friede for the update provided and discussed
a number of the issues raised. It was pointed out that international standards
and reference reagents were now available in the case of both Zika and Ebola
but were seemingly not well used. There was thus a need to consider how best to
advertise their availability and encourage their implementation. The importance
of having reference materials available during early vaccine development and
evaluation was also highlighted, and could be one of the goals of the Coalition
on Epidemic Preparedness Innovations (CEPI). The Committee heard that CEPI
activities would indeed involve the promoting and funding of the development of
standards, reagents and assays, initially for a limited number of priority pathogens.
It was considered that collaboration between CEPI and the Committee would
be vital in achieving a coordinated and timely outcome at the global level. The
need for developing WHO guidance documents for priority pathogen vaccines
or further guidance on prime-boost vaccines should also be kept under review

as the R&D Blueprint evolves. In addition, consideration should be given to
the development of more flexible and dynamic approaches for developing and
establishing standards for the quality, safety and efficacy of products intended for
use in public health emergencies.
10
WHO Informal Consultation on options to improve regulatory preparedness to address public health
emergencies. Geneva, 17–19 May 2017. Meeting report. Geneva: World Health Organization; 2017
(http://www.who.int/medicines/news/2017/PHEmeeting-reportIK-EG16_Nov_2017.pdf, accessed 8 April
2018).
26
General
2.5.2 International standards and reference preparations

– revision of TRS 932 Annex 2
Dr Clare Morris informed the Committee that discussions held during the
2017 meeting of the Standardisation of Genomic Amplification Techniques
(SoGAT) group had highlighted a number of issues relevant to the ways in
which WHOCCs produce, evaluate and distribute international standards and
reference preparations. Current approaches to developing and replacing WHO
international standards are based upon guidance provided by WHO in 2004.11
Dr Morris indicated that the points raised could impact upon both the content
of the current guidance and the procedures established in different WHOCCs.
The question had been raised as to whether manufacturers have to
recalibrate their (otherwise unchanged) systems when a replacement international
standard becomes available. Current approaches vary, with some manufacturers
keeping their system calibrated to previous versions of the standard while
others recalibrate to the most current. However, WHO assures the “continuity
of unitage” and further guidance in this area is sought. Another point raised
was whether replacement standards should be calibrated against a stockpile of
the first international standard or against the most recent batch of international
standard. Although calibrating against the first (or other early) standard would
likely prevent potential drift of the IU, current practice is to assess candidates
against the current standard, potentially favouring drift in the IU across batches.
One implication of recommending the first option would be the need for the long-
term storage of ampoules of the first international standard at low temperature
(for example, at −80 °C) for future replacement studies. This requirement would
need to be communicated in a guidance document and implemented.
The proposal was also made that that when a standard is replaced, only
commercial assays should be included in the replacement collaborative study
on the basis of their assumed higher consistency, and the possibility of a drift
in IU introduced by “less sensitive” laboratory-developed tests. The argument
against this approach is that it would not allow for a check to be made on the
harmonization of locally developed tests, nor on assays that may have been
developed after the assessment of the previous standard.
The Committee thanked Dr Morris for her report, following which there
was considerable discussion of these and related issues. It was widely agreed that
the current WHO Recommendations required revision and updating to include
11
Recommendations for the preparation, characterization and establishment of international and other
biological reference standards (revised 2004). In: WHO Expert Committee on Biological Standardization:
fifty-fifth report. Geneva: World Health Organization; 2006: Annex 2 (WHO Technical Report Series, No. 932;
http://www.who.int/immunization_standards/vaccine_reference_preparations/TRS932Annex%202_
Inter%20_biol%20ef%20standards%20rev2004.pdf?ua=1, accessed 8 April 2018).
27
more relevant examples. Currently the document makes reference only to the
development of reference standards for vaccine potency assessment, and no
reference to topics such as commutability assessment and calibration of secondary
standards. It was urged that any revision process take into consideration all the
different target audiences and seek their views and inputs.
After highly detailed and wide-ranging technical discussion, the
Committee concluded that further guidance was evidently needed on all
of the many issues raised. It would be very timely to now review in detail the
current WHO Recommendations and to develop up-to-date guidance on the
production and evaluation of international standards for IVDs, blood products,
biotherapeutics and vaccines. The Committee also considered it worthwhile to
explore the need for a companion document primarily directed towards the
users of international standards and other reference preparations which could
include guidance on the calibration of secondary standards, as well as broader
metrological considerations. The Committee requested that the broad range of
issues raised be discussed by WHOCCs and that specific proposals be presented
to the Committee for consideration at its next meeting.
28
3. International Recommendations, Guidelines and
other matters related to the manufacture, quality
control and evaluation of biological substances
3.1 Biotherapeutics other than blood products
3.1.1 Guidelines on procedures and data requirements for
changes to approved biotherapeutic products
Changes are essential for the continual improvement of the manufacturing
process and for maintaining state-of-the-art controls on biotherapeutic products,
and such changes often need to be implemented after the product has been
approved (that is, when it has been licensed or when marketing authorization has
been received). Changes may be made for a variety of reasons including: (a) to
maintain routine production (for example, replenishment of reference standards
or change of raw materials); (b) to improve product quality, or the efficiency and
consistency of manufacture (for example, changes in the manufacturing process,
equipment or facility, or adding a new manufacturing site). Changes may also
need to be made to the product labelling information to reflect, for example, a new
indication, a change in the dosage regimen, information on co-administration
with other medicines or improvement in the management of risk by the addition
of a warning statement for a particular target population.
Biotherapeutic products are an increasingly important component of
global health care and several WHO guidelines on their regulatory evaluation are
available. During international consultations on the development of these WHO
guidelines, and during their implementation, it became clear that there was a
need to develop specific WHO guidelines on changes to approved biotherapeutic
products in order to help address the complexity and other challenges associated
with the global life-cycle management of such products. In May 2014, the 67th
World Health Assembly adopted two relevant resolutions: one on promoting
access to biotherapeutic products and ensuring their quality, safety and efficacy
(WHA67.21) and the other on regulatory systems strengthening (WHA.67.20).
In support of these resolutions, WHO had been requested to provide guidance,
particularly on how to deal with increasingly complex biotherapeutic products,
including SBPs. In addition, it had been recommended during the 16th ICDRA
that WHO assist Member States in ensuring regulatory oversight throughout the
life-cycle of biotherapeutic products.
A WHO Guidelines document had therefore been prepared to provide
guidance to NRAs and manufacturers on the regulation of changes to already
licensed biotherapeutic products, including SBPs, in order to assure their
continued quality, safety and efficacy, as well as continuity of supply and access.
These WHO Guidelines note that the implementation of new regulations
should not adversely affect product supply and accessibility. Therefore, NRAs are
29
strongly encouraged to establish requirements that are commensurate with their

own regulatory capacity, experience and resources, and to apply the concepts of
reliance or work sharing, or to use collaborative approaches, when reviewing
post-approval changes. The NRAs of procuring countries are encouraged to
consider the establishment of procedures for the expedited approval of changes
based on previous expert review and approval of the same changes by the NRAs
of the countries in which the products are licensed, or based on the decision of a
recognized regional regulatory authority.
The Committee was informed that the latest version of the proposed
WHO Guidelines (WHO/BS/2017.2311) was the result of three rounds of
international consultations during 2016–2017 and one informal consultation.
Although no major issues had been raised during the final public consultation
(since these had already been addressed) a number of points for clarification had
been identified and addressed. The view expressed by industry was that these
WHO Guidelines would be extremely valuable at the global level and were very
much welcomed.
The Committee reviewed the document WHO/BS/2017.2311 and
reflected upon the points raised in the final public consultation. Following
discussion, the Committee concluded that no major issues remained to be
resolved but indicated that a number of minor amendments to the document
be made as these were considered to be helpful clarifications. Subject to these
changes being made to the text, the Committee recommended that the guidelines
be adopted and attached to its report (Annex 3).
3.2 Cellular and gene therapies

3.2.1 Global activities in cell therapy products
The Committee was reminded of the outcome of its discussions on this topic
during its previous meeting and provided with an update on the progress made
since then. At that time, the Committee had recognized that new cell-based
medicinal products – referred to as cell therapy products (CTPs) – have great
potential in the treatment of various diseases and would become important
future public health interventions. There was also a clear consensus within the
Committee that global harmonization in the cell therapy field is needed and that
WHO should become engaged in this area. The Committee had recommended
that WHO collaborate with a range of international groups active in cell therapy,
with the goal of providing a common guideline document.
It was felt that the document should focus on somatic and not stem cell
therapy and should include quality considerations. An agreed definition of cell
therapy would also be helpful, along with clarification of whether genetically
modified cells should be included or considered under gene therapy. At the 16th
ICDRA in 2014, it had been concluded that products containing genetically
30
International Recommendations, Guidelines and other matters
modified viable cells should be considered CTPs. However, products containing

viable cells which are used in transfusion medicine (for example, thrombocyte,
erythrocyte or granulocyte concentrates) or for haematopoietic reconstitution
should not be considered to be CTPs. Conversely, there were many cases where
genetically modified cell therapy and tissue-engineered products had been
excluded from the area of CTPs. In this context, the Committee considered
that the development of harmonized definitions and terminology would be
particularly helpful for countries now setting their own national requirements in
this area. Although deliberations on the development of measurement standards
for CTPs was considered to be premature at this time, an analysis of licensed
and clinical trials of CTPs in various countries since the previous meeting of the
Committee showed the field to be extremely active worldwide.
The Committee also heard that WHO had been involved in discussions
organized by the International Pharmaceutical Regulatory Forum Cell Therapy
Working Group on the preparation of a draft reflection paper entitled General
principles to address the nature and duration of follow-up for subjects of clinical
trials using cell therapy products. Furthermore, an overview of the current
regulatory landscape, along with an outline of the common principles that may
facilitate future discussions on the regulatory evaluation of these products,
had been developed at a 2016 meeting organized by the International Alliance
for Biologicals. Many experts saw no reason to exclude stem cell therapy from
standardization activities and had proposed that WHO include both stem cell
and somatic cell therapies in future WHO standardization activities.
The Committee discussed these and other developments and agreed that
WHO standardization activities should include stem cells. The Committee also
recommended that WHO urgently establish a small working group of experts to
consider further the most appropriate approach and timeframe for developing
WHO guidelines for CTPs and to update the Committee on the further progress
made in this complex and rapidly developing field at its next meeting.
3.3 In vitro diagnostics

3.3.1 WHO IVD prequalification: update report
The WHO prequalification (PQ) programme for IVD devices aims to promote
and facilitate equitable access to safe, appropriate and affordable IVDs of good
quality. WHO IVD PQ involves a comprehensive assessment of individual IVDs
using a standardized procedure to determine whether or not a product meets
the necessary requirements. This approach is based on international regulatory
practice with a particular focus placed on IVDs for priority diseases. The
Committee was informed that the activities of the IVD PQ group are coordinated
through EMP, with its scope of work currently lying primarily in the area of rapid
diagnostic tests (RDTs), enzyme immunoassays (EIAs), flow cytometry and NAT-
31
based assays for the management of a number of bloodborne diseases (including

malaria), with IVDs for HPV having recently been added to the programme.
IVD manufacturers can submit a dossier to the PQ group at any time for
either a full or abridged PQ assessment. The abridged protocol is used in cases
where a stringently assessed version of the product is submitted for PQ or where
a non-stringently assessed (rest-of-world) version is submitted but a stringently
assessed version also exists that is not substantially different. Inspections of IVD
manufacturers are performed, with the frequency and degree of scrutiny of
inspections determined using a risk-based approach.
During 2016–2017 the IVD PQ group experienced a surge in demand
for malaria RDT assessment, with the benefits of WHO PQ being increasingly
recognized by manufacturers in light of changes in WHO procurement policy
criteria. To date, the group had assessed 20 applications in 2017, with a clear
trend towards submissions from new manufacturers.
The Committee was then provided with an outline of an alternative
approval process designed to speed up PQ assessment. In this scenario, a
manufacturer could select a laboratory from the list of Prequalification Evaluating
Laboratories. The selected laboratory then informs the IVD PQ group that an
evaluation has been commissioned. The manufacturer would then bear the cost
of the evaluation and would be responsible for coordinating directly with the
chosen laboratory. This process was expected to provide greater flexibility as
manufacturers can choose between following the standard PQ pathway in which
WHO mandates the evaluation or mandating a WHO-assessed laboratory to
evaluate the product directly. It is envisaged that such an approach will save time
in cases where dossier screening is straightforward, and will reduce the need for
WHO coordination, bring evaluations closer to the countries of use, create a
broader network of laboratories and reduce the overlapping and duplication of
activities. As mechanisms for greater transparency are now in place, a number
of IVD PQ group documents are being revised to clarify issues of eligibility, fees
and scrutiny of assessment.

To assist manufacturers, the group also produces guidance documents
that appear either in its Technical Guidance Series or Technical Specification
Series. These documents are intended to provide clear directions on the
extent of validation and verification required of IVD manufacturers, as well
as guidance on the formulation of a suitable design dossier. Among its range
of collaborative and other activities, the IVD PQ group provides inputs to a
number of external agencies, including ISO and CLSI, in the development of
standards and guidelines, evaluates new IT business solutions, participates in
working groups and develops and expands assessor pools, including through
the training of assessors. There were also plans to extend the scope of the work
to include cholera IVDs with technical specifications and laboratory protocols
having been developed and technical expert assessors identified.
32
During discussions the Committee raised the issue of the degree of

awareness among new start-up companies of the activities of the WHO IVD
PQ group. It was suggested that members of the group could attend relevant
meetings in order to raise the profile of this work. Discussion then moved on
to the proportion of IVDs that were rejected. The Committee was informed
that this varied considerably according to the maturity of the testing method
involved. For example, whereas most EIAs and NAT-based assays are approved,
the rejection rate for RDTs, especially for malaria, was much higher. However,
it was also noted that there has been an improvement in the quality of
manufacturer assessments in this field as the commercial benefits of WHO PQ
were increasingly being recognized.
3.3.2 Human immunodeficiency virus rapid diagnostic

tests for professional use and/or self-testing
The Committee was provided with an overview of the main elements required
in a technical dossier submitted by a manufacturer for the purpose of WHO
IVD PQ, with particular reference to the performance studies required. Current
WHO guidance 12 instructs that for each study the manufacturer should provide
a study description, study identifier, product identifier, IFU version, study/report
dates (and summary of findings), conclusions reached in regard to meeting the
predefined objectives, a study protocol and a full report.
Examples were then given of the type of TSS and TGS documents
available from the IVD PQ group. Each of these documents addresses specific
aspects of IVD validation to help manufacturers improve the quality of their
IVDs. It was intended that such documents would be read in conjunction with
relevant international and national standards and guidance.
The presentation then focused on the details of the proposed new TSS
document on HIV RDTs intended for professional use and/or self-testing. The
need for such guidance was first identified in 2015 and a small drafting group
consisting of PQ dossier assessors and external experts was assembled to produce
a first draft. This draft was first published on the WHO website in September
2016 with comments invited over a 3-month period from regulatory agencies,
manufacturers and professional societies. Following review of all comments
received, a revised version was produced in December 2016 in order to elicit
further comments prior to the presentation of the document for consideration
by the Committee.
12
Instructions for compilation of a product dossier. Prequalification of In Vitro Diagnostics Programme.
Geneva: World Health Organization; 2014 (PQDx_018 v3, 27 August 2014; http://www.who.int/entity/
diagnostics_laboratory/evaluations/141015_pqdx_018_dossier_instructions_v4.pdf?ua=1, accessed 7
April 2018).
33
The Committee was informed that most of the further comments

received related to minor editorial changes which for the most part had been
incorporated into the document WHO/BS/2017.2305. One comment questioning
the necessity to validate kit stability once opened was not incorporated as the
immediate use of kits could not always be assumed in field settings in LMIC. The
Committee considered the document WHO/BS/2017.2305 and recommended
that it be adopted and attached to its report (Annex 4). The question was then
raised of whether the future oversight of such documents would fall to the newly
established SAGE IVD. Clarification was provided that, although its precise
remit had yet to be established, it was envisaged that SAGE IVD would oversee
WHO PQ activities from a more strategic aspect.
3.3.3 Establishing stability of in vitro diagnostic medical devices

Although the stability of a diagnostic device is an essential characteristic, many
RDT manufacturers were not sufficiently familiar with suitable procedures for
assessing this. The lack of appropriate stability studies noted by the WHO IVD
PQ group indicated that manufacturers were not taking into account the actual
environmental and other conditions of use of products in LMIC. As a result,
many stability studies had been undertaken under optimal conditions that had
satisfied regulatory requirements in high-income countries but which did not
reflect conditions in a field setting in LMIC, where the cold chain supply may be
limited and where conditions such as dust and extreme humidity are a reality.
It was therefore proposed in 2015 that a WHO IVD PQ TGS document
be developed that explicitly outlined the necessary requirements for establishing
the stability of IVD medical devices. Although other guidelines and standards
were available on this topic, it was felt that these were often written in language
that was not well understood by manufacturers in LMIC. It was therefore
intended that the TGS document would highlight the implicit principles required
to address stability evaluation using examples relevant to the intended audience,

such as assessing extremes of temperature, humidity and the effect of light on
repeated opening. There was also a need to highlight the requirement to generate
stability data for each critical component, and to provide greater clarity on the
minimum number of lots that should be tested. Guidance was also to be given on
assessing the suitability of specimens used for stability assessment.
Guided by these considerations, a first draft was produced in August
2015 and a round of public comments invited in December 2015. A version
of the document incorporating all comments (WHO/BS/2017.2304) was then
produced. The Committee was informed that most comments received during
the consultation phase were editorial and had been accepted and incorporated. It
was indicated that although the document was being submitted for consideration
by The Committee, an additional annex was to be finalized over the coming
34
months comprising case studies for IVD medical devices. Clarification was
sought and confirmation received that the additional annex would constitute a
“how to” guide complementing the principles provided in the main document.
The possibility of separately reviewing and adopting the additional annex at a
later date via a WebEx meeting of the WHO network of collaborating centres
for blood products and in vitro diagnostics was raised and it was agreed that this
suggestion would be presented to the Committee during its closed session.
The Committee considered the document WHO/BS/2017.2304 and
recommended that it be adopted and attached to its report (Annex 5).
Consideration of the additional annex with a view to its adoption would be
undertaken prior to the next meeting of the Committee in 2018.
3.3.4 WHO consultation on the First WHO International

Standard for anti-rubella immunoglobulin
The Committee was informed that a WHO consultation had been held in June
2017 to discuss a number of issues associated with the use of the First WHO
International Standard for anti-rubella immunoglobulin. Since 1966, a sequence
of three WHO measurement standards for anti-rubella had been used, with
the current material (RUBI-1-94) having been in use since 1996. This material
had been derived from human normal immunoglobulin obtained from healthy
Danish volunteers attending blood donation centres.
Although data from the establishment study are limited, it appears that
the current standard gives different results across laboratories using a range of
methodologies. It is also widely acknowledged that the use of the material had
evolved over time from its initial established purpose in therapeutic monitoring
to its use now in calibrating diagnostics to establish vaccine-mediated protection.
In essence, a shift has occurred away from the measurement of functional
antibody activity to evaluation of their binding ability in high-throughput assays.
Problems in the standardization of such assays are widely acknowledged.
Furthermore, since the 1980s, the protective cut-off for immune
protection has been changed from 15 IU/ml to 10 IU/ml. This figure was largely
derived from values close to the limit of detection in neutralization assays.
However, it has been demonstrated that once vaccinated, individuals often
have titres < 10 IU/ml and yet are known to be protected due to the absence
of reported rubella cases amongst vaccinated individuals. Additionally,
epidemiological studies have demonstrated that there have been no cases of
congenital rubella in individuals with values < 10 IU/ml, suggesting that values
< 10 IU may be protective.
The Committee was provided with a summary of the main outcomes of
the consultation. There was agreement that the current international standard
should continue to be made available as a well-characterized reference material.
35
However, IVD manufacturers, regulators and assay users should be made aware
(through the amending of the IFU) of its potential lack of commutability when
used as a calibrant. At the same time, diagnostic expert committees, vaccine
efficacy evaluators and regulators should be encouraged to reconsider the
appropriateness of quantitative anti-rubella determinations. Other discussion
points raised included the continuing relevance of using 10 IU/ml as an assumed
immunity threshold and the potential need for further studies to resolve this and
other issues. In general, the question remains of whether the current standard
should continue to be made available following changes to the IFU and if so what
mechanisms should be used to ensure that stakeholders are made aware of the
relevant issues and WHO recommendations.
The Committee agreed that the First WHO International Standard
for anti-rubella immunoglobulin should continue to be made available as a
well-characterized reference material. The Committee further agreed that
manufacturers, regulators and assay users should be made aware of its potential
lack of commutability and other limitations in the IFU, and that stakeholders in
the diagnostic field should be encouraged to reconsider both the appropriateness
of quantitative anti-rubella measurement for the determination of immune
status and the use of 10 IU/ml as a cut-off point for assessing immune protection.
To establish the protection status of individuals, anti-rubella determination
using high-specificity qualitative assays should be considered as an alternative
approach to antibody quantitation. In conclusion, the Committee proposed that
the outcomes of the consultation and its own subsequent conclusions should
be disseminated to technical and relevant clinical audiences, for example in a
scientific publication in a high-impact journal and through targeted distribution
of this Committee report.
3.4 Vaccines and related substances

Guidelines on the quality, safety and efficacy of Ebola vaccines
3.4.1
The Committee was reminded that as part of ongoing WHO efforts to support
the development of Ebola vaccines, draft WHO Guidelines had been prepared
on the scientific and regulatory considerations relating to their quality, safety
and efficacy. The proposal to develop such Guidelines had been endorsed by
the Committee in 2014 and various drafts had now been prepared and reviewed
during a series of WHO informal consultations and public consultation, and by
the Committee itself. The Committee was reminded that work had started on
the Guidelines during the evolving Ebola epidemic. With the end of the large-
scale outbreak in Africa in 2016, Ebola disease had returned to its previous
sporadic pattern. This epidemiological situation made the evaluation of Ebola
vaccine efficacy and licensing more challenging. Interest also shifted away
from monovalent Ebola Zaire vaccines to multivalent preparations directed
36
against more than one Ebola strain as well as the Marburg virus. Developing the
Guidelines had therefore presented many challenges, not least in keeping up to
date in a rapidly evolving situation.
At its meeting in 2016, the Committee had considered a version of the
WHO Guidelines for adoption but after extensive discussion agreed that the
guidance given on multivalent Ebola vaccines and on the clinical evaluation
of vaccine candidates using innovative clinical trial designs would benefit
from expansion. There was also a need to provide guidance on how to evaluate
and license Ebola vaccines subsequent to the potential licensure of one of the
advanced vectored vaccines. Further revision of the document was therefore
undertaken to address the comments received from the Committee and other
experts. These revisions were reviewed through a process of international public
consultation through the WHO website in 2017 and no further key issues were
identified. The majority of comments received concerned improvements in
clarity or the updating of cited publications. Respondents generally considered
the document to be very comprehensive and of value in the evaluation of
vaccines other than Ebola vaccines.
The Committee was reminded of the overall structure and content of the
Guidelines which include guidance on regulatory expectations in relation to the
quality, nonclinical and clinical aspects of vaccines submitted for full licensure.
Additional text considers those aspects of Ebola vaccine development which
might be accelerated during a public health emergency. Particular attention
is given to viral-vectored vaccines as these are currently the most advanced
vaccine candidates. The latest revision of the Guidelines (WHO/BS/2017.2327)
also takes into account the fact that Ebola vaccine development had been
discussed by the WHO SAGE on Immunization. This allowed for a number of
streamlining enhancements, notably the replacement of an earlier appendix on
Ebola vaccines currently in clinical trials with a reference to the report of the
SAGE Ebola Working Group which includes a detailed listing of such vaccines.
The Committee noted the comments and suggestions which had been
received by WHO and expressed its agreement on the way in which these had
been addressed. After making a number of further clarifications to the text, the
Committee recommended that the document WHO/BS/201.2327 be adopted
and attached to its report (Annex 2). The Committee also commended the WHO
Secretariat and the drafting group for all their efforts in developing the document
under such difficult and rapidly changing circumstances.
37
4. International reference materials – antibiotics
All reference materials established at the meeting are listed in Annex 6.
4.1 Proposed new projects and updates – antibiotics

4.1.1 Proposed Third WHO International Standard for erythromycin
Erythromycin is used globally as an antibiotic and is listed as a “watch group
antibiotic” in the WHO Model List of Essential Medicines (March 2017). The
international standard for erythromycin is used to calibrate regional and
national secondary standards, as well as manufacturers’ in-house standards, all
of which are routinely used to guarantee the appropriate filling and dosing of
erythromycin preparations. There is therefore a global need for this standard.
The Committee was informed that stocks of the Second WHO
International Standard for erythromycin were running low and a replacement
standard was now required. Bulk material would be obtained from a major
manufacturer and then suitably formulated and processed by EDQM. The
collaborative study required to calibrate the replacement would involve
pharmacopoeias, NCLs and manufacturers, and would be followed by
appropriate statistical evaluation using the current international standard as the
material against which the replacement standard would be calibrated. Around
12 laboratories from different regions of the world were expected to participate
in the study.
Mindful of the issue of widespread and increasing resistance to antibiotics
worldwide, the Committee endorsed the proposal (WHO/BS/2017.2328) to
establish a Third WHO International Standard for erythromycin. In addition,
the Committee recommended that the current WHO listing of international
standards for antibiotics be reviewed and stocks updated as required to ensure
their ready availability.
38
5. International reference materials –
biotherapeutics other than blood products
5.1 WHO International Standards and Reference Reagents –

biotherapeutics other than blood products
5.1.1 Second WHO International Standard for parathyroid
hormone 1-34 (recombinant, human)
Human parathyroid hormone 1-34 is the N-terminal biologically active fragment
of parathyroid hormone. The recombinant form of this peptide (rhPTH1-34),
expressed in Escherichia coli cells, is commonly known as teriparatide and is
prescribed in the USA and Europe as a treatment for osteoporosis. The product is
under patent protection until 2019, after which it is anticipated that teriparatide
SBPs will reach the market.
The First WHO International Standard for parathyroid hormone 1-34
(recombinant, human) was established in 2007 for the calibration of therapeutic
teriparatide preparations. This material contained rhPTH 1-34 donated by
Eli Lilly & Co. The Committee was informed that this same manufacturer had
again donated rhPTH1-34 for the purpose of producing a replacement for the
current standard, stocks of which were now running low.
The candidate material (NIBSC code 15/304) had been formulated and
distributed into ampoules for evaluation in an international collaborative study.
Twelve participating laboratories in eight countries were asked to determine
the mass content of the candidate standard using high-performance liquid
chromatography (HPLC) in order to calibrate the candidate material against
the same primary calibrant used to calibrate the first international standard.
Mean estimated rhPTH1-34 content of the candidate standard was 0.914 mg/
ampoule (CV = 2.16%; n = 28; 95% confidence interval of 0.902–0.926 mg/
ampoule). Assessments were also made of the purity, bioactivity and stability
of the candidate standard. Mean estimated purity was 99.08% (CV = 0.59%,
n=11) with bioassay data showing good agreement between the potencies of the
candidate material and the current international standard. Results also indicated
that the candidate material was sufficiently stable to serve as an international
standard and plans were in place to assess its stability again in 12 months.
However, the Committee was also informed that the estimated content
of the candidate material in terms of the current international standard was
0.936 mg/ampoule – 2.4% higher than the estimate of 0.914 mg/ampoule
obtained using the primary calibrant. HPLC assays of the current international
standard suggested a lower content than had been assigned on establishment,
almost certainly due to lower precision and greater uncertainty during content
39
determination compared to current assays. Furthermore, to maintain continuity

with the historical unitage it was proposed that the candidate material 15/304
also be assigned a content of 9140 IU (1 IU = 100 ng).
The Committee considered the report of the study (WHO/BS/2017.2312)
and recommended that the candidate material 15/304 be established as
the Second WHO International Standard for parathyroid hormone 1-34
(recombinant, human) with an assigned content of 0.914 mg/ampoule and
9140 IU/ampoule.
5.1.2 First WHO International Standard for rituximab

Rituximab is a chimeric mouse-human mAb used in the treatment of CD20-
positive B-cell lymphoproliferative malignancies, transplant rejection and
autoimmune disorders. Rituximab is administered as a monotherapy or in
combination with chemotherapy regimens. The exact anti-tumour mechanism
of rituximab remains unclear. However, it is assumed that it exerts its effects by
various mechanisms involving the binding of its Fab domain to CD20-positive B
lymphocytes and the induction of apoptosis – either directly or by the immune
effector functions of its Fc domain.
Rituximab appears on the WHO Model List of Essential Medicines for
a basic health-care system. The Committee was informed that large sales of the
innovator product had driven the rapid growth of SBP development which is
expected to widen market competition and increase patient access worldwide.
Numerous rituximab SBPs were now under development, with some being at
late stages of development. A number of non-originator versions had also been
approved in some countries under local regulatory pathways that did not appear
to involve the rigorous comparability exercise required.
Because mAbs derived by recombinant DNA technology are structurally
complex molecules sensitive to small changes in the manufacturing process there
is a recognized global need for their standardization to ensure the quality, safety
and efficacy of such products. A proposal to develop an international standard
for rituximab had been endorsed by the Committee in 2014. The Committee
was reminded that WHO international standards for the biological activity of
therapeutic mAbs were intended for the evaluation of bioassay performance,
including the calibration and validation of potency assays and must be clearly
differentiated from the reference product mAb used to determine biosimilarity.
In the case of rituximab, the proposed WHO international standard would
be expected to facilitate assessment of the biological activities of products by
different stakeholders and thus enable the development of rituximab SBPs that
are consistent in terms of quality and efficacy. The proposed standard would
define bioactivity units for rituximab but would not define specific activity (IU/
mg) requirements.
40
International reference materials – biotherapeutics other than blood products
A preparation of recombinant chimeric rituximab expressed in Chinese

hamster ovary (CHO) cells had been donated by Sandoz GmbH. The material had
been formulated and lyophilized at NIBSC prior to evaluation in an international
collaborative study of its suitability to serve as an international standard. The
candidate material (NIBSC code 14/210) was tested in 16 laboratories in nine
countries alongside a coded duplicate, a second rituximab lyophilized preparation
and an in-house reference standard where available. Comparator rituximab had
been purchased and reformulated by NIBSC. All preparations were tested for their
complement dependent cytotoxic activity (CDC) with 11 laboratories also testing
for their antibody-dependent cytotoxic activity (ADCC). A limited number of
laboratories also performed cell-based antibody binding and apoptosis assays.
Stability monitoring the candidate material 14/210 showed no loss of CDC or
ADCC activity upon storage at the recommended storage temperature of −20 °C.
Nevertheless, stability monitoring and prediction studies over a further extended
period were ongoing. The study results indicated that the candidate preparation
would be suitable to serve as an international potency standard for rituximab
and that its use would help harmonize the reporting of rituximab bioactivities by
different laboratories using their in-house potency assays.
The Committee considered the report of study (WHO/BS/2017.2309)
and following discussion and clarification of a number of points recommended
that the candidate material 14/210 be established as the First WHO International
Standard for rituximab with the following assigned values:
■■ 1000 IU of CDC activity per ampoule
■■ 1000 IU of ADCC activity per ampoule
■■ 1000 IU of cell-binding activity per ampoule
■■ 1000 IU of apoptotic activity per ampoule.
The Committee emphasized the importance of explaining very clearly
in the IFU that this international standard was to be used solely to standardize
bioassays. It is not intended to form the basis of any revised product labelling
or dosing requirements as any decisions regarding the use of the IU for specific
activity specifications are solely the responsibility of the relevant competent
regulatory authorities. The international standard should also be very clearly
distinguished from the reference product mAb to be used in the comparability
studies of SBPs.
5.1.3 First WHO International Standard for infliximab

Infliximab, the first anti-TNF-α mAb to be developed, is a chimeric mAb
consisting of human IgG1 heavy chain and kappa light chain constant regions
with fused mouse variable regions. Infliximab was first approved in the USA in
41
1998 and has been extremely successful in the treatment of various autoimmune
diseases or disorders associated with increased TNF-α and resultant excess
inflammation. Current therapeutic indications include rheumatoid arthritis (in
combination with methotrexate), Crohn’s disease, ulcerative colitis, ankylosing
spondylitis, psoriatic arthritis and psoriasis.
Following recent patent expiration in Europe and imminent expiry in
the USA, infliximab is an important target for SBP manufacturers with several
such products already approved in the European Union, the USA and several
other countries worldwide. The availability of a WHO international standard
for infliximab with a bioactivity expressed in IU would facilitate determination
of the biological activity of infliximab products and enable its harmonization
worldwide, thus ensuring patient access to products which are consistent in
quality and effectiveness.
The Committee was informed that a proposed WHO international
standard had now been developed in collaboration with the European
Pharmacopoeia following endorsement of the project by the Committee in 2012.
The Committee was further informed that despite its clinical and commercial
success, there are a number of safety and efficacy issues surrounding its use and
that monitoring to rationalize treatment strategies was now being considered.
Studies had shown that monitoring infliximab serum trough levels as a basis for
clinical decision-making had increased both therapeutic and cost effectiveness
in a number of indications. Commercially available ELISAs or newly developed
mass spectrometry methods were currently used to monitor serum drug trough
levels. For each of these methods the availability of an international standard
would serve to qualify the in-house reference standards used thus globally
harmonizing therapeutic infliximab monitoring.
A preparation of recombinant infliximab expressed in SP2/0 cells had
been donated by Celltrion. This was then filled at NIBSC following standardized
procedures to produce the candidate material (NIBSC code 16/170). Commercial

infliximab had also being purchased and reformulated for use as a comparator
product. An international collaborative study was then conducted to evaluate
the suitability of candidate material 16/170 to serve as an international standard.
Twenty eight laboratories in 16 countries participated in the study using in
vitro cell-based bioassays and binding assays. Human serum samples, spiked
with differing amounts of the two infliximab preparations, were also assessed
to evaluate the suitability of the candidate material in harmonizing currently
used methods for determining serum trough levels of infliximab. Stability
studies over 9.5 months indicated that the candidate material 16/170 was stable
during long-term storage at −20 °C. Furthermore, potency was not diminished
after 1 week of storage at either 4 °C or 20 °C following reconstitution, or after
repeated freeze-thaw cycles. As no loss in activity was detected at any of the
42
International reference materials – biotherapeutics other than blood products
elevated temperatures, no predicted loss in activity could be calculated. Stability

monitoring was reported to be ongoing.
Study results indicated that the candidate material 16/170 was suitable
to serve as an international standard for the in vitro determination of infliximab
potency. No overall consensus regarding unitage assignment for either ADCC
or CDC was reached. In addition, and on the basis of limited data, the candidate
material 16/170 also appeared suitable for use in the qualification of in-house
standards for tests used in therapeutic drug monitoring of infliximab. However,
a further study may be required to facilitate harmonization in clinical practice.
and following discussion recommended that the candidate material 16/170
be established as the First WHO International Standard for infliximab, with
assigned values per ampoule of 500 IU of TNF-neutralizing activity and 500 IU
of binding activity. In addition, the candidate material 16/170 was assigned a
content of 50 µg/ampoule for use in therapeutic drug monitoring. No unitage
was assigned to ADCC or CDC activities.
The Committee noted that this international standard was intended
to support assay calibration by defining international units of bioactivity.
It is not intended to form the basis of any revised product labelling or dosing
requirements as any decisions regarding the use of the IU for specific activity
specifications are solely the responsibility of the relevant competent regulatory
authorities. The international standard should also be very clearly distinguished
from the reference product mAb to be used in the comparability studies of SBPs.
43
6. International reference materials – blood
products and related substances
6.1 WHO International Standards and Reference Reagents –

blood products and related substances
6.1.1 First WHO Reference Reagent for activated
blood coagulation factor X (human)
Activated blood coagulation factor X (FXa) is a trypsin-like serine protease that
plays a crucial role in the coagulation cascade. FXa is regarded as an inherent
impurity of factor eight inhibitor bypassing activity (FEIBA), an activated
prothrombin complex concentrate used in the treatment of haemophilia in
patients with inhibitory antibodies against FVIII. In the absence of an international
reference standard for FXa, direct measurements of FXa activity in FEIBA have
been made relative to working standards calibrated against the non-WHO NIBSC
Reference Material for blood coagulation factor Xa (NIBSC code 75/595). This
reference material had been sourced from bovine plasma, prepared by an external
group and arbitrarily assigned a potency of 1 U/ampoule. With no information
on the uniformity of the fill or on the stability of the material, its continued use
had been questioned and the development of a replacement material proposed.
Although the measurement of FXa levels in FEIBA is the primary
regulatory use for an FXa reference standard, the current standard is also used
routinely to calibrate local standards. Potential future uses for a replacement
FXa reference material also include the standardization of measurement of
FXa contamination in non-activated products such as prothrombin complex
concentrates used in the reversal of anticoagulant therapy, and for a recently
licensed FX concentrate product used in the treatment of congenital FX

deficiency. Standardizing the biological activity of direct FXa inhibitors (as used
in anticoagulation therapies) is another significant potential use which would
require a human FXa standard due to key differences between bovine and human
FXa. The development of a WHO international standard for human FXa would
require a large-scale study at a time when the full range of its intended uses were
not yet known. It had therefore been proposed that a WHO reference reagent be
established as an interim measure.
Donated source material had been formulated and lyophilized, and
the resulting candidate material (NIBSC code 15/102) calibrated against
75/595 using direct chromogenic assays. Based on the results of a total of 12
independent assays conducted by two laboratories, a potency estimate for
15/102 was assigned. Intra-laboratory variability was low (geometric coefficient
44
International reference materials – blood products and related substances
of variation (GCV) < 4%) with inter-laboratory geometric mean estimates of

potency also in close agreement (6.5 and 6.8 U/ampoule). The overall geometric
mean estimate of potency of 6.7 U/ampoule was therefore assigned to candidate
material 15/102. Long-term stability was assessed in accelerated-degradation
studies, with data indicating that the candidate material 15/102 remained stable
after 10 months, with no measurable degradation. Predicted activity loss per
year at normal storage temperature (−20 °C) was 0.008 % by activated partial
thromboplastin time (APTT) assay and 0.012 % by prothrombin time (PT)
assay. On-bench stability (after reconstitution) was assessed over 6 hours using
PT and APTT assays. No measurable loss of potency was detected within 4 hours
of reconstitution.
and recommended that the candidate material 15/102 be established as the First
WHO Reference Reagent for activated blood coagulation factor X (human), with
an assigned potency of 6.7 U/ampoule. During discussion, the Committee raised
a number of issues in relation to the original selection of bovine material and its
continued use. It was further noted that since the change from bovine to human
material may require the additional calibration of clotting assays, a statement to
this effect should be included in the IFU.
6.1.2 Second WHO International Standard for activated

blood coagulation factor IX (human)
Activated blood coagulation factor IX (FIXa) is a highly thrombogenic process-
related impurity found in therapeutic prothrombin complex concentrates and
monocomponent plasma-derived and recombinant FIX concentrates. As new-
generation modified FIX products are now licensed for replacement therapy,
there has been a recent increase in demand for the current WHO international
standard. As stocks of this international standard were now close to exhaustion,
a replacement standard was urgently required.
Bulk starting material prepared by the activation of recombinant
human FIX followed by size exclusion chromatographic purification had been
donated by Pfizer. The purity of the material was assessed and confirmed by
PAGE with silver staining. The estimated specific activity of the bulk was 612 IU/
mg. Following formulation at NIBSC, 18 000 ampoules were produced of the
candidate material (NIBSC code 14/316). During an international collaborative
study, 19 laboratories participated in the value assignment of the proposed
Second WHO International Standard for activated blood coagulation factor IX
(human) relative to the first international standard. Data sets were generated
using purified reagent chromogenic/fluorogenic based assays, one-stage clotting
assays based on APTT, one-stage clotting assays based on NAPTT and a thrombin
generation test (TGT).
45
With the exception of one laboratory, similar potencies were obtained for
the coded duplicates provided. The overall geometric mean potency determined
by purified reagent assays was 10.48 IU/ampoule, with values of 11.67 and 12.10
IU/ampoule produced by assays based on APTT and NAPTT respectively. The
results obtained using TGT were similar to those obtained using purified reagent
assays. Given some degree of uncertainty concerning the influence of other
components involved in clot- and plasma-based assays on the measurement
of FIXa it was proposed by NIBSC that the value assigned to the replacement
standard should be based on the results of purified reagent assays only – as had
been the case with the current WHO international standard.
and recommended that the candidate material 14/316 be established as the
Second WHO International Standard for activated blood coagulation factor IX
(human) with an assigned potency of 10.5 IU/ampoule.
6.1.3 First WHO International Standard for blood coagulation

factor XII (plasma, human) via assignment of additional
analytes to the current Second WHO International Standard
for blood coagulation factor XI (plasma, human)
The role of blood coagulation factor XII (FXII) in haemostasis was not previously
considered important because its deficiency is not associated with bleeding.
However there is now emerging interest in FXII. The finding that it is activated
by agents such as mast cells, platelet polyphosphates and clinically used materials
such as stents and mechanical valves suggests that it may play a significant role
in thrombogenesis, especially in patients with prothrombotic conditions. FXII
inhibition may therefore present an attractive option for antithrombotic therapy
and various antibodies and inhibitors are in development. There is therefore now
a need for supporting assays for FXII, for which there is currently no international
standard. A plasma international standard for FXII functional activity (FXII:C)

and antigen (FXII:Ag) would support the development of such assay methods
and the clinical monitoring of patients.
The 2015 collaborative study required to replace the First WHO
International Standard for blood coagulation factor XI (plasma, human) had
presented an opportunity to assess the feasibility of establishing an international
standard for FXII. Since similar handling and processing conditions (such as
avoidance of cold activation and contact with negatively charged surfaces) are
needed for both contact factors, the same candidate material could therefore be
assigned both FXI and FXII values. The feasibility of this was assessed based on
the number and type of assays performed by participating laboratories and on the
precision of the data returned. The results indicated that there were a sufficient
number of laboratories capable of performing functional and antigenic assays
46
International reference materials – blood products and related substances
with reasonable precision, and that this data set could be used for assignment of
both FXII:C and FXII:Ag values to the candidate material.
Twenty laboratories took part in a collaborative study to assign FXII:C
and FXII:Ag values to the Second WHO International Standard for blood
coagulation factor XI (plasma, human) (NIBSC code 15/180). Value assignment
was against local normal pooled plasmas which were assumed to have 1 U/ml of
functional activity or antigen content. For FXII:C, 28 sets of results from one-
stage clotting assays using 13 different APTT reagents against local plasma pools
(total number of donors = 566) were returned. Intra-laboratory GCVs were in
the range 1–20%, with the majority being < 10%. The overall geometric mean
was 0.86 IU/ampoule, with an inter-laboratory GCV of 10%. For FXII:Ag, nine
sets of results obtained using three different commercial kits/paired antibody
sets and one in-house reagent were analysed against local plasma pools (total
number of donors = 216). Intra-laboratory GCVs were in the range 4–12%, with
the majority being < 10%. The overall geometric mean was 0.80 IU/ampoule,
with an inter-laboratory GCV of 11%.
and recommended that the First WHO International Standard for blood
coagulation factor XII (human, plasma) be established via assignment of an
FXII:C unitage of 0.86 IU/ampoule and an FXII:Ag unitage of 0.80 IU/ampoule to
the current Second WHO International Standard for blood coagulation factor XI
(plasma, human).
47
7. International reference materials – in vitro diagnostics
7.1 WHO International Standards and Reference

Reagents – in vitro diagnostics
7.1.1 First WHO Reference Reagent for lupus anti-dsDNA serum
Systemic lupus erythematosus (SLE) is a severe autoimmune connective tissue
disease in which autoantibodies are generated to a range of antigens. Among
these autoantibodies, antibody to double-stranded DNA (anti-dsDNA) is highly
specific to SLE, occurring in 70% of cases of SLE against a non-SLE background
of < 5%. The level of anti-dsDNA can also be used to monitor disease activity.
As a result, the measurement of anti-dsDNA is a widely used diagnostic test for
SLE and a range of kits and diagnostic tests are available.
In 1985, a freeze-dried preparation of plasma obtained from a patient with
definite SLE had been established as the First WHO International Standard for
anti-double-stranded DNA serum and had been used to assign units to diagnostic
tests. The Committee was informed that this material was now exhausted and
that calls had been made for its replacement with a suitable preparation.
Oligo-specific SLE plasma from a single donor was therefore obtained
and following appropriate processing was prepared as a lyophilized candidate
standard for lupus anti-dsDNA serum. The candidate material (NIBSC code
15/174) was evaluated in an international collaborative study involving 36
laboratories in 17 countries. Comparisons of the candidate material were made
against both local standards (some of which were previously calibrated against
the First WHO International Standard for anti-double-stranded DNA serum)
and three individual plasma donations from patients with SLE in order to support
the evaluation of commutability of the candidate material.
In all laboratories and for all test methods used, the candidate material
exhibited anti-dsDNA reactivity. In approximately half of the laboratories,
the material behaved in an apparently similar way to local standards and, by
inference, to the previous international standard. However in a similar number of
laboratories there was observable non-parallelism and no quantitative traceability
to the unitage of the previous international standard could be established.
Moreover, across the entire study, it was not possible to establish commutability,
as a consistent ranking order for the three patient samples was not obtained.
Given the apparent lack of qualitative comparability of this candidate
material with the previous international standard, it was considered unwise to
establish it as a replacement international standard with a defined unitage in IU
based on the previous international standard. It was proposed that candidate
material 15/174 be established de novo as the First WHO International Standard
48
International reference materials – in vitro diagnostics
for lupus anti-dsDNA serum with a nominal potency of 100 IU/ampoule, noting
the name change from the previous standard for anti-dsDNA serum. Moreover,
it was proposed that information be provided to users emphasizing that caution
would be needed in transferring the new unitage to existing assay methods.
The Committee questioned why plasma from only one patient had been
used for candidate material generation. Despite this also being the case for the
establishment of the previous international standard it was suggested that as
each patient has different autoantibodies, the use of a pooled sample would be
better for the preparation of an international standard for anti-dsDNA serum.
The Committee also concluded that it would not be appropriate to establish a
first WHO international standard with a similar reagent for the same analyte
based on a name change alone and that the previous international standard
could not be replaced by any preparation due to the inability to maintain
continuity of unitage. The Committee considered the report of the study
(WHO/BS/2017.2306) and recommended instead that the candidate material
15/174 be established as the First WHO Reference Reagent for lupus anti-
dsDNA serum with a nominal value of 100 U/ampoule. The Committee further
indicated that labelling should inform users of the lack of continuity to the First
WHO International Standard for anti-double-stranded DNA serum, and that
parallelism and commutability had not been established.
7.1.2 Third WHO International Standard for hepatitis A

virus RNA for NAT-based assays
Hepatitis A virus (HAV) nucleic acid amplification technique (NAT)-based
assays are primarily used in the safety testing of plasma intended for use in
medicinal products. It is the recommended method as directed by the European
Pharmacopeia monograph, with assays required to detect 100 IU/ml HAV RNA.
The WHO international standard is used by blood product manufacturers,
clinical laboratories, control authorities and IVD manufacturers to calibrate the
secondary standards used in such assays.
The First WHO International Standard for hepatitis A virus RNA for
NAT-based assays (NIBSC code 00/560) was established by the Committee in
2003. Although a second batch (NIBSC code 00/562) had also been made from
the same material (but lyophilized at a different time point) its stability was
brought into question at the time of replacement of 00/560. In 2013, a proposed
replacement material was made (NIBSC code 12/234). However, the stability of
this material had also been found to be questionable and in 2014 the Committee
recommended that the previous candidate material 00/562 be established for an
interim period as the Second WHO International Standard for hepatitis A virus
RNA for NAT-based assays, and shipped on dry ice to allow continuity of supply,
but that an investigation into a stable replacement should begin immediately.
49
Following a successful pilot study, two candidate materials were taken

forward for evaluation in an international collaborative study. One of the
candidate materials consisted of human plasma spiked with HAV (NIBSC code
15/276) and the other of human plasma spiked with HAV with the addition
of hepes buffer and trehelose (NIBSC code 15/278). Eleven laboratories in
10 countries assessed a total of eight panel members which also included
the current international standard. Variability in reported laboratory mean
estimates for a number of samples was higher for qualitative assays compared
to quantitative assays. Stability data at a 12-month time point suggested that
both candidate materials were stable and would therefore be shipped at ambient
temperature. It was proposed that the candidate material 15/276 (consisting only
of human plasma spiked with HAV) should considered for establishment as the
replacement standard, with a potency of 4.49 log 10 IU/ml based on the qualitative
assay results.
There was some discussion regarding the reasons for the lack of stability
observed in previous preparations, which remains unexplained. It was noted
that lyophilization of the previous international standard had taken place off
site from NIBSC and that the specifics of the process used were not completely
known. The candidate materials used for this study had been lyophilized
at NIBSC. It was also clarified that the rationale for using only qualitative
data sets was based on these being the predominant assay type in the field.
However, it was agreed that given the observed high level of consistency of the
comprehensive data set that the value assigned to the international standard
would be 4.42 log 10 IU/ml.
Third WHO International Standard for hepatitis A virus RNA for NAT-based
assays with an assigned potency of 4.42 log 10 IU/ml.
7.1.3 Fourth WHO International Standard for HIV‑1 RNA for NAT-based assays
The advent of NAT-based assays in the 1990s allowed for the direct detection of
HIV, thus providing a positive indication of infection weeks in advance of the
traditionally used serological tests. However, inter-assay sensitivity varied greatly
and the need for harmonization across this new technology was recognized,
including in the field of blood transfusion safety where concerns had been raised
regarding the risk of transfusion transmitted infections occurring through
false-negative screening results.
In 2015, the Committee had been informed that stocks of the current
international standard were diminishing and the proposal to develop a
replacement standard was endorsed. A candidate material (NIBSC code 16/194)
was therefore developed using an HIV-1 primary isolate (subtype B) which
derived from the same viral stocks used for the production of the two most
50
recent international standards. As with the current international standard the

viral stocks were heat-inactivated using established procedures. Inactivation
was then confirmed by cell culture and p24 antigen expression. More than
9000 vials of candidate material were produced and the material assessed in a
collaborative study which also included the current international standard.
Twenty one laboratories from 11 countries participated in the study
and returned a total of 23 data sets (17 from quantitative assays and 6 from
qualitative assays). Analysis of the data for both the candidate material and
current international standard revealed two distinct data groups – one consisting
of qualitative data and the other of quantitative data. When expressing data
as a relative potency to the current international standard this disparity was
not repeated. It was also noted that four laboratories that had used the same
quantitative assay (Siemens) reported higher results more consistent with the
qualitative assays. Stability of the candidate material up to a 12-month time point
was satisfactory, with no loss of titre to suggest degradation.
There was some discussion as to the possible reasons for the observed
discrepancies. It was highlighted that the only difference between the materials
evaluated in this study and previous materials was that the former had been heat-
inactivated. However, insufficient earlier data was available to investigate this
further. It was also reiterated that limited conclusions regarding commutability
could be drawn from the inclusion of only one patient sample.
The Committee considered the report of the study (WHO/BS2017.2314)
Fourth WHO International Standard for HIV-1 RNA for NAT-based assays with
an assigned potency of 5.10 log 10 IU/ml.
7.1.4 First WHO International Standard for Ebola virus

antibodies (plasma, human); and First WHO Reference
Panel for Ebola virus antibodies (plasma, human)
The Committee was reminded that in response to the 2014–2016 Ebola
epidemic, a First WHO Reference Reagent for Ebola virus antibodies had been
developed and established in 2015 as an interim standard with an assigned
potency of 1 U/ml. The standard comprised a preparation of convalescent
plasma obtained from a patient recovering from Ebola virus disease (EVD)
and had been chosen as it could quickly be formulated into an urgently needed
standardization material.
Further work had since been undertaken to fully evaluate and
develop EVD convalescent plasma samples for evaluation in an international
collaborative study with the aim of establishing materials to serve as the First
WHO International Standard and First WHO Reference Panel for Ebola virus
antibodies (plasma, human). The study assessed six different anti-Ebola samples
comprising convalescent plasma pools from Africa, Italy, Norway, the United
51
Kingdom and the USA, along with a negative human plasma sample, for inclusion
in the panel. A total of 17 laboratories in four countries (predominantly in the
United Kingdom and the USA) returned 26 data sets from three categories of
assay – neutralization of live Ebola virus, neutralization of Ebola pseudotypes and
enzyme immunoassays (EIAs). It was highlighted that a number of laboratories
using neutralization methods reported the negative plasma sample as positive,
particularly laboratories using a lentivirus-vector system. In addition, as potency
values derived for convalescent plasma samples from Norway, Italy and the
United Kingdom were near the detection limit for some assays, some values
were reported as negative. EIA data were analyzed separately and presented as
relative potency expressed against the candidate international standard material
(NIBSC code 15/262) and the current reference reagent (NIBSC code 15/220).
GCVs were lower in all samples when expressed against the candidate material
and were in the range 41–92%. The stability of the candidate material 15/262
was demonstrated to be suitable for its use as an international standard and
for its shipment at ambient temperatures. Due to the paucity of materials no
stability data could be generated for the proposed reference panel members
(NIBSC code 16/344).
During discussion, the Committee expressed concern that the stability
of the proposed reference panel members had not been assessed, and suggested
that their real-time stability should be assessed annually to ensure fitness for
purpose. Concern was also expressed regarding a specific proposal made to
assign an IU value to a single panel member as an international standard would
also be available, and as stability data were lacking.
and recommended that the candidate material 15/262 (a freeze-dried pool
of convalescent plasma from Sierra Leone) be established as the First WHO
International Standard for Ebola virus antibodies (plasma, human) with an
assigned potency of 1.5 IU/ml. The Committee further recommended that the
candidate material 16/344) be established as the First WHO Reference Panel for
Ebola virus antibodies (plasma, human) without assigned units. The Committee
highlighted that the labelling of the reference panel should clearly indicate the
potential for a false-positive result for the negative panel member in some assay
types. The Committee also agreed that the current First WHO Reference Reagent
for Ebola virus antibodies should remain available for use with its originally
assigned potency of 1 U/ml.
7.1.5 First WHO Reference Panel for genomic KRAS

codons 12 and 13 mutations
Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations are present in
approximately 20% of all cancers and are especially prevalent in colorectal, lung
and pancreatic cancers, with 90% of such mutations being located in codons
52
12 and 13. Patients with KRAS mutations do not respond to anti-Epidermal

Growth Factor Receptor antibody therapies, and accurate and reproducible
KRAS mutation testing is essential in ensuring that suitable and effective
treatment is administered.
Following endorsement of the project by the Committee in 2015, an
international collaborative study had been conducted to assess the suitability
of a panel of genomic DNA materials as a proposed WHO reference panel for
genomic KRAS codons 12 and 13 mutations (NIBSC code 16/250) for use in
diagnostics standardization. The panel comprised eight freeze-dried purified
genomic DNAs representing the seven most-common KRAS mutations
associated with colorectal cancer plus wild-type KRAS codons 12 and 13.
The materials were produced from cancer cell lines and from a wild-type
lymphoblastoid cell line, respectively.
A total of 56 laboratories from 34 countries participated in the
collaborative study with each sent triplicate coded samples of the eight panel
members. Laboratories were requested to test these materials using their routine
diagnostic assays. Of the 68 data sets returned 36 were derived from quantitative
methods, with a wide variety of different methodologies used. Details were
provided on the calculation performed to calculate KRAS copy number for each
panel member. It was proposed that each panel member should be established
with a value relating to genotype, consensus mutation percentage, consensus
mutant KRAS copy number and consensus total KRAS copy number. These
would be clearly stated in a table in the IFU.
During discussion, it was noted that the replacement of formalin fixation
would not happen in the near future and that many laboratories use blood
to study mutations. However, for fixed tissue and blood samples the levels of
genomic fragmentation would be high, whereas the genomic DNA proposed in
this study would be of higher quality than is routinely used in these methods. It
was indicated that the IFU should highlight the potential for poor harmonization
between assays other than NGS and digital PCR. It was also noted that the
genomic DNA panel would provide an improved means of standardizing methods
and that going forward it may be possible to produce secondary standards in a
different format (such as fragmented DNA) back-calibrated to this high-quality
primary reagent. It was further noted that cell line stability may be a problem for
replacement panels in the future. With only 2000 panels available, it may also be
necessary to consider restricting their distribution to laboratories producing kits,
assays or secondary standards to be made available to others. It was also noted
that due to the requirement for extended KRAS analysis in all patients presenting
with colorectal cancer, the development of panel members representing codons
61, 117 and 146 was also planned.
53
WHO Reference Panel for genomic KRAS codons 12 and 13 mutations. The
value details of each panel member, relating to genotype, consensus mutation
percentage, consensus mutant KRAS copy number and consensus total KRAS
copy number were to be provided in the IFU.
7.1.6 First WHO International Standard for human

herpes virus 6B DNA for NAT-based assays
Human herpes virus 6A (HHV-6A) and HHV-6B are members of the β-subfamily
of herpes viruses and share ~90% sequence homology. First isolated in the
1980s, HHV-6A and B are now recognized as separate viruses. Seroprevalance
in adults is high (> 90%) with primary infection occurring in the first 2 years
of childhood. HHV-6B predominates in Europe, Japan and the USA while
HHV‑6A is common in sub-Saharan Africa. Latency is established in monocytes
with transplant recipients at increased risk from HHV-6B reactivation linked to
organ dysfunction, myelosuppression, encephalitis and graft-versus-host disease.
A number of previous studies have highlighted considerable variability in the
detection of HHV-6, highlighting the need for a measurement standard.
Two candidate standards were prepared (> 5000 vials each for HHV-6A
and HHV-6B) and evaluated for their suitability in an international collaborative
study alongside seven other materials comprising the corresponding liquid
bulk materials and various clinical samples in different matrices, including two
chromosomally integrated samples. Twenty six laboratories from 12 countries
returned 36 data sets, the majority of which (34) were from quantitative assays.
Raw data estimates for both candidate materials were very similar showing an
approximate 2.6 log 10 variation across the different quantitative assays, which
increased by a further one log 10 with the inclusion of qualitative assays in the
data set. Agreement between laboratories was improved by using the liquid
preparation of the candidate (HHV-6B) material (NIBSC code 15/266) to derive
a relative potency assessment. This effect was also mirrored in the HHV-6A
candidate material. Evaluation of the stability of candidate material 15/266 up to
a 6-month time point indicated no loss in potency. Stability data indicated that
this material was stable at −20 °C and at higher ambient temperatures that reflect
global shipment. Although study data showed that both candidate materials
performed equally well, HHV-6B has the greater clinical diagnostic significance.
It was therefore proposed that the candidate material 15/266 be established as the
international standard with a value assignment of 7.75 log 10 IU/ml. It was pointed
out that this value had been derived only from the quantitative estimates.
WHO International Standard for human herpes virus 6B DNA for NAT-based
assays with an assigned potency of 7.75 log 10 IU/ml.
54
7.1.7 First WHO International Standard for Plasmodium falciparum antigens

Malaria is a mosquito-borne disease endemic to 91 countries and territories. In
2015, there were an estimated 212 million cases of malaria worldwide resulting
in 429 000 deaths, mainly among children under 5 years of age in Africa. Malaria
is caused by protozoan Plasmodium parasites, with the majority of morbidity
and mortality due to the species P. falciparum. The Committee was informed of
the growing market for rapid detection tests (RDTs) for malaria antigens, which
are now routinely used for both the diagnosis and management of malaria. The
main targets of such products are histidine-rich protein 2 (HRP2), plasmodial
lactate dehydrogenase (pLDH) and aldolase.
Although numerous malaria RDTs are currently available, their validation
and quality control are limited as there is currently no international standard for
the above antigens. At present, clinical isolates or culture-derived materials from
the United States Centers for Disease Control and Prevention and the WHO
Malaria Specimen Bank are typically used in the evaluation of new assays and
technologies. However, demand for these materials is heavy and recent RDTs are
characterized by improved sensitivity requiring appropriate reference materials.
A suitable reference material produced from a single source could be used
worldwide for the quality control and standardization of malaria RDTs.
Following provision of source material by the United States Centers
for Disease Control and Prevention, an international collaborative study was
conducted to assess the suitability of an in vitro-derived P. falciparum candidate
material (NIBSC code 16/376) for use as a WHO international standard. The
performance of 19 different RDTs from a range of manufacturers was assessed
by 13 laboratories in 11 countries using 14 clinical isolates. Data sets obtained
from laboratories which had additionally used ELISAs showed variable results
with wide-ranging GCVs for both HRP2 and pLDH. However, when these
values were evaluated as relative potencies to the proposed candidate material
a significant reduction in variability was observed for both analytes. Separate
analysis of the results obtained using RDTs showed that all participating
laboratories detected HRP2 and pLDH end-point titres of the candidate material
using all of the RDTs tested, while HRP2 and pLDH end-point titres of clinical
isolates were detected in the majority of RDT assays carried out, indicating that
HRP2/pLDH could be more reliably detected in the candidate material than in
the clinical isolates tested. Stability evaluation of the candidate material 16/376
indicated no detectable degradation of HRP2 with the pLDH antigen predicted
to be stable at −70 °C storage. However, some degradation was predicted at
higher storage temperatures and accelerated and real-time stability studies were
ongoing. Based on these findings it was recommended that ampoules should be
stored at −70 °C and shipped at −20 °C or lower.
55

First WHO International Standard for Plasmodium falciparum antigens with
the individually assigned unitages of 1000 IU/ampoule for HRP2 and 1000 IU/
ampoule for pLDH.
7.1.8 First WHO International Standard for anti-

cytomegalovirus immunoglobulin G
Cytomegalovirus (CMV) poses a major health threat to immunocompromised
people, and to transplant recipients and other patients undergoing
immunosuppressive therapy. Moreover, CMV is the leading viral cause of
birth defects. Measurement of anti-CMV immunoglobulin G (IgG) is used for
screening, determining serological status, assessing immunity and evaluating the
risk of CMV disease. Despite there being no known correlation with protection,
the quantitative determination of anti-CMV IgG is considered useful in
identifying reactivation.
Due to the lack of an international standard, anti-CMV immunoglobulin
tests differ widely in a number of key aspects and the values obtained using
different tests are not comparable. The reliability of serological diagnosis
therefore depends heavily on the assay used. In light of increasing recognition
of the need for an anti-CMV IgG reference material for diagnostic purposes, a
proposal to evaluate such a material in an international collaborative study had
been endorsed by the Committee in 2013.
A freeze-dried anti-CMV IgG candidate material (PEI code A1) was
produced from a pool of three human disease state plasmas purchased from
Biomex GmbH – a commercial supplier in Germany. During pre-testing, the
candidate material displayed, among other characteristics, both a high IgG level
and high IgG avidity, and was shown to be anti-CMV IgM negative and CMV
DNA negative. The candidate material was lyophilized to produce 1900 ampoules.
Sixteen laboratories from nine countries participated in the collaborative

study, including reference, regulatory and research laboratories, IVD manufacturers
and blood banks. A total of 10 study samples were used, with all samples diluted
until the test cut-off to determine the endpoint titre. Calibration of the tests using
the candidate material A1 was effective in harmonizing the results.
During discussions, it was noted that the test kits used in the study did
not appear to correlate very well. It was clarified that whilst the raw data output
from each assay did show differences this was not the case when Spearman
Rank analysis was carried out. It was further clarified that IgG-only assays are
designed for the quantitative interpretation of infectious stage and for following
titre development. Such kits may be used for staging in neonatal infections and in
post-transplantation situations but for acute infection the measurement of IgM
(rather than IgG) or DNA was used.
56

and recommended that the candidate material A1 be established as the First
WHO International Standard for anti-cytomegalovirus immunoglobulin G
with an assigned unitage of 46.4 IU/vial. The Committee also recommended
that a number of statements should be made in the IFU regarding issues of
commutability, the lack of analytical sensitivity in a minority of tests when
diluting low-avidity anti-CMV IgG samples and the unsuitability of the standard
for harmonizing anti-CMV IgG/IgM assays.
7.1.9 First WHO International Standard for chikungunya

virus RNA for NAT-based assays
Chikungunya virus (CHIKV) is a member of the Alphavirus genus in the
Togaviridae family and was first identified as a human pathogen in the early
1950s. The virus is present not only in Africa but also in Asia and the Indian
subcontinent and, since 2013, has spread to the Americas, particularly central
and southern areas. Small outbreaks have also occurred in Europe. For the
laboratory diagnosis of acute CHIKV infections and blood screening, NAT-
based assays are considered the most sensitive detection method. There is
however currently no standardization of NAT-based assays for the detection
of CHIKV RNA. Following endorsement by the Committee in 2010 of the
proposal to establish an international standard for CHIKV RNA, an international
collaborative study was conducted.
The candidate material chosen was a CHIKV isolate that had been
imported into the USA in 2006 and which was characterized as the East-Central-
South-African genotype. The virus was propagated in Vero cells and then heat-
inactivated. A total of 3524 vials containing 0.5 ml of the candidate material (PEI
code 11785/16) were produced in 2016.
Twenty five laboratories from 14 countries participated in the
international collaborative study, including IVD manufacturers, regulatory
authorities, and clinical, reference and research laboratories. The methods
used involved automated or manual extraction followed by testing in a range
of qualitative and quantitative in-house and commercial NAT-based assays. In
total, 31 data sets (20 qualitative and 11 quantitative) were received from 24
laboratories. The majority of the methods used were real-time RT-PCR assays
which targeted several different regions of the CHIKV genome. The lyophilized
candidate material 11785/16 was detected by all assays evaluated in the study and
was estimated to have a potency of 6.39 log 10 U/ml. No significant titre loss was
detected at 20 °C, 4 °C, or 20 °C for up to 6 months. However, under accelerated
conditions at 37 °C, a log reduction of 0.5 was observed after 6 months. Both
real‑time and accelerated stability studies were currently ongoing.
57
First WHO International Standard for chikungunya virus RNA for NAT-based
assays with an assigned unitage of 2.5 x 10 6 IU/ml. It was noted that although
shipping on dry ice was proposed, the data suggested that shipping at ambient
temperature might be acceptable. It was suggested that the stability of the
material at ambient temperature should be further monitored in the follow-up
stability studies.
7.1.10 First WHO International Standard for Zika virus antibodies

(immunoglobulin G and immunoglobulin M) (human)
The accurate diagnosis of Zika virus (ZIKV) infection, particularly in pregnant
women, is a crucial step in making appropriate health-care decisions. Following
a negative PCR result, both serum immunoglobulin G and immunoglobulin M
are measured to determine prior exposure, where immunoglobulin M would
indicate a recent infection. The standardization of serological tests is now required
to improve both their sensitivity and specificity. Following its endorsement of
a proposal to develop an international standard for use in the calibration and
control of ZIKV antibody assays in 2016 the Committee was provided with an
update of the progress made.
A candidate material for establishment as an international standard
had been formulated by NIBSC and sent out as part of a panel of samples for
evaluation in an international collaborative study. Participating laboratories had
indicated that they would be using a range of different serological assay types to
assess the panel of anti-Zika-positive samples and negative specimens, along with
samples containing antibodies to other flaviviruses. Samples were dispatched to
laboratories in Central America, Europe, the Far East and the USA. It had been
hoped that sufficient data would have been received to permit establishment of
the standard during the current meeting of the Committee. However, data sets
from a significant number of laboratories were still to be received and no study
report was available.

The Committee discussed the possibility of establishing the candidate
material as an interim standard but agreement was reached that this would be
ill advised prior to the collaborative study data becoming available for analysis.
The Committee then considered the possibility of establishing the international
standard prior to its next annual meeting. It was however recognized that the
Committee currently lacked any mechanism for recommending the establishment
of reference preparations between its meetings. In view of these constraints, the
Committee recommended that establishment of the international standard be
deferred. An understanding was however reached that NIBSC would continue
to make the candidate material available with an arbitrary interim unit defined
by the institute.
58
7.2 Proposed new projects and updates – in vitro diagnostics

7.2.1 Proposed First WHO Reference Panel for cancer mutation detection
Cancer is the second leading cause of death globally and new cases are expected to
increase by 70% in the next 20 years. There is therefore a strong need to improve
the quality of cancer diagnostics and treatment worldwide. Drug therapies are
advancing, with multiple globally licensed anti-cancer drugs now targeting
specific tumour biomarkers. Despite the progress made, there remains a gap
in the translational research between the identification of new biomarkers and
the development and commercialization of genetic diagnostic tests. Standards
for assay validation and verification are needed to ensure the consistency of
these diagnostics.
To support standardization efforts in this rapidly advancing field, the
development of a First WHO Reference Panel for cancer mutation detection
is proposed. This would be the first panel of its kind and would support the
standardization of multiple mutation detection in tumour DNA samples by
next-generation sequencing (NGS). The application of NGS to the personalized
diagnosis, treatment and monitoring of cancer is becoming more widespread,
and is based on the parallel analysis of multiple genomic markers. However, it is
recognized that NGS is a complex technology and variability can be introduced
at many stages. The proposed reference panel is intended to serve as a benchmark
across different platforms.
It is proposed that the panel would comprise approximately four
genomic DNAs of varying mixtures derived from approximately six cancer
cell lines. This will provide coverage for up to 300 mutations in around 60
key oncogenes. Approximately 2500 ampoules will be produced and the panel
assessed in a variety of NGS platforms representing a range of input variables. It
was emphasized that the development of such a panel will not negate the need
to develop other panels covering additional genes and mutation types and that
standards will continue to be proposed for certain single cancer mutations.
It was envisaged that the final panel would be presented to the Committee for
establishment in 2019.
Discussion topics included the amount of DNA that should be present in
each ampoule. Previous materials contain 5 µg which would be overly sufficient.
In view of this and the anticipated popularity of the panel it was suggested
that consideration be given to increasing the anticipated production of 2500
ampoules by reducing the DNA content in each, while remaining mindful of
potential stability issues. The possibility of increasing DNA volume through
multiple cell passages was also discussed and it was concluded that this would
not be a suitable approach to expanding production due to practical limitations.
Instead, consideration could be given to limiting the number of ampoules that
59
laboratories could receive and ensuring that the material was only used in the
calibration of kits, assays or secondary reference materials which would be made
available to others. It was suggested that such an approach would also promote
alignment to the primary standard.
The Committee endorsed the proposal (WHO/BS/2017.2320) to develop
a First WHO Reference Panel for cancer mutation detection.
7.2.2 Proposed Third WHO International Standard

for prekallikrein activator
The international standard for prekallikrein activator (PKA) is used to determine
the level of this impurity in albumin and is thus highly important in promoting
patient safety. Albumin solutions contaminated with PKA can result in vasodilation
thus complicating the use of albumin to manage hypovolemia.
The Committee was informed that there is frequent and worldwide
demand for the current international standard, stocks of which were now running
low. Therefore, a replacement standard was needed to ensure continuity of supply
and ongoing comparability of test results across laboratories. The proposed
candidate material was an albumin solution with a high level of PKA that had
been donated by a manufacturer and filled into ampoules. The replacement
preparation will be calibrated against the current international standard by end
users in a joint NIBSC/EDQM international collaborative study scheduled for
November 2017. During discussion, it was noted that both antithrombin activity
and temperature need to be well controlled when performing PKA assays.
a Third WHO International Standard for prekallikrein activator.
7.2.3 Proposed First WHO Reference Reagent for

anti-human platelet antigen 15b
The detection of alloantibodies against human platelet antigens (HPAs) in

patient serum is important in the diagnosis and treatment of thrombocytopenias,
including fetal and neonatal alloimmune thrombocytopenia, platelet refractoriness
and post-transfusion purpura. Thrombocytopenias relating to alloantibodies to
HPA-15b have been reported in both Caucasian and Japanese populations. As
with HPA-1a, -3a and -5b (for which WHO potency references exist) HPA‑15b
is therefore also of clinical importance, with studies indicating that it is as
immunogenic as HPA-5. Currently, the methods used to detect anti-HPA-15b
antibodies can be unreliable and vary in their sensitivity.
Following requests made to NIBSC for a reference material for anti-
HPA-15b alloantibodies, the Committee was asked to endorse a proposal to
develop a First WHO Reference Reagent for anti-human platelet antigen 15b. The
proposed reference reagent would be used to validate the minimum sensitivity
60
of anti-HPA-15b antibody test methods used by clinical laboratories, test kit

manufacturers, research institutions and proficiency testing organizations. This
will allow users to determine the sensitivity of their antibody detection assays,
thus providing confidence that a negative result is not simply due to poor assay
sensitivity. The proposed reference reagent would also improve the comparability
of results obtained using different tests.
The intended source material is a serum containing anti-HPA-15b
antibody obtained from one or more donors. Currently approximately 2 litres
of serum from one donor is available containing low titres of anti-HPA-15b. The
material requires further evaluation and the anti-HPA-15b antibodies may need
concentrating. However, as anti-HPA-15b antibody is relatively rare, additional
material could be difficult to source. Testing for infection markers will also be
required. The Committee was informed that submission of the proposed reference
reagent for establishment is scheduled for 2019.
During discussion it was clarified that although the current serum also
contained other anti-HPA antibodies, the high specificity of testing methods
meant that this would not affect its suitability as a reference reagent. In addition,
although material from only one patient would not represent patient variability
in the real world, this would have to be accepted due to the rarity of the antibody.
It was suggested that contacting experts in the field could be very helpful in
determining whether the IgG subclasses of the proposed source material required
characterization and whether or not further source materials could be obtained.
a First WHO Reference Reagent for anti-human platelet antigen 15b.
7.2.4 Proposed First WHO International Standard for anti-

cyclic citrullinated peptide antibodies
Anti-cyclic citrullinated peptide (anti-CCP) is an autoantibody found in
patient sera that is widely used as a diagnostic and prognostic marker, along
with rheumatoid factor (RF), for rheumatoid arthritis, a common systemic
autoimmune disease. Unlike RF determination, anti-CCP measurement can
provide a differential diagnosis of rheumatoid arthritis from other diseases such
as SLE. Furthermore, anti-CCP is now recommended by The European League
Against Rheumatism as a marker for the identification of rheumatoid arthritis
patients with erosive disease, thus aiding in its therapeutic management.
A number of qualitative and quantitative first-, second- and third-
generation test kits are commercially available and use different combinations
of cyclic citrullinated peptides for capturing anti-CCP antibodies. However, the
controls and calibrators provided with the kits only have an arbitrarily assigned
unitage (U/ml). As a result, quantitative values obtained using different test kits
are not comparable and cut-off values for a positive result vary significantly
between kits. The calibration of test kits using an international standard for
61
anti-CCP antibodies would allow for meaningful comparisons to be made,

facilitate the use of anti-CCP antibodies as prognostic and diagnostic markers
of rheumatoid arthritis, and improve disease monitoring. Several organizations
have now highlighted the need for such an international standard and a
proposal was made to establish a First WHO International Standard for anti-
cyclic citrullinated peptide antibodies. The international standard would be used
by diagnostic test kit manufacturers, research institutions, proficiency testing
organizations and clinical laboratories. It was further proposed that the same
preparation used for this international standard could also serve as the Second
WHO International Standard for rheumatoid factor (see section 7.2.5 below).
If successfully sourced, the candidate material will comprise pooled
serum from rheumatoid arthritis patients free from infection markers and with
a sufficient concentration of anti-CCP and RF. International units would be
assigned based on results generated by end users in an international collaborative
study, with submission of the study report to the Committee in 2019. The
Committee was informed that a reference serum had already been prepared
by the Autoantibody Standardisation Committee (ASC) of the International
Union of Immunological Societies. Consideration had been given to possible
approaches to making the unitages of the two materials consistent. During
discussion it was pointed out that the ASC standard was not a WHO standard.
However, this standard should be included in the collaborative study to ensure
consistency of unitage.
The Committee endorsed the proposal (WHO/BS/2017.2320) to
develop a First WHO International Standard for anti-cyclic citrullinated peptide
antibodies.
7.2.5 Proposed Second WHO International Standard for rheumatoid factor

Rheumatoid factor (RF) is an autoantibody found in the serum of patients with
rheumatoid arthritis, a common, systemic autoimmune disease which affects at
least 1.3 million adults in the USA and approximately 1–2% of adults worldwide.
RF binds to IgG Fc resulting in immune-complex formation, inflammation and
joint damage and is a diagnostic marker of rheumatoid arthritis.
The current First WHO International Reference Preparation for
rheumatoid arthritis serum (lyophilized) has been distributed worldwide and
is used to calibrate assays and diagnostic test kits which measure RF levels in
patient serum. It is used by clinical laboratories, test kit manufacturers and
research institutes to calibrate their working standards. Stocks of this material
are now running low and a proposal was made to develop a replacement
standard to ensure the continuity of test result comparability across laboratories.
The replacement preparation will be calibrated against the current reference
preparation by end users in a collaborative study using methods which are not
RF-isotype specific. In addition, nominal values for specific RF isotypes (IgM,
62
IgA and IgG) may be assigned based on the results obtained using isotype-
specific detection methods. It was pointed out that different antibody subclasses
have different prevalences in different geographical areas of the world.
a Second WHO International Standard for rheumatoid factor.
7.2.6 Proposed Second WHO reference reagents

for dengue virus subtypes 1–4
The Committee was informed that the previous reference materials had proved
very useful during the 2015–2016 ZIKV epidemic in understanding flavivirus
serological cross-reactivity with dengue viruses. As these materials were now
fully depleted a replacement was urgently required, not only for continuity but
also to support the initiation of a new dengue vaccination programme.
It was intended that the replacement materials would represent the
same four serotypes (dengue serotypes 1–4) as the previous reference reagents.
However, due to insufficient stocks of the material used to produce the reference
materials a direct replacement cannot be produced and new material is required.
Sourcing suitable replacement material may prove to be problematic. In addition,
previous issues encountered during efforts to standardize neutralization assays
had not been resolved and may arise again during the replacement study. It is
acknowledged that there is some urgency concerning the need to replace the
previously used materials. For example, it is expected that the forthcoming ZIKV
vaccine roll-out may initiate increased anti-dengue testing, while the possibility
has been raised that prior exposure to dengue viruses increases the likelihood
of haemorrhagic fever following ZIKV vaccination. Therefore every effort was
being made to present the finalized study report to the Committee in 2018–2019.
During discussion it was pointed out that the National Institutes of
Health had supported studies of both dengue and Zika in Brazil and may have
access to positive samples. Other possibilities were also highlighted along with
the importance of ensuring that any candidate source material would need to be
free of antibodies to ZIKV and yellow fever virus.
replacements for the First WHO reference reagents for dengue virus serotypes
1–4.
7.2.7 Proposed First WHO International Standard for cutaneous leishmaniasis;

and First WHO Reference Panel for cutaneous leishmaniasis
Cutaneous leishmaniasis is a protozoan parasitic disease transmitted via the
infectious bite of sand flies belonging to the genus Phlebotomus in Europe, North
Africa, the Middle East and Asia and to the genus Lutzomyia in the southern USA
and Central and South America. The epidemiology of the disease is complex
63
with different protozoan species present in the eastern and western hemispheres.
It is estimated that there are over 1 million new cases each year with varying
levels of clinical manifestation. Early detection and diagnosis improves treatment
efficacy and it was proposed that the development of both an international
standard and a species-specific reference panel would facilitate this.
It is intended that preparations will be formulated from cultured pathogens
and a range of species types assessed in an international collaborative study. The
candidate material demonstrating the largest degree of assay harmonization
would be used as the proposed WHO international standard, while the other
species types would be used in a WHO reference panel. Depending on the
availability of source materials, it is anticipated that the study report will be
presented to the Committee in either 2019 or 2020. During discussion the issue
of using a common matrix for testing was discussed. It was clarified that as
laboratories often use tissue preparations instead of whole blood the suitability of
the reference materials should be evaluated in both matrix types.
a First WHO International Standard for cutaneous leishmaniasis and a First
WHO Reference Panel for cutaneous leishmaniasis.
7.2.8 Proposed First WHO International Standard for

Plasmodium vivax antigens; and First WHO Reference
Reagent for anti-malaria (Plasmodium vivax) serum
The need to standardize RDTs for malaria detection is well recognized. While
there are a range of reference preparations, both molecular and serological, to
support Plasmodium falciparum assay standardization, significant differences
between P. falciparum and P. vivax necessitate the development of separate
materials. The Committee was provided with an outline of the challenges
inherent in P. vivax diagnosis. These include the lower parasite densities
characteristic of the clinical stage and the inability of diagnostic tests to detect
the hypnozoite stage of the life-cycle. The availability of standards that would
allow for the development of assays with improved sensitivity to P. vivax LDH
and the inclusion of serological markers for hypnozoites would significantly
improve the diagnostic field.
A proposal was made to develop a First WHO International Standard
for Plasmodium vivax antigens and a First WHO Reference Reagent for anti-
malaria (Plasmodium vivax) serum for evaluation in two separate collaborative
studies. Each of the projects would be undertaken in conjunction with the
Foundation for Innovative New Diagnostics (FIND). It was pointed out that
obtaining P. vivax other than from clinical isolates was problematic and such
isolates may be difficult to source in the quantities required. In addition, the
standards developed would need to appropriately address the need for assay
64
standardization in the areas of diagnosis, seroepidemiology and immunogenicity

of potential vaccines. A realistic timeframe was being allocated to the sourcing
of materials and it was anticipated that the completed study report would be
presented to the Committee in 2020.
During discussion the possibility of developing antigen standards for
P. vivax using recombinant proteins was raised given the difficulty of acquiring
high-titre clinical source material. A further suggestion was made to acquire
blood specimens during the acute, convalescent and recrudescent phases of
P. vivax infection.
a First WHO International Standard for Plasmodium vivax antigens, and a First
WHO Reference Reagent for anti-Malaria (Plasmodium vivax) serum.
7.2.9 Proposed First WHO International Standard

for anti-MERS-CoV serum
Despite the rising global incidence of infection with the Middle East respiratory
syndrome coronavirus (MERS-CoV) there is currently no international standard
for use in diagnostics and research. Following a pilot study carried out at NIBSC
to assess a small number of materials for their potential suitability as a candidate
international standard it was proposed that an international collaborative
study now be conducted. This collaborative study would involve a range of
different laboratories and methods and would focus on the establishment of
an international standard for use in harmonizing diagnostic assays for MERS-
CoV infection. It was envisaged that the biggest issue would be the sourcing of
suitable volumes and titres of material. The ideal source material would be serum
obtained from convalescent patients that had confirmed MERS-CoV infection.
It was pointed out that in the pilot study a transchromosomal bovine IgG
sample had been included even though this type of material had never been used
previously for such a purpose. Were it to be selected as the candidate reference
material its suitability and commutability would need to be rigorously assessed.
a First WHO International Standard for anti-MERS-CoV serum.

MERS-CoV RNA for NAT-based assays
The rapid diagnosis and quarantining of individuals infected with MERS-CoV
is extremely important in preventing the spread of infection to health-care
workers and the wider population. As the use of molecular methods to detect
MERS‑CoV infection increases there is a growing need for standardization to
ensure their accuracy and sensitivity. The Committee was provided with an
overview of the findings of an external quality assessment study of MERS-CoV
65
RNA assays. The study had highlighted wide variability in reporting based on
different MERS-CoV assays. In addition, there was a high possibility of under-
reporting of MERS incidence owing to the patient sampling sites utilized.
In some infected individuals MERS-CoV was not always detected in upper
respiratory tract samples. Furthermore, upper and lower respiratory tract swabs,
potentially containing only low concentrations of MERS-CoV RNA, are the most
commonly used diagnostic samples.
A proposal was made to develop a first WHO international standard
for MERS-CoV RNA. Following the earlier identification of a potential source
of suitable stock material the intention was to provide this to NIBSC as a heat-
inactivated high-titre tissue culture that could further be diluted into a suitable
matrix. This material would then be evaluated in an international collaborative
study. Although sourcing suitable clinical material for the study may be
problematic, the group responsible for conducting the external quality assessment
study outlined above had indicated that they may be able to assist with this. It is
anticipated that the results of the collaborative study would be presented to the
Committee in 2019.
During discussion it was noted that MERS-CoV has a rapidly changing
genome and attention would need to be given to ensuring the relevance of the
chosen sample to circulating strains. The sequence of the candidate material
would have to be determined and a broad range of variants included in the study
to better understand the efficiency of variant detection.
a First WHO International Standard for MERS-CoV RNA for NAT-based assays.
7.2.11 Proposed Sixth WHO International Standard for

hepatitis C virus RNA for NAT-based assays
The WHO international standard for HCV RNA is used to calibrate secondary
reference materials for use in NAT-based assays. In addition to its use in the safety
testing of blood donations, and in testing for HCV in cells, tissues and organs,
NAT-based assays are also widely used in the management of HCV infection,
particularly in the diagnosis of disease and the initiation and monitoring of
antiviral therapies.
Despite efforts to ensure its exclusive use in the calibration of secondary
reference materials, demand for this international standard remains high. As
HCV cannot be propagated in tissue culture, standard development relies upon
sourcing HCV-positive plasma of suitable volume and titre. Historically, this has
proved problematic and has led to the production of small batches (< 2000 vials).
The proposal to replace the current international standard was being brought to
the Committee with sufficient lead time to allow for an attempt to be made to
source higher volumes of starting material. At the current rate of dispatch, there
66
is approximately a 3-year supply of the current international standard, which

should allow sufficient time to source suitable replacement materials with a view
to presenting the outcome of their evaluation to the Committee in either 2019
or 2020.
During discussion it was pointed out that the improvement of HCV
treatment protocols could lead to an upsurge in demand for HCV testing. This
should be borne in mind when deciding upon the volume of material to be
produced. It was also recommended that the genotype 1a should continue to
be used for the international standard. It was agreed that material does not have
to be from a single source and that pooling could be used to ensure sufficient
volumes. The suggestion was made that suitable source material might be
obtained from commercial plasma-collection establishments.
a Sixth WHO International Standard for hepatitis C virus RNA for NAT-based
assays.
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8. International reference materials –
vaccines and related substances
8.1 WHO International Standards and Reference

Reagents – vaccines and related substances
8.1.1 First WHO international standards for oral poliomyelitis vaccines
As the WHO Global Polio Eradication Initiative approaches completion a number
of changes in vaccination policy have been necessary. Following the certified
global eradication of wild-type poliovirus serotype 2 in 2015, the highly successful
trivalent oral poliomyelitis vaccine (tOPV) used for routine immunization was
replaced with a bivalent OPV (bOPV) containing only poliovirus serotypes 1
and 3. Monovalent versions of the three poliovirus serotypes (mOPV1, mOPV2
and mOPV3) have also been produced and maintained as stockpiles to respond
to polio outbreaks now and in the post-eradication era. The Committee was
informed that all of these products will be required for the foreseeable future.
Accordingly, the manufacture and control of these vaccines must be
maintained to the highest level in support of polio eradication. Relevant
standard preparations must therefore be available to ensure that testing
meets appropriate worldwide regulatory requirements. The Second WHO
International Standard for the potency testing of trivalent OPV was established
in 2004 and has been used for the calibration of regional working references,
manufacturer in-house references and NCL references worldwide. However,
due to the raised containment requirements for poliovirus serotype 2 following
its eradication, this standard is no longer suitable for testing bOPVs at lower
containment levels.
The Committee was informed that candidate WHO international

standards for bOPV, mOPV1, mOPV2 and mOPV3 had been produced from
three commercially produced monovalent bulks. A collaborative study had then
been conducted to establish the first WHO international standards for bOPVs
and mOPVs. The NIBSC codes 16/164 (bOPV1+3), 16/196 (mOPV1), 15/296
(mOPV2) and 16/202 (mOPV3) were assigned and the candidate materials
tested in an international collaborative study involving 14 laboratories. The
bOPV 1+3, mOPV1 and mOPV3 candidate materials were tested in duplicate
by all laboratories, while the mOPV2 candidate material was tested by the
four laboratories that were able to meet the raised containment requirements
for poliovirus serotype 2. Overall levels of intra-assay and intra-laboratory
variation in the results obtained were in line with the previous study to establish
the Second WHO International Standard for the potency testing of trivalent
68
International reference materials – vaccines and related substances
OPV, thus demonstrating a high level of consistency within laboratories. This

was supported by the range of values obtained between laboratories for all the
candidates which were within 0.5 log 10 TCID50 of the mean, indicating that the
methods are well standardized. All of the candidate materials exhibited good
stability at −70 °C indicating their suitability for use as international standards. It
was expected that under long-term storage they would behave in a very similar
way to the current second international standard. Data also indicated that the
materials are stable for 1 month at 4 °C. Annual monitoring of the potency of
these materials will be carried out. The recommended shipping and storage
requirements will be in dry ice and −70 °C respectively and will be reflected in
the IFU.
and recommended that: (a) candidate material 16/164 be established as the
First WHO International Standard for the potency testing of bivalent OPV, with
potencies of 7.19, 6.36 and 7.32 log 10 TCID50 /ml for serotypes 1, 3 and total virus
content respectively; (b) candidate material 16/196 be established as the First
WHO International Standard for the potency testing of monovalent serotype 1
OPV, with a potency of 7.32 log 10 TCID 50 /ml; (c) candidate material 15/296 be
established as the First WHO International Standard for the potency testing
of monovalent serotype 2 OPV, with a potency of 6.74 log 10 TCID 50 /ml; and
(d) candidate material 16/202 be established as the First WHO International
Standard for the potency testing of monovalent serotype 3 OPV, with a potency
of 6.66 log 10 TCID 50 /ml. The Committee noted that NIBSC should address
storage and distribution needs in relation to mOPV2 in view of its increased
containment level and should reflect such needs in the IFU.
8.1.2 Second WHO International Standard for pertussis toxin

Reference preparations of pertussis toxin (PTx) are required for the quality
control and assessment of pertussis vaccines. Acellular pertussis vaccines contain
pertussis toxin in its detoxified form and regulatory safety tests are required to
ensure that residual levels of pertussis toxin activity and reversion to toxicity
do not exceed levels shown to be safe in clinical lots. This is usually evaluated
using the murine histamine sensitization test (HIST) for final formulated lots
and/or CHO cell clustering assay for purified pertussis toxoids. Biochemical
assays comprising fetuin-binding ELISA and enzymatic reaction coupled to
HPLC have also been developed to measure PTx activity in vitro. However, most
manufacturers and control laboratories routinely use only the HIST and CHO
cell tests to monitor residual PTx activity in acellular pertussis vaccines and
whole cell vaccines. As all of these assays require active PTx as a control, a WHO
international standard is required and the First WHO International Standard for
pertussis toxin was established in 2003. Stocks of this international standard were
69
now low, with the development of a replacement standard having been endorsed
by the Committee in 2014.
The Committee was informed that a frozen preparation of PTx
manufactured by the Serum Institute of India had been donated to NIBSC where it
had been formulated and freeze-dried in sealed glass ampoules. An international
collaborative study had then been conducted to determine the suitability of the
candidate material (NIBSC code 15/126) to replace the current international
standard. A total of 14 laboratories from 12 countries took part in the study
with 11 performing HIST, 14 performing CHO cell clustering assay and three
performing biochemical assays to measure the enzymatic and carbohydrate-
binding activities of the toxin.
Study data confirmed that the candidate material 15/126 exhibited
biological activity both in HIST and CHO cell assays. However, unlike the current
international standard, the levels of activity in HIST and CHO cell assays did not
accord and the use of an individual unitage for each assay type was proposed.
The candidate material also exhibited activity in the biochemical assays used.
Accelerated degradation studies indicated that the material would be stable at
recommended storage conditions with further stability studies showing that the
material is less stable once reconstituted – a characteristic of other PTx materials.
and after discussion recommended that the candidate material 15/126 be
established as the Second WHO International Standard for pertussis toxin with
an assigned unitage of 1881 IU/ampoule for HIST and 680 IU/ampoule for the
CHO cell clustering assay.
8.1.3 First WHO international standards for Citrobacter freundii

and Salmonella Typhi Vi polysaccharides
Typhoid fever is caused by an infection with Salmonella enterica subspecies
enterica serovar Typhi (Salmonella Typhi). Salmonella Typhi expresses a capsular
polysaccharide – Vi polysaccharide (Vi PS) – which is a virulence factor and

considered the main protective antigen. A Vi PS capsule which has similar
physicochemical and immunological characteristics to that of Salmonella Typhi
is expressed by the soil bacterium Citrobacter freundii. Vaccination is the most
cost-effective preventative strategy to control typhoid, especially in areas where
multidrug resistant strains are endemic. Since plain Vi PS vaccines are unable
to provide immunoprotection for young children or infants, conjugate Vi PS
vaccines have been developed. The Committee was informed that two Vi PS-
tetanus toxoid conjugate vaccines had been licensed in India and that several
more Vi PS conjugate vaccines (using a variety of carrier proteins) were in
development. Vi PS-based typhoid vaccine production relies on physicochemical
and serological methods for estimating polysaccharide content as a measure of
potency and to ensure that batches are consistently manufactured. However,
70
the methods used are currently not well standardized between individual
laboratories and different reference materials are in use. The intended use of
WHO international standards for Vi PS was to provide globally standardized
Vi PS quantification with the aim of harmonizing measurement of the Vi PS
content of typhoid vaccines.
The standards would be used to facilitate calibration of the various assays
and in-house reference materials, and were likely to be in considerable demand,
particularly by manufacturers and NCLs in low- and middle-income countries.
The Committee was reminded that at its meeting in 2015 it endorsed a project
to develop WHO international standards for Salmonella Typhi Vi PS and for
C. freundii Vi PS, since both have been used to produce conjugate vaccines.
Source materials had been donated by the Novartis Vaccines Institute
for Global Health (now the GSK Vaccines Institute for Global Health) and
by GlaxoSmithKline Biologicals for the development of C. freundii and
Salmonella Typhi Vi PS international standards respectively. These materials
had been filled into ampoules at NIBSC and assigned the NIBSC codes 12/244
(C. freundii Vi PS) and 16/126 (Salmonella Typhi Vi PS). An international
collaborative study had then been conducted involving 20 laboratories from
12 countries. During the study, two separate evaluations were made – one to
assign unitage using qNMR to quantitate Vi PS/ampoule using the N-acetyl and
O-acetyl resonances and the second to assess the suitability of the candidate
materials in determining the Vi PS and O-acetyl content of vaccine samples using
commonly used physicochemical assays and immunoassays and in comparison
with in‑house standards. Stability studies performed over 6 months indicated
that both candidate materials were stable at temperatures used for storage
(−20 °C) and during laboratory manipulation (4 °C). Accelerated degradation
studies showed no observable size reduction for either material following storage
at up to 56 °C for 6 months. The amount of polysaccharide per ampoule remained
constant under all conditions. Further real-time and accelerated stability studies
were ongoing.
The Committee considered the report of the study (WHO/BS/
2017.2310) and after further discussion and clarification of the need for the
two international standards recommended that: (a) the candidate material
12/244 be established as the First WHO International Standard for Citrobacter
freundii Vi polysaccharide with a content of 1.94 ± 0.12 mg/ampoule; and
(b) the candidate material 16/126 be established as the First WHO International
Standard for the Salmonella Typhi Vi polysaccharide with a content of 2.03 ±
0.10 mg/ampoule. The Committee further noted that the intended use of
both WHO international standards was for the quantification of the Vi PS
component of vaccines containing Vi PS using the HPAEC-PAD, ELISA, rate
nephelometry or rocket immuno-electrophoresis assays, with the observation
that the latter may not be suitable for all bulk Vi PS conjugates.
71
8.1.4 First WHO International Standard for anti-typhoid capsular

Vi polysaccharide immunoglobulin G (human)
The Committee was provided with a brief overview of the history of efforts
to establish a WHO international standard for anti-typhoid capsular Vi
polysaccharide IgG. These efforts had led to the conducting of an international
collaborative study to evaluate the performance of a number of candidate
reference materials using the commercial assay VaccZyme ELISA as well as in-
house ELISAs. The study found considerable variation in the results obtained
using in-house assays, and the Committee at its 2014 meeting recommended
against the establishment of the proposed candidate material (NIBSC code
10/126) as an international standard at that time. Possible causes of the variation
included differences in ELISA procedures, in the quality of Vi PS used or
differences in the anti-Vi IgG.
The Committee was informed that a new IgG preparation had been
obtained and a collaborative study of its performance completed. The new
candidate material (NIBSC code 16/138) had been obtained from healthy
volunteers immunized with licensed typhoid vaccines, and been donated by the
Oxford Vaccine Group with full ethical approval. The candidate material was filled
into ampoules, freeze-dried and evaluated for stability. The collaborative study
involved seven laboratories (including vaccine manufacturers, NCLs and research
laboratories) in six countries. The suitability of candidate material 16/138 for use
as a reference material for anti-Vi IgG serum (human) was evaluated alongside a
previously used reference reagent (Vi-IgG R1, 2011), the previous candidate material
10/126, three post-vaccination sera and one pre-vaccination serum in several Vi
ELISA formats.
In the majority of cases, valid estimates were obtained for the potency of
coded samples relative to both the candidate material 16/138 and Vi-IgG R1, 2011
and for the relative potency of the two standards to each other. Despite giving
valid estimates, the NIBSC ELISA (using biotinylated Vi) showed no concordance
with the VaccZyme ELISA, suggesting that the modification of Vi makes these
types of ELISAs unsuitable as an alternative to the VaccZyme ELISA. Study
data indicated that the candidate material 16/138 would be suitable for use as a
reference standard for anti-Vi IgG serum (human) in the VaccZyme ELISA and
in in-house ELISAs where the commutability of 16/138 with the coded samples
and Vi-IgG R1, 2011 was evident. Stability evaluation of candidate material 16/138
indicated adequate stability when stored at −20 °C.
and, following considerable discussion and clarification of a number of points,
recommended that the candidate material 16/138 be established as the First
WHO International Standard for anti-typhoid capsular Vi polysaccharide IgG
(human) with an assigned unitage of 100 IU/ampoule. Relative unitages of 54 IU/
72
ampoule and 163 IU/vial were also assigned to candidate material 10/126 and
Vi‑IgG R1, 2011 respectively. The Committee agreed that a follow-up collaborative
study might be helpful in clarifying whether in-house ELISAs based on
poly‑L‑Lysine and native Vi could be suitable alternatives to the commercial
VaccZyme ELISA.
8.1.5 First WHO International Standard for antiserum

to respiratory syncytial virus
The development of a respiratory syncytial virus (RSV) vaccine is a widely
recognized global health priority. The Committee was informed that activity in
this area had increased significantly in recent years, with numerous RSV vaccine
candidates in development – 14 of which were now in human clinical trials. RSV-
neutralizing activity in serum has been reported to correlate with protection
against acute lower respiratory infection with RSV in both rodent models and
human infants. Accurately quantifying this neutralizing activity will be vital in
the development of RSV vaccines.
There are multiple formats of RSV-neutralization assays, and accurately
comparing the neutralization titres in sera from multiple clinical trials using
different neutralization assay formats is challenging. A reference antiserum is
therefore urgently needed to standardize clinical trial data and outcomes. Sixty
serum samples obtained from healthy adults were provided by PATH for use
as source material for a proposed WHO international standard. Samples with
high and medium RSV antibody titres were selected and two candidate pools
were prepared, processed, filled, freeze-dried and assigned the NIBSC codes
16/284 and 16/322. A collaborative study was then conducted to characterize
the performance of the two candidate materials in a range of diverse RSV-
neutralization assays and to assess their suitability for use as international
standards for anti-serum to RSV. The study involved 21 laboratories from
9 countries, including university laboratories, manufacturers/developers of RSV
vaccines and public health laboratories. All participants used their own in-house
virus-neutralization assay and their own virus stocks. Study samples comprised
the two candidate materials 16/284 and 16/322, naturally infected adult sera,
age-stratified naturally infected paediatric sera, sera from RSV vaccine clinical
trials in maternal and elderly subjects, a mAb to RSV (palivizumab), two cotton
rat serum samples, and samples from the BEI Resources Panel of Human
Antiserum and Immune Globulin to Respiratory Syncytial Virus.
Study results indicated that inter-laboratory variability in neutralization
titres was significantly reduced when values were expressed relative to those of
either of the two candidate materials. The collaborative study also indicated that
the standards were useful for multiple sample types across a wide variety of assay
formats. However, analysis suggested that the cotton rat serum samples and mAb
73
sample behaved differently from the human serum samples and that a more
suitable standard should be considered for these sample types. This would not
be an issue for the establishment of a WHO international standard as its main
role would be to look at neutralizing antibody activity in human serum mostly
produced during RSV vaccine clinical trials.
Stability data for 16/284 indicated a low predicted loss in activity per
year (< 0.01%) when stored at −20 °C, suggesting that it is sufficiently stable
to serve as a WHO international standard. A long-term stability-monitoring
programme will be needed to show that candidate material 16/284 remains
stable over its lifetime. Stability data for 16/322 were not currently available
but it too will be monitored for stability over its lifetime. Furthermore,
stability analysis indicated that the candidate materials were also stable after
reconstitution. Both candidate materials showed loss of activity at 37 °C after
2 weeks, with 16/322 showing a greater loss than 16/284.
First WHO International Standard for antiserum to respiratory syncytial virus
with an assigned unitage of 1000 IU/vial. The Committee also assigned a unitage
of 960 IU/vial to candidate material 16/322 as a potential future replacement
standard.
8.2 Proposed new projects and updates –

vaccines and related substances
8.2.1 Proposed First WHO Reference Panel for Vibrio cholera
O1 and O139 lipopolysaccharides
Vibrio cholera O1 and O139 are leading causes of bacterial diarrhoea and
bacteraemia in Africa, South-East Asia and the Caribbean. Cholera outbreaks
occur frequently in refugee camps and following natural disasters. Young
children and infants are particularly vulnerable. Oral cholera vaccine (OCV),
inactivated, is the most cost-effective measure to contain and prevent the disease
in such settings. Three such vaccines have now been prequalified by WHO
and are part of a WHO-maintained stockpile. To be effective, cholera vaccines
require appropriate regulation at the national level to ensure their efficacy. As
standardization is a prerequisite for achieving appropriate quality control of
these vaccines the availability of WHO standards for the lipopolysaccharides
(LPS) of O1 Inaba, O1 Ogawa and O139 V. cholera would be expected to support
the assay development needed for the technology transfer and quality control
of inactivated OCV.
Purified LPS O1 Inaba, O1 Ogawa and O139 will be obtained from the
International Vaccine Institute, Republic of Korea, and the material filled and
74
freeze-dried at the NIBSC. Approximately 1000 ampoules of each serotype LPS

would be produced. A collaborative study would then be required to assess the
suitability of the three candidate materials for use in a WHO reference panel
for the inhibition ELISA assay used to determine the potency of OCVs. The
study would also compare the reactivity of the candidate materials and in-house
standards using in-house inhibition ELISAs. Collaborative study participants
are expected to include vaccine developers/manufactures and NCLs, with the
total number likely to be quite small. However, three or four new manufacturers
from low- and middle-income countries were expected to enter the field. It
was anticipated that the reference panel would be used by NCLs and vaccine
manufacturers to calibrate the immunoassays used to determine OCV potency,
with a predicted demand of approximately 10 vials per year.
Following discussion of timelines, funding and details of the proposed
assay, as well as issues related to the donation of reference material, the
Committee endorsed the proposal (WHO/BS/2017.2319) to develop a First
WHO Reference Panel for Vibrio cholera O1 and O139 lipopolysaccharides and
requested that feedback be provided on progress.
8.2.2 Proposed First WHO Reference Panel for anti-Vibrio cholera

O1 and O139 lipopolysaccharide serums (rabbit)
The Committee was informed that this proposal was part of a joint effort with
the International Vaccine Institute, and the Bill & Melinda Gates Foundation to
produce three sets of antisera in rabbits – anti-O1 Inaba serum, anti-O1 Ogawa
serum and anti-O139 serum. The materials would be filled and freeze-dried at
NIBSC to produce approximately 500 ampoules, each containing 1 ml of freeze-
dried antiserum. An international collaborative study would then be undertaken
to assess the suitability of the candidate materials for use in a WHO reference
panel for anti-O1 Inaba, anti-O1 Ogawa and anti-O139 for the inhibition ELISA
used to determine the potency of OCVs. The collaborative study would also
compare the reactivity of the candidate materials and in-house standards using
in-house inhibition ELISAs.
It was envisaged that the resulting rabbit antisera panel would be used
by NCLs and vaccine manufacturers, primarily in low- and middle-income
countries, to calibrate the immunoassays used to determine the potency of OCV
batches. The panel was also expected to be used to compare the potency and
stability of vaccines with respect to a proposed new standard OCV (see section
8.2.3 below). The predicted level of demand was approximately 10 vials per year.
The Committee endorsed the proposal (WHO/BS/2017.2319) to
develop a First WHO Reference Panel for anti-Vibrio cholera O1 and O139
lipopolysaccharide serums (rabbit).
75

Vibrio cholera vaccine (oral, inactivated)
The Committee was informed that the availability of a mixture of killed Vibrio
cholera O1 Inaba, O1 Ogawa and O139 whole cells in a composition similar to
that of OCVs would enhance and support the further development of assays
required to ascertain vaccine quality and facilitate technology transfer for cholera
vaccine manufacturing. A candidate material will be evaluated in a collaborative
study to assess its suitability for use as an international standard vaccine for use
in in-house inhibition ELISAs to determine the OCV potency. Approximately
1000 ampoules containing 1 ml of freeze-dried cells were expected to be made
available. This proposal is part of a joint effort with the International Vaccine
Institute and the Bill & Melinda Gates Foundation to ensure sufficient supplies
of inactivated OCV of appropriate quality, safety and efficacy.
a First WHO International Standard for Vibrio cholera vaccine (oral, inactivated).
8.2.4 Proposed First WHO International Standard for antibody

to the influenza virus haemagglutinin stem domain
The need for improved, longer lasting and more broadly protective influenza
vaccines has long been recognized. Such vaccines would have a public health
impact worldwide, in both low- and high-income countries, and would
potentially improve the public health response to seasonal and pandemic
influenza. The Committee was informed that many antibodies binding to the
stem domain of the haemagglutinin (HA) of influenza A viruses have been
found to be cross-reactive between drifted viruses of the same subtype, and in
some cases between viruses of different subtypes. As a result, attempts were now
under way to develop broadly reactive and protective influenza vaccines that
elicit HA stem-binding antibodies, with clinical trials expected soon. In addition,
a number of laboratories are developing therapeutic mAbs against the HA stem

domain, and such efforts might also benefit from the development of a WHO
international standard.
Various assays are used to measure HA stem-binding antibodies, including
virus neutralization assays and binding assays. As vaccine candidates progress
through pre-clinical and clinical testing, the harmonization of serological read-
outs would be beneficial. A WHO international standard would help achieve such
harmonization thus improving comparability both between laboratories and
between studies. The optimal format such a standard was not yet clear and it is
not known whether a single mAb, an oligoclonal mixture of mAbs or a polyclonal
antiserum (of human or animal origin) would perform best. Obtaining mAbs
from commercial entities may involve lengthy MTA negotiations.
76
The Committee was informed that the proposed project would therefore
be conducted in two phases and would take more time than a more typical
standardization project. In the first phase of the project, candidate materials
will be sent to a small number of laboratories and tested for HA stem-binding
antibodies using their in-house assays. The materials will also be evaluated in
terms of their ability to harmonize assay results. Based on the outcome of this
first study, a second larger study would then be undertaken which would include
the best performing standard types identified in the first study, as well as samples
from human clinical trials.
The Committee considered this to be an exciting and forward-looking
project and expressed its support in principle. However, it also noted the
exploratory nature of the first phase of the project and therefore endorsed the
first exploratory phase of the proposal (WHO/BS/2017.2319) to develop a First
WHO International Standard for antibody to the influenza virus haemagglutinin
stem domain. The Committee asked that NIBSC report back at its next meeting
on the progress made, as well as on the state of the influenza vaccine field, to
allow for further review and consideration of the proposal by the Committee.
8.2.5 Proposed First WHO international standards for

influenza virus pathogenicity for safety testing
The safety testing of an influenza candidate vaccine virus (CVV) is based upon
comparison against the parental wild-type virus in ferret studies. Although the
CVV must be attenuated relative to the corresponding wild-type virus there are
currently no accepted criteria for attenuation. The Committee was informed
that the WHO biosafety risk assessment and guidelines for the production and
quality control of human influenza pandemic vaccines – which specifies safety-
testing procedures for CVVs – was currently under review. It had been proposed
that standards of pathogenicity could be used instead of reliance upon wild-type
viruses specific to each CVV during the development of CVVs for influenza
vaccines for pandemic preparedness purposes. Such CVVs are generated on a
continual basis and need to be assessed for attenuation. It was proposed that
guidance on the use of the standard viruses would be included in the revised
version of the WHO guidelines.
CVVs are crucial in the production of influenza vaccines and in the case
of a pandemic, must be generated and tested very rapidly. Having the proposed
standard viruses available before a pandemic would enable laboratories to
harmonize their safety testing. In the event of a pandemic – when access to a
newly emerging wild-type virus may be difficult – tests could be conducted faster
and CVVs released sooner without compromising safety. Ultimately with fewer
animals being required for testing, a greater range of CVVs could be tested in a
pandemic situation if required.
77
Animal tests are also subject to variability and the ferret safety test is no
exception. Having standards that allow for the bench-marking of the test will lead
to better understanding of test results and greater confidence in them. Moreover,
this proposal is in line with moves towards reducing the use of animals in the
research and development of biological medicines. Instead of comparing every
new CVV with its respective wild-type parental virus, the occasional testing of
the standard viruses would be conducted, thus reducing the overall number of
ferrets required. The use of standard viruses in this way – in combination with
defined cut-off values/ranges – will not only reduce the number of animals used
but will also provide a means of assessing CVVs derived from parental wild-
type viruses which are non-pathogenic in ferrets, which cannot currently be
demonstrated to be attenuated as per the existing WHO guidance.
Following discussion and clarification of how the proposed standards
would be used the Committee endorsed the proposal (WHO/BS2017.2319) to
develop the First WHO international standards for influenza virus pathogenicity
for safety testing. However, in view of ongoing discussions on the revision of the
existing WHO biosafety risk assessment and guidelines for the production and
quality control of human influenza pandemic vaccines, the Committee requested
that it be updated on the progress made in this project and on all other relevant
developments at its next meeting.
8.2.6 Proposed Third WHO International Standard for

anti-rabies immunoglobulin (human)
Rabies is a neglected zoonosis with substantial public health and economic impacts
worldwide. Rabies antibody assays are used to evaluate the immunogenicity
of human rabies vaccines and the potency of immunoglobulins used in post-
exposure prophylaxis. The standardization of assays used for the detection and
quantification of rabies antibodies is crucial. The Committee was informed that
stocks of the current international standard are nearing depletion and are now
under restricted sales. A replacement standard was now urgently needed as
many national regulatory requirements for rabies IgG potency assays state that a
reference preparation calibrated in IU must be used.
The development of a new international standard would require the
donation of human immunoglobulin preparations from manufacturers. As the
ability of manufacturers to donate such preparations is currently uncertain there
is concern that the current international standard may become depleted before its
replacement is established. The calibration of a candidate replacement standard
would require a collaborative study in the usual way. In this case, the aim of the
collaborative study would be to calibrate the candidate material in IUs against
the current international standard in assays such as the rapid fluorescent focus
inhibition test, the mouse neutralization test and the plaque reduction assay.
78
A rabies challenge virus standard 11 (G protein) pseudotyped lentiviral particle

neutralization assay was also available and was currently undergoing a feasibility
study for adoption into the European Pharmacopoeia.
The collaborative study would involve up to 12 laboratories worldwide,
performing a range of rabies vaccine assays, and representing manufacturers
of rabies vaccines and NCLs. Study samples would include the candidate
replacement standard, the current international standard and, if possible, EDQM
reference material.
The Committee noted that the availability of IgG to serve as the proposed
third international standard was uncertain but was assured that sufficient
quantities of the current international standard would be retained for calibrating
its replacement. The Committee therefore endorsed the proposal (WHO/
BS/2017.2319) to develop a Third WHO International Standard for anti-rabies
immunoglobulin (human).
79
Annex 1
WHO Recommendations, Guidelines and other documents
related to the manufacture, quality control and evaluation
of biological substances used in medicine
WHO Recommendations, Guidelines and other documents are intended to
provide guidance to those responsible for the production of biological substances
as well as to others who may have to decide upon appropriate methods of
assay and control to ensure that products are safe, reliable and potent. WHO
Recommendations (previously called Requirements) and Guidelines are scientific
and advisory in nature but may be adopted by an NRA as national requirements
or used as the basis of such requirements.
Recommendations concerned with biological substances used in
medicine are formulated by international groups of experts and are published
in the WHO Technical Report Series 1 as listed below. A historical list of
Requirements and other sets of Recommendations is available on request from
the World Health Organization, 20 avenue Appia, 1211 Geneva 27, Switzerland.
Reports of the WHO Expert Committee on Biological Standardization
published in the WHO Technical Report Series can be purchased from:
WHO Press
World Health Organization
20 avenue Appia
1211 Geneva 27
Switzerland
Telephone: + 41 22 791 3246
Fax: +41 22 791 4857
Email: [email protected]
Website: www.who.int/bookorders
Individual Recommendations and Guidelines may be obtained free of
charge as offprints by writing to:
Technologies Standards and Norms
Department of Essential Medicines and Health Products
World Health Organization
20 avenue Appia
1211 Geneva 27
Switzerland
1
Abbreviated in the following pages to “TRS”.
81
Recommendations, Guidelines and other Reference

documents
Animal cells, use of, as in vitro substrates for the Revised 2010, TRS 978 (2013)
production of biologicals
BCG vaccines (dried) Revised 2011, TRS 979 (2013)
Biological products: good manufacturing Revised 2015, TRS 999 (2016)
practices
Biological standardization and control: Unpublished document
a scientific review commissioned by the UK WHO/BLG/97.1
National Biological Standards Board (1997)
Biological substances: International Standards Revised 2004, TRS 932 (2006)
and Reference Reagents
Biotherapeutic products, changes to approved Adopted 2017, TRS 1011 (2018)
biotherapeutic products: procedures and data
requirements
Biotherapeutic products, similar Adopted 2009, TRS 977 (2013)
Biotherapeutic protein products prepared by Revised 2013, TRS 987 (2014);
recombinant DNA technology Addendum 2015, TRS 999 (2016)
Blood, blood components and plasma Revised 1992, TRS 840 (1994)
derivatives: collection, processing and quality
control
Blood and blood components: management Adopted 2016, TRS 1004 (2017)
as essential medicines
Blood components and plasma: estimation of Adopted 2016, TRS 1004 (2017)
residual risk of HIV, HBV or HCV infections
Blood establishments: good manufacturing Adopted 2010, TRS 961 (2011)

practices
Blood plasma (human) for fractionation Adopted 2005, TRS 941 (2007)
Blood plasma products (human): viral Adopted 2001, TRS 924 (2004)
inactivation and removal procedures
Blood regulatory systems, assessment criteria Adopted 2011, TRS 979 (2013)
for national
Cholera vaccines (inactivated, oral) Adopted 2001, TRS 924 (2004)
Dengue tetravalent vaccines (live, attenuated) Revised 2011, TRS 979 (2013)
Diphtheria, tetanus, pertussis (whole cell), and Revised 2012, TRS 980 (2014)
combined (DTwP) vaccines
82
Annex 1

documents
Diphtheria vaccines (adsorbed) Revised 2012, TRS 980 (2014)
DNA vaccines: assuring quality and nonclinical Revised 2005, TRS 941 (2007)
safety
Ebola vaccines Adopted 2017, TRS 1011 (2018)
Haemophilus influenzae type b conjugate Revised 1998, TRS 897 (2000)
vaccines
Haemorrhagic fever with renal syndrome (HFRS) Adopted 1993, TRS 848 (1994)
vaccines (inactivated)
Hepatitis A vaccines (inactivated) Adopted 1994, TRS 858 (1995)
Hepatitis B vaccines prepared from plasma Revised 1987, TRS 771 (1988)
Hepatitis B vaccines made by recombinant DNA Revised 2010, TRS 978 (2013)
techniques
Human immunodeficiency virus rapid diagnostic Adopted 2017, TRS 1011 (2018)
tests for professional use and/or self-testing
Technical Specifications Series for WHO
Prequalification – Diagnostic Assessment
Human interferons prepared from Adopted 1988, TRS 786 (1989)
lymphoblastoid cells
Influenza, biosafety risk assessment and safe Adopted 2005, TRS 941 (2007)
production and control for (human) pandemic
vaccines
Influenza vaccines (inactivated) Revised 2003, TRS 927 (2005)
Influenza vaccines (inactivated): labelling Addendum to TRS 927;
information for use in pregnant women TRS 1004 (2017)
Influenza vaccines (live) Revised 2009, TRS 977 (2013)
Influenza vaccines, human, pandemic, Adopted 2007, TRS 963 (2011)
regulatory preparedness
Influenza vaccines, human, pandemic: Adopted 2016, TRS 1004 (2017)
regulatory preparedness in non-vaccine-
producing countries
In vitro diagnostic medical devices, establishing Adopted 2017, TRS 1011 (2018)
stability of,
Technical Guidance Series for WHO
83

documents
Japanese encephalitis vaccines (inactivated) Revised 2007, TRS 963 (2011)
for human use
Japanese encephalitis vaccines (live, attenuated) Revised 2012, TRS 980 (2014)
for human use
Louse-borne human typhus vaccines (live) Adopted 1982, TRS 687 (1983)
Malaria vaccines (recombinant) Adopted 2012, TRS 980 (2014)
Measles, mumps and rubella vaccines and Adopted 1992, TRS 848 (1994);
combined vaccines (live) Note TRS 848 (1994)
Meningococcal polysaccharide vaccines Adopted 1975, TRS 594 (1976);
Addendum 1980, TRS 658 (1981);
Amendment 1999, TRS 904 (2002)
Meningococcal A conjugate vaccines Adopted 2006, TRS 962 (2011)
Meningococcal C conjugate vaccines Adopted 2001, TRS 924 (2004);
Addendum (revised) 2007,
TRS 963 (2011)
Monoclonal antibodies Adopted 1991, TRS 822 (1992)
Monoclonal antibodies as similar biotherapeutic Adopted 2016, TRS 1004 (2017)
products
Papillomavirus vaccines (human, recombinant, Revised 2015, TRS 999 (2016)
virus-like particle)
Pertussis vaccines (acellular) Revised 2011, TRS 979 (2013)
Pertussis vaccines (whole-cell) Revised 2005, TRS 941 (2007)
Pharmaceutical products, storage and transport Adopted 2010, TRS 961 (2011)
of time- and temperature-sensitive
Pneumococcal conjugate vaccines Revised 2009, TRS 977 (2013)
Poliomyelitis vaccines (inactivated) Revised 2014, TRS 993 (2015)
Poliomyelitis vaccines (inactivated): guidelines Adopted 2003, TRS 926 (2004)
for the safe production and quality control of
inactivated poliomyelitis vaccine manufactured
from wild polioviruses
Poliomyelitis vaccines (oral) Revised 2012, TRS 980 (2014)
Quality assurance for biological products, Adopted 1991, TRS 822 (1992)
guidelines for national authorities
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Annex 1

documents
Rabies vaccines for human use (inactivated) Revised 2005, TRS 941 (2007)
produced in cell substrates and embryonated
eggs
Reference materials, secondary: for NAT-based Adopted 2016, TRS 1004 (2017)
and antigen assays: calibration against WHO
International Standards
Regulation and licensing of biological products Adopted 1994, TRS 858 (1995)
in countries with newly developing regulatory
authorities
Regulatory risk evaluation on finding an Adopted 2014, TRS 993 (2015)
adventitious agent in a marketed vaccine:
scientific principles
Rotavirus vaccines (live, attenuated, oral) Adopted 2005, TRS 941 (2007)
Smallpox vaccines Revised 2003, TRS 926 (2004)
Snake antivenom immunoglobulins Revised 2016, TRS 1004 (2017)
Sterility of biological substances Revised 1973, TRS 530 (1973);
Amendment 1995, TRS 872 (1998)
Synthetic peptide vaccines Adopted 1997, TRS 889 (1999)
Tetanus vaccines (adsorbed) Revised 2012, TRS 980 (2014)
Thiomersal for vaccines: regulatory expectations Adopted 2003, TRS 926 (2004)
for elimination, reduction or removal
Thromboplastins and plasma used to control Revised 2011, TRS 979 (2013)
oral anticoagulant therapy
Tick-borne encephalitis vaccines (inactivated) Adopted 1997, TRS 889 (1999)
Transmissible spongiform encephalopathies Revised 2005, WHO (2006)
in relation to biological and pharmaceutical
products 2
Tuberculins Revised 1985, TRS 745 (1987)
Typhoid vaccines, conjugated Adopted 2013, TRS 987 (2014)
Typhoid vaccines (live, attenuated, Ty21a, oral) Adopted 1983, TRS 700 (1984)
Typhoid vaccines, Vi polysaccharide Adopted 1992, TRS 840 (1994)
2
Available online at: http://www.who.int/biologicals/publications/en/whotse2003.pdf
85

documents
Vaccines, changes to approved vaccines: Adopted 2014, TRS 993 (2015)
procedures and data requirements
Vaccines, clinical evaluation: regulatory Revised 2016, TRS 1004 (2017)
expectations
Vaccines, clinical evaluation: use of human Adopted 2016, TRS 1004 (2017)
challenge trials
Vaccines, lot release Adopted 2010, TRS 978 (2013)
Vaccines, nonclinical evaluation Adopted 2003, TRS 926 (2004)
Vaccines, nonclinical evaluation of vaccine Adopted 2013, TRS 987 (2014)
adjuvants and adjuvanted vaccines
Vaccines, prequalification procedure Adopted 2010, TRS 978 (2013)
Vaccines, stability evaluation Adopted 2006, TRS 962 (2011)
Vaccines, stability evaluation for use under Adopted 2015, TRS 999 (2016)
extended controlled temperature conditions
Varicella vaccines (live) Revised 1993, TRS 848 (1994)
Yellow fever vaccines Revised 2010, TRS 978 (2013)
Yellow fever vaccines, laboratories approved Revised 1995, TRS 872 (1998)
by WHO for the production of
Yellow fever virus, production and testing of Adopted 1985, TRS 745 (1987)
WHO primary seed lot 213-77 and reference batch
168-736
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Annex 2
Guidelines on the quality, safety and efficacy of Ebola
vaccines
Introduction 91
Purpose and scope 92
Terminology 94
General considerations 97
Part A. Guidelines on the development, manufacture and control of
Ebola vaccines 102
A.1 General manufacturing guidelines 103
A.2 Control of source materials 105
A.3 Control of Ebola vaccine production 112
A.4 Filling and containers 122
A.5 Control tests on final lot 123
A.6 Records 125
A.7 Retained samples 125
A.8 Labelling 125
A.9 Distribution and transport 126
A.10 Stability testing, storage and expiry date 126
Part B. Nonclinical evaluation of Ebola vaccines 128
B.1 General remarks 128
B.2 Product characterization and process development 128
B.3 Pharmacodynamic studies 129
B.4 Nonclinical safety studies (toxicity testing) 132
B.5 Pharmacokinetic (biodistribution) studies 133
B.6 Environmental risk 133
Part C. Clinical evaluation of Ebola vaccines 134
C.1 General considerations 134
C.2 Demonstration of effectiveness of candidate Ebola vaccines 138
C.3 Safety evaluation of candidate Ebola vaccines 145
C.4 Ethical considerations 147
C.5 Statistical considerations 147
C.6 Serological and diagnostic assays 149
C.7 Special populations 151
C.8 Post-marketing surveillance 154
87
Part D. Guidelines for NRAs 156

D.1 General 156
D.2 Release and certification 156
Authors and acknowledgements 157
References 160
Appendix 1 Model protocol for the manufacturing and control of viral-vectored
Ebola vaccines 171
Appendix 2 Model NRA Lot Release Certificate for viral-vectored Ebola vaccines 178
Guidelines published by the World Health Organization (WHO) are

intended to be scientific and advisory in nature. Each of the following
sections constitutes guidance for national regulatory authorities
(NRAs) and for manufacturers of biological products. If an NRA so
desires, these WHO Guidelines may be adopted as definitive national
requirements, or modifications may be justified and made by the NRA.
It is recommended that modifications to these Guidelines are made
only on condition that such modifications ensure that the product is
at least as safe and efficacious as that prepared in accordance with the
guidance set out below.
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Annex 2
Abbreviations
Ad human adenovirus
AESI adverse event of special interest
AR attack rate
ARU attack rate in unvaccinated individuals
ARV attack rate in vaccinated individuals
BCG bacillus Calmette–Guérin
BDBV Bundibugyo ebolavirus
BSL biosafety level
CBER Center for Biologics Evaluation and Research
CEF chick embryo fibroblast
ChAd3 chimpanzee adenovirus type 3
DCVMN Developing Countries Vaccine Manufacturers Network
EBOV Ebola virus
ELISA enzyme-linked immunosorbent assay
ELISpot enzyme-linked immunospot
ERA environmental risk assessment
EUAL WHO emergency use assessment and listing (procedure)
EVD Ebola virus disease
GMO genetically modified organism
GP glycoprotein
HVAC heating, ventilation and air conditioning
& Associations
ICH International Council for Harmonisation of Technical
Requirements for Pharmaceuticals for Human Use
89
ICP immune correlate of protection

ICS intracellular cytokine staining
Ig immunoglobulin
LAL Limulus amoebocyte lysate
LVV lentiviral vector
MARV Marburg virus
MVA modified vaccinia Ankara
NAT nucleic acid amplification technique
PCR polymerase chain reaction
PDL population doubling level
qPCR quantitative polymerase chain reaction
RESTV Reston ebolavirus
RNA ribonucleic acid
RR relative risk
RT reverse transcriptase
rVSV recombinant vesicular stomatitis virus
SAE serious adverse event
SAGE WHO Strategic Advisory Group of Experts
SPF specific pathogen-free

SUDV Sudan ebolavirus
SWRCT stepped wedge randomized cluster trial
TAFV Tai Forest ebolavirus
VLP virus-like particle
VSV vesicular stomatitis virus
ZEBOV Zaire ebolavirus
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Annex 2
Introduction
The unprecedented scale and severity of the Ebola virus disease (EVD) epidemic
in West Africa in 2014–2016 led to calls for the urgent development and licensing
of an Ebola vaccine (1, 2). A considerable amount of work was subsequently
undertaken over a short period of time and a series of international consultations
held on related public health issues and on Ebola vaccine development, evaluation
and licensing (2–4). The development of Ebola vaccines and implications for
future immunization policy recommendations are being monitored by the WHO
Strategic Advisory Group of Experts (SAGE) on Immunization (5). In addition,
as part of ongoing WHO measures to support the development of Ebola vaccines,
guidance was prepared on the scientific and regulatory considerations relating
to their quality, safety and efficacy. In March 2015, WHO convened an informal
consultation in Geneva attended by scientific experts, regulatory professionals
and other stakeholders involved in Ebola vaccine development, production,
evaluation and licensure. The purpose of this consultation was to review initial
draft guidelines prepared by a drafting group, and to seek consensus on key
technical and regulatory issues (6). The draft guidelines were revised in the light
of comments made, and then underwent public consultation which resulted in a
large number of further comments and suggestions. The draft guidelines, together
with the comments, were discussed by the WHO Expert Committee on Biological
Standardization at its meeting in October 2015. During 2016, further revisions
were made following public consultations and working group discussions. One
major challenge during the development of these Ebola vaccine guidelines was
that they were initially prepared during the rapidly evolving epidemic situation
when the need for a vaccine was most urgent. With the end of the large-scale EVD
outbreak in Africa, declared by WHO in June 2016, EVD returned to its previous
sporadic pattern – an epidemiological situation which made the evaluation of
Ebola vaccine efficacy, and thus licensing, more challenging. Interest also shifted
from the development of monovalent Ebola virus (EBOV) Zaire vaccines to
multivalent preparations directed against more than one EBOV strain, as well as
against the Marburg virus (MARV).
The WHO Expert Committee on Biological Standardization reviewed the
draft document again in October 2016 and after extensive discussion agreed that
the guidance should be extended to include multivalent Ebola vaccines and the
clinical evaluation of candidate vaccines using innovative clinical trial designs.
There was also a recognized need to provide guidance on how to evaluate and
license Ebola vaccines subsequent to the potential licensure of one of the advanced
vectored vaccines. These WHO Guidelines are the result of these discussions.
This document provides information and guidance on the development,
production, quality control and evaluation of candidate Ebola vaccines in the
form of WHO Guidelines rather than WHO Recommendations. This allows
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for greater flexibility with respect to the expected future of Ebola vaccine
development, production, quality control and evaluation. Given that this is a very
dynamic field both in terms of technologies and clinical trial designs, these WHO
Guidelines should be read in conjunction with other relevant recent guidelines.
A model protocol for the manufacturing and control of viral-vectored
Ebola vaccines is provided in Appendix 1 of these WHO Guidelines. This
protocol outlines the information that should be provided as a minimum by a
manufacturer to the NRA in support of a request for the release of a vaccine
for use. The protocol is not intended to apply to material intended for clinical
trials. A Lot Release Certificate signed by the appropriate NRA official should be
provided if requested by a manufacturer, and should certify whether or not the
lot of vaccine in question meets all national requirements and/or Part A of these
WHO Guidelines. The purpose of this is to facilitate the exchange of vaccines
between countries, and should be provided to importers of the vaccines. A model
NRA Lot Release Certificate is provided in Appendix 2.
Purpose and scope

These WHO Guidelines provide scientific and regulatory guidance for national
regulatory authorities (NRAs) and vaccine manufacturers on the quality,
nonclinical and clinical aspects of Ebola vaccines relevant to marketing
authorizations. In particular, the document deals with Ebola vaccines based on
viral vectors, which are currently at the most advanced stage of development and
for which no specific WHO guidance is available. The document also discusses
opportunities to accelerate vaccine development and product availability during
a public health emergency.
The document does not address access programmes or regulatory
pathways for making investigational Ebola vaccines available for situations
where their use is not primarily intended to obtain safety and efficacy or
effectiveness information.
Although recombinant viral-vectored Ebola vaccines are the main
category of vaccine considered in this document, some aspects of the guidance
provided are relevant to other approaches. General guidance on other technologies
relevant to Ebola vaccine development has been published elsewhere by WHO,
including guidance on:
■■ inactivated vaccines (7–9)
■■ protein antigens produced by recombinant technology (10–13)
■■ DNA vaccines (14, 15).
In the past 10 years, WHO has convened two consultations to consider
the development, production and evaluation of viral-vectored vaccines in general,
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and the reports of those meetings provide useful discussion and opinions on the
quality, safety and efficacy aspects of such vaccines (16, 17). A regional guideline
is also available for live recombinant viral-vectored vaccines (18).
Although recombinant viral-vectored Ebola vaccines are by far the most
advanced candidates, other approaches to the development of Ebola vaccines are
also being investigated. These include different production platforms, such as
recombinant DNA vaccines expressing an EBOV antigen produced in Escherichia
coli (19), Ebola virus-like particles (VLPs) expressed from recombinant baculovirus
in insect cells, and other forms of subunit vaccines. Most developmental
approaches to Ebola vaccines involve recombinant DNA technology.
Part A of this document focuses on the development, manufacturing
and quality control issues relevant to viral-vectored vaccines against EBOV.
Although the key principles related to nonclinical development (Part B) and
clinical development (Part C) may apply to vaccine approaches other than those
based on viral vectors, special considerations and guidance would be required
for such products – and they are therefore not elaborated upon in this document.
Any mention of specific vaccines is for information only and should not be
considered as an endorsement of a particular candidate.
Parts A, B and C provide guidance in general terms on the full quality,
nonclinical and clinical requirements for a license submission for viral-vectored
Ebola vaccines. The document also considers the principles which may be
applied to product development, manufacturing and control – and to nonclinical
and clinical evaluation – during a public health emergency to allow for the rapid
introduction of an Ebola vaccine. Wherever appropriate, discussions on the
minimum dataset required are highlighted and aspects of vaccine development
which may be accelerated during a public health emergency are indicated. These
context-specific discussions and indications are shown as indented smaller text
in Parts A, B and C. In addition, special considerations regarding the quality
requirements at different stages of clinical development are discussed in sections
A.2.4, A.3.6, A.3.7 and A.3.8.
These WHO Guidelines should be read in conjunction with other
relevant WHO guidelines such as those on nonclinical (20, 21) and clinical (22)
evaluation of vaccines, as well as relevant documents that describe the minimum
requirements for an effective National Pharmacovigilance System (23). Other
WHO guidance, such as that on the evaluation of animal cell cultures as substrates
for the manufacture of biological medicinal products and for the characterization
of cell banks (24), should also be consulted as appropriate.
It should be noted that there remain knowledge gaps in the scientific
understanding of EVD and Ebola vaccines which are being addressed by
ongoing research and development. This document has been developed in the
light of the available knowledge to date, and with regard to the currently most
advanced candidate Ebola vaccines.
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Terminology
The definitions given below apply to the terms as used in these WHO Guidelines.
These terms may have different meanings in other contexts.
Adventitious agents: contaminating microorganisms of a cell culture
or source materials, including bacteria, fungi, mycoplasmas/spiroplasmas,
mycobacteria, Rickettsia, protozoa, parasites, transmissible spongiform
encephalopathy (TSE) agents and viruses that have been unintentionally
introduced into the manufacturing process of a biological product.
Adverse event of special interest (AESI): an adverse event (serious
or non-serious) that is of scientific and medical concern specific to the
sponsor’s product or programme, and for which ongoing monitoring and rapid
communication by the investigator to the sponsor can be appropriate. Such
an event might warrant further investigation in order to be characterized and
understood. Depending on the nature of the event, rapid communication by the
trial sponsor to other parties (for example, regulators) might also be warranted.
Attenuated virus: a strain of virus in which pathogenicity has been
reduced so that the virus strain will initiate an immune response without
producing the disease.
Benefit–risk assessment: a decision-making process for evaluating
whether or not the benefits of a given medicinal product outweigh the risks.
Benefits and risks need to be identified from all parts of a dossier – that is, the
quality, nonclinical and clinical data – and integrated into the overall assessment.
Candidate vaccine: an investigational vaccine which is in the research and
clinical development stages and has not been granted marketing authorization or
licensure by a regulatory agency.
Cell bank: a collection of appropriate containers whose contents are of
uniform composition, stored under defined conditions. Each container represents
an aliquot of a single pool of cells.

Cell substrate: cells used to manufacture a biological product.
Expression construct: an expression vector containing the genetic coding
sequence of the recombinant protein.
Expression system: the host cell containing the expression construct
and the cell culture process that is capable of expressing protein encoded by the
expression construct.
Final bulk: a formulated vaccine preparation from which the final
containers are filled. If applicable, the final bulk may be prepared from one
or more monovalent antigen bulks and, in this case, mixing should result in a
uniform preparation to ensure that final containers are homogenous.
Final lot: a collection of sealed final containers of formulated vaccine
that is homogeneous with respect to the risk of contamination during the filling
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Annex 2
process. A final lot must therefore have been filled from a single vessel of final
bulk or prepared in one working session.
Heterologous gene: a transgene from the disease-causing organism that
is integrated into the genomic sequence of the viral vector.
Immune correlate of protection (ICP): an immunological response
that correlates with vaccine-induced protection from disease and is considered
predictive of clinical efficacy. The ICP may be mechanistic (that is, causative for
protection) or it may be non-mechanistic (that is, an immune response that is
present in persons protected by vaccination but that is not the cause of protection).
Immunogenicity: the capacity of a vaccine to elicit a measurable immune
response.
Marketing authorization: a formal authorization for a medicine
(including vaccines) to be marketed. Once an NRA approves a marketing
authorization application for a new medicine, the medicine may be marketed
and may be available for physicians to prescribe and/or for public health use (also
referred to as product licensing, product authorization or product registration).
Master cell bank (MCB): a quantity of well-characterized cells of animal
or other origin, derived from a cell seed at a specific population doubling level
(PDL) or passage level, dispensed into multiple containers, cryopreserved and
stored frozen under defined conditions (such as the vapour or liquid phase of
liquid nitrogen) in aliquots of uniform composition. The MCB is prepared from
a single homogeneously mixed pool of cells. In some cases, such as genetically
engineered cells, the MCB may be prepared from a selected cell clone established
under defined conditions. Frequently, however, the MCB is not clonal. It is
considered best practice for the MCB to be used to derive working cell banks.
Monovalent vaccine: a vaccine containing immunizing antigen, or a gene
encoding an immunizing agent, against a single strain or type of disease agent.
Platform technology: a production technology in which different viral-
vectored vaccines are produced by incorporating heterologous genes for different
proteins into an identical viral vector backbone.
Multivalent vaccine: a vaccine containing a mixture of more than one
immunizing antigen or genes encoding several immunizing agents active against
more than one strain or type of disease agent.
Pooled virus harvest: a homogeneous pool of two or more single virus
harvests.
Public health emergency: an extraordinary event that is determined, as
provided in the International Health Regulations (25), to: (a) constitute a public
health risk to other States through the international spread of disease; and
(b) potentially require a coordinated international response.
Seed lot: a system according to which successive batches of viral-vectored
vaccine are derived from the same virus master seed lot of viral vector at a given
95
passage level. For routine production, a virus working seed lot is prepared from
the virus master seed lot. The final product is derived from the virus working
seed lot and has not undergone more passages from the virus master seed lot
than the vaccine shown in clinical studies to be satisfactory with respect to safety
and efficacy.
Single virus harvest: a quantity of virus suspension of one virus strain
harvested from cell cultures derived from the same working cell bank and
prepared from a single production run.
Vaccine efficacy: measures direct protection (that is, protection induced
by vaccination in the vaccinated population sample). Vaccine efficacy is most
commonly a measure of the proportionate reduction in disease attack rate (AR)
between the control group that did not receive vaccination against the infectious
disease under study (ARU) and the vaccinated group (ARV). Vaccine efficacy
(expressed as a percentage) can be calculated from the relative risk (RR = ARV/
ARU) of disease when comparing the vaccinated group to the unvaccinated
control group as [(ARU-ARV)/ARU] x 100 – that is, as (1-RR) x 100. This estimate
may be referred to as absolute vaccine efficacy. Alternatively, vaccine efficacy may
be defined as a measure of the proportionate reduction in disease AR in a group
vaccinated with the candidate vaccine relative to a control group vaccinated with
a licensed vaccine against the infectious disease under study. This estimate may
be referred to as relative vaccine efficacy (22).
Vaccine effectiveness: an estimate of the protection conferred by
vaccination. It is usually obtained by monitoring the disease to be prevented by
the vaccine during routine use in a specific population. Vaccine effectiveness
measures both direct and indirect protection (for example, the estimate may in
part reflect protection of unvaccinated persons secondary to the effect of use of
the vaccine in the vaccinated population) (22). Evidence for vaccine effectiveness
may also be derived from challenge-protection studies conducted in animal
models or from a vaccine-induced immune response (for example, pre-specified

antibody threshold induced by the vaccine in vaccinated persons).
Virus master seed: a collection of appropriate containers whose contents
are of uniform composition, stored under defined conditions. Each container
represents an aliquot of a single pool of virus vector particles of defined passage
from which the virus working seed is derived.
Virus pre-master seed: a single pool of virus vector particles of defined
passage from which the virus master seed is derived.
Virus working seed: a collection of appropriate containers whose contents
are of uniform composition, stored under defined conditions. Each container
represents an aliquot of a single pool of virus vector particles of defined passage
derived directly from the virus master seed lot and which is the starting material
for individual manufacturing batches of viral-vectored vaccine product.
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Annex 2
Working cell bank (WCB): a quantity of cells of uniform composition

derived from one or more ampoules of the MCB at a finite passage level, stored
frozen at −70 °C or below in aliquots, one or more of which is used for vaccine
production. All containers are treated identically and once removed from storage
are not returned to the stock.
General considerations
Ebola viruses, Ebola virus disease and epidemiology
Ebola viruses belong to the Filoviridae family of filamentous, negative-stranded
RNA, enveloped viruses consisting of three genera: Ebola virus, Marburg virus
and Cueva virus – the latter being a pathogen of bats in Spain (26). There are five
distinct species of EBOV: Zaire ebolavirus (ZEBOV), Sudan ebolavirus (SUDV),
Tai Forest ebolavirus (TAFV), Reston ebolavirus (RESTV) and Bundibugyo
ebolavirus (BDBV) (26, 27). Marburg virus (MARV) appears to be antigenically
stable and at present there is only a single species. The first recognized MARV
outbreak in humans was in 1967 and was linked to infected monkeys imported
from Uganda that infected laboratory workers in Marburg and Belgrade (28).
Bats are believed to be the natural reservoir of all filoviruses. EBOV and MARV
cause severe haemorrhagic fever in humans and non-human primates alike, with
high morbidity and mortality rates (29, 30). Outbreaks of infection with Ebola
filoviruses have been noted since 1976, mainly in Central Africa, and recur at
intervals. Prior to the 2014–2016 EVD epidemic in West Africa there had not
been such a large-scale outbreak and the disease had not been recorded in West
Africa, apart from a single infection with TAFV.
The incubation period following infection with EBOV and prior to
the onset of symptoms is believed to be approximately 2–21 days, with initial
symptoms being similar to diseases such as influenza or malaria (31, 32).
Patients then progress rapidly to a life-threatening disease (33). From a practical
perspective, infected individuals rarely if ever become infective before symptoms
appear, but those who survive remain infective until the virus is cleared from
their blood and other bodily fluids. It has been reported that viable EBOV can
persist in ocular fluid for at least 9 weeks following clearance of viraemia (34).
EBOV has also been detected in semen for months following recovery from EVD,
which is consistent with the possible persistence of the virus within immune-
privileged tissue sites in the body (35, 36). Presumptive sexual transmission of
EBOV from recovered individuals has also been reported (37, 38). Individuals
suffering from EVD have been treated aggressively with oral and intravenous
fluids, including electrolyte replacements, to combat severe diarrhoea and
dehydration, with some surviving the infection (33).
97
Filoviruses are high-risk agents classified as biosafety level 4 (BSL-4)

pathogens. They consist of a non-segmented RNA genome of approximately
19 kb containing 7 genes encoding viral proteins VP24, VP30, VP35, VP40, a
nucleoprotein, a glycoprotein (GP) and a polymerase (39). The GP is a type‑1
transmembrane GP that is cleaved into disulphide-linked GP1 and GP2
subunits. The mature GP forms homotrimers that are presented as spikes on
the surface of infected cells and virions, and is responsible for receptor binding,
viral entry and, most likely, immunity (40, 41). Most of the vaccines currently
under development are based on the EBOV GP and have been shown to
confer protection from lethal EBOV challenge in animal models – including,
importantly, in non-human primates (42, 43).
Natural immune responses to Ebola viruses

Filovirus infection in humans elicits innate, cellular and humoral responses.
Immunoglobulin M (IgM) and IgG antibodies have been reported to develop
early in infected patients who survive, whereas fatal cases are associated with
immune dysregulation and high viraemia (44). Some cross-reactive immune
responses across the five EBOV species have been reported (45). Cellular
responses can also be detected. The generation of neutralizing antibodies during
filovirus infection and the passive transfer of neutralizing monoclonal antibodies
or monkey convalescent immunoglobulin preparations have been shown to
sometimes protect non-human primates against lethal filovirus challenge –
though overall the data are somewhat conflicting (44, 46). Data suggest that
antibodies play a significant role in protection against filovirus infection but
correlates of protection have not been established and the importance of cellular
immunity is uncertain (47, 48).
Ebola vaccines development

A large number of candidate Ebola vaccines are under development. Some

of these vaccines had already been in preclinical development prior to the
2014–2016 EVD epidemic and are significantly more advanced than the
others. To date, several candidate vaccines (including monovalent, bivalent
and multivalent candidate vaccines) have undergone or are undergoing clinical
development at different trial phases. The Phase III trial for a recombinant
vesicular stomatitis virus (rVSV)-vectored candidate vaccine (rVSVΔG-
ZEBOV-GP), undertaken in Guinea, is the only study that has reported clinical
efficacy and effectiveness for any candidate Ebola vaccine. This candidate
vaccine was granted access to the Priority Medicine (PRIME) scheme by the
European Medicines Agency, and Breakthrough Therapy designation by the
United States Food and Drug Administration (5). Examples of Ebola vaccines
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Annex 2
currently under clinical development are provided in a WHO Working Group

background paper (49).
The most advanced Ebola vaccines are based on live recombinant virus
vector platforms. Such vaccines have been developed in Canada, China, Europe,
Russia and the USA. Five of the most advanced platforms used to engineer these
vaccines are rVSV (50, 51), chimpanzee adenovirus type 3 (ChAd3) (52), human
adenovirus type 26 (Ad26) (53), human adenovirus type 5 (Ad5) (51, 54) and
the modified vaccinia Ankara (MVA) strain (55). To date, the virus vectors have
been produced in a wide variety of cell lines including PER.C6 (Ad26.ZEBOV),
chick embryo fibroblasts (MVA-BN-Filo), Procell-92.S (ChAd3-EBOZ), Vero
(rVSV-ZEBOV) and HEK 293 (Ad5-EBOV). Monovalent candidate vaccines
have been constructed to express the EBOV GP of one EBOV strain, such as
the Zaire strain responsible for the epidemic in West Africa. Others have been
developed as multivalent vaccines expressing the GP of more than one EBOV
strain and/or MARV and/or the TAFV nucleoprotein. Multivalent vaccines have
also been produced by blending monovalent bulks expressing glycoproteins
from different EBOV and/or MARV strains. These candidates are currently
under study in non-human primates and in humans, either as single vaccines
or for use in heterologous prime-boost vaccine schedules where priming is done
with one vaccine and boosting with another – as for example, Ad26.ZEBOV/
MVA-BN-Filo (56, 57) and rVSV/Ad5 (51).
The viral-vectored vaccines under development include those that are
replication-incompetent in the human host or in human cells as well as those
that are replication-competent but likely to be highly attenuated because of their
recombinant gene inserts and cell culture passage. Replication-incompetent
vectors include adenoviral vectors derived both from human adenoviruses
(such as Ad26 and Ad5) and from non-human primate adenoviruses (such as
ChAd3), as well as MVA. MVA is a highly attenuated vaccinia strain, derived
by more than 500 passages in hens’ eggs. The non-recombinant MVA was
used as a human smallpox vaccine in Germany in the 1970s and a derivative
has now been licensed for use in a future smallpox emergency in Canada and
Europe. Vectors that are replication-competent but attenuated include rVSV
(a negative-stranded RNA virus animal pathogen) in which attenuation is due
to the insertion of a recombinant heterologous gene such as the EBOV GP in
place of the VSV GP. These viral-vector platforms have been used to produce
other investigational products – including gene therapy products, and both
prophylactic and therapeutic vaccines – and data from their quality, nonclinical
and clinical evaluations provide supporting safety data for their use in Ebola
vaccine production (50, 58, 59).
The need for careful clinical studies using candidate vaccines in the
target population will be of paramount importance. WHO has developed
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a document – Ebola virus disease (EVD) vaccine target product profile (60)
– which provides guidance on WHO preferred options in relation to two
categories of Ebola vaccine (reactive use and prophylactic use). Encouraging
results on the immunogenicity and safety of these candidate options, as well
as on their clinical efficacy based on disease end-points, have already been
generated and their evaluation in larger Phase II and Phase III trials is ongoing.
This includes novel trial design clinical studies (ring vaccination) using the
rVSV-ZEBOV vaccine (32, 54, 61–64). A prime-boost approach, using a two-
dose schedule with different vector vaccines, is also being explored. Boosting
of Ad26.ZEBOV responses by MVA-BN-Filo resulted in sustained elevation of
specific immunity with no vaccine-related serious adverse responses reported
(56, 57). Administration of rVSV-ZEBOV vaccine resulted in low-level viraemia
detectable by polymerase chain reaction (PCR) during the first and sometimes
second week after vaccination (63). The vaccine virus was also detected by PCR
in the urine and saliva of a minority of the recipients. An unexpected safety
signal was detected in one study when mild-to-moderate and generally short-
lived arthritis developed during the second week following immunization in a
minority of recipients and at one site in particular (63). In subsequent studies
in healthy North American and European adults which carefully assessed
joint-related adverse events, transient post-vaccination arthritis was noted in
approximately 5% of vaccine recipients (65, 66). However, the epidemiological
situation has now changed significantly. Using strict infection control and public
health measures, the EBOV epidemic has been ended – though there will still
be a risk of new Ebola cases or clusters occurring through, for example, sexual
transmission or new introduction of the virus into the human population. WHO
declared Sierra Leone free of EBOV transmission in March 2016 and Guinea
and Liberia free of EBOV transmission in June 2016, bringing to an end the
large-scale Ebola outbreak in the three African countries mainly affected (67).
In the absence of ongoing disease transmission, the assessment of Ebola vaccine
efficacy will now be more challenging. Nevertheless, it is expected that current

clinical trials of candidate vaccines will provide key data on safety, reactogenicity
and immunogenicity to inform licensure.
Accelerated availability of vaccines during a public

health emergency – general principles
The quality of a vaccine must always be taken into account during the process
of evaluating whether the benefit derived from its administration is greater than
any risks which might be associated with its use. This is a principle by which all
pharmaceuticals, whether they are chemical or biological, medicine or vaccine,
are evaluated to decide whether they should be made available for use or not.
The principle applies equally to a product intended for use in a clinical trial or
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as a licensed product, or to be made available through emergency procedures.

In addition, there is an obligation to provide full assurance that the vaccine
will not cause harm to the recipient due to a failure of manufacture and control
that results in contamination of the product with unwanted components such
as microorganisms or toxic materials. This requirement is absolute, regardless
of the stage of development of the product or the urgency of the need for
its availability.
Beyond this, the process and product characterization requirements
will depend on the prevailing clinical situation and the urgency of need for
the product. However, it is generally accepted that in order to gain marketing
authorization for a vaccine the usual standards for quality development,
manufacture and control will apply. During the assessment of a marketing
authorization application, the balance of benefits and risks of the vaccine to
the intended population is taken into consideration and must be found to be
positive if the product is to be granted marketing approval. The specific findings
related to the assessment of product quality are taken into account in this
benefit–risk assessment.
It is not possible to provide a “road map” of the minimum process and
product characterization and control requirements for a viral-vectored vaccine
against EVD, or against any other disease with the potential to cause a public
health emergency, since the requirements will be partially dependent on the
ongoing epidemic situation in the affected countries.
In the case of viral-vectored vaccines, many of the opportunities to
accelerate development and product availability during a public health emergency
are likely to involve exploiting the knowledge gained from similar products
manufactured with the same vector backbone (that is, platform technology). If
a new vaccine is based on a well-characterized platform technology, then key
aspects of manufacture and control (but not stability) can be based on the specific
platform with only confirmatory information required for the new vaccine. This
principle is especially applicable during the phase of clinical trial development.
For licensure, product-specific data will be required but supportive platform-
derived data may decrease the requirement for some product data if it can be
shown that the benefit–risk assessment remains positive. Scientific advice should
be sought from relevant regulatory authorities.
During product development, it might be possible to defer certain tests
and development procedures provided it can be justified that their deferral
does not affect product safety – and if it can also be argued that performing the
tests or development procedures would hinder the availability of the product
(for example, where performing the tests are on the critical path for product
availability, or where large quantities of scarce material required for clinical
purposes would need to be used). Such deferrals should be identified on a case-
by-case basis and discussed with the NRA.
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In some cases, even if the nature of a public health emergency affects the
benefit–risk balance in such a way as to justify the accelerated development and
approval of a vaccine for use in a public health emergency, the manufacturer
would still be responsible for completing the full development work to the same
standard required for a new vaccine under non-emergency conditions should it
be decided to subsequently submit the product for full licensure. The required
supplementary data and timelines for submission should be agreed between the
applicant and the NRA.
Similar considerations apply to the nonclinical evaluation of candidate
Ebola vaccines. For nonclinical evaluation during a public health emergency,
it is paramount to determine a minimum nonclinical package (see section B.4)
that can reasonably support initiation of early Phase I clinical trials. This should
take into account the characteristics and novelty of candidate vaccines and the
supportive information derived from the platform technology on which the
vaccine is based. For example, the presence of nonclinical data and/or clinical
experience gained with the same vector may support the omission of a specific
safety test or toxicity testing programme. For a candidate vaccine derived from
a novel platform, a certain amount of toxicity data (see section B.4) should at
a minimum be obtained, and should focus on unexpected direct and indirect
consequences that might result from vaccination.
In general, the use of a minimum safety package during nonclinical
evaluation should be backed up by the continuous assessment of additional data
collected during clinical development. At the time of the licensing application,
the complete nonclinical programme data appropriate for a particular vaccine
should be submitted, or the application should be otherwise adequately justified.
Clinical development of an Ebola vaccine in the setting of an outbreak is
complex, and close collaboration between public health authorities, NRAs, the
community, clinical investigators and the vaccine developer is essential to ensure
that studies will meet authorization requirements, including requirements for
ethical study conduct.

A WHO emergency use assessment and listing (EUAL) procedure (68)
has been developed to expedite the availability of unlicensed vaccines needed
during a public health emergency of (usually) international concern.
Part A. Guidelines on the development, manufacture

and control of Ebola vaccines
At the time of writing this document, no WHO guidance on viral-vectored
vaccines was available. Consequently, this section focuses on issues relevant to
the development, manufacturing and quality control steps leading to the licensing
of such vaccines developed to protect against EVD.
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The lead viral-vectored vaccines and their replication abilities are

summarized in a WHO document (49). The relevance of aspects of the guidance
provided in this document should be considered with respect to the replication
status of the products. For example, tests for reversion to competency apply to
replication-incompetent viral vectors where genes required for replication are
not present in the vector. On the other hand, for replication-competent viral-
vectored vaccines, the level of attenuation of the parent and recombinant viral
vectors should be considered.
A.1 General manufacturing guidelines

The WHO Target Product Profile (60) prioritizes the development of multivalent
vaccines from 2016 onwards and seeks at a minimum coverage for MARV and
for both Zaire and Sudan species of EBOV.
The general manufacturing requirements contained in the WHO good
manufacturing practices for pharmaceutical products: main principles (69) and
WHO good manufacturing practices for biological products (70) should apply to
the design, establishment, operation, control and maintenance of manufacturing
facilities for recombinant Ebola vaccines.
Quality control during the manufacturing process relies on the
implementation of quality systems, such as good manufacturing practice (GMP),
to ensure the production of consistent vaccine lots with characteristics similar
to those of lots shown to be safe and effective in clinical trials. Throughout
the process, a number of in-process control tests should be established (with
acceptable limits) to allow quality to be monitored for each lot from the beginning
to the end of production. It is important to note that most release specifications
are product specific and should be agreed with the NRA as part of the clinical
trial or marketing authorization.
Manufacturers should present a risk assessment regarding the biosafety
level of their manufacturing facility and of the vaccine product. The principles
presented in the WHO Laboratory biosafety manual (71) should be followed to
justify the classification. Approval for the classification should be sought from
the relevant authority in the country/region in which the manufacturing facility
is located.
A.1.1 International reference materials

The highly pathogenic nature of EBOV raises particular concerns for the
preparation of international reference materials as they must be both safe for use
and representative of clinical samples to be analysed. Generally, plasma reference
preparations are used for the standardization of assays for evaluating immune
response, and artificial RNA viruses containing part of the EBOV genome are
used for the standardization of nucleic acid assays for assessing viraemia.
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Plasma from a recovered repatriated patient who contracted Ebola in

West Africa one month before the plasma was collected was established by
the 2015 WHO Expert Committee on Biological Standardization as the First
WHO Reference Reagent for Ebola virus antibodies, with an assigned unitage
of 1 U/ml (72). As the reference material resulted from a natural infection it is
likely to have relevant antibody specificities. It is considered to be of acceptable
safety for three reasons: (a) the patient was fully recovered clinically; (b) the
plasma was negative for EBOV nucleic acid in PCR assays performed in various
laboratories; and (c) the plasma was treated with solvent/detergent (an established
method used in the blood products industry for decades for the inactivation of
enveloped viruses).
Following evaluation and characterization of candidate materials (73,
74), the First WHO International Standard for Ebola virus antibodies
(assigned unitage = 1.5 IU/ml) and the First WHO Reference Panel for Ebola
virus antibodies were established by the 2017 WHO Expert Committee on
Biological Standardization. The First WHO International Standard for Ebola
virus antibodies is intended for standardizing assays used in the detection and
quantitation of EBOV antibodies. It is not intended to be used to set a protective
threshold, which is currently unknown (see section C). The First WHO Reference
Reagent for Ebola virus antibodies and the First WHO Reference Panel for
Ebola virus antibodies can be used in the assessment of factors that affect assay
variability (75).
Following evaluation and characterization of candidate materials (76,
77), two EBOV RNA preparations were also established as reference reagents by
the 2015 WHO Expert Committee on Biological Standardization for use in the
standardization of nucleic acid amplification technique (NAT)-based assays. One
of these materials (Ebola NP-VP35-GP-LVV) consists of the RNA encoding the
nucleoprotein VP35 and GP genes and is intended for use in standardizing assays
directed at these genes only. The second (Ebola VP40-L-LVV) consists of the
RNA encoding the VP40 and L genes and again is intended to standardize assays
directed only at these genes. Both preparations are packaged in non-replicating
lentiviral vectors (LVVs) with the EBOV genes incorporating mutations that
make them inactive. Collectively the two materials were established as the First
WHO reference reagents for Ebola virus RNA for NAT-based assays with assigned
unitages of 7.5 log 10 U/ml and 7.7 log 10 U/ml respectively.
The First WHO Reference Panel for Ebola virus VP40 antigen was
established by the 2016 WHO Expert Committee on Biological Standardization
(78). The panel consists of different recombinant VP40 antigens and may be
suitable for the evaluation and quality control of Ebola antigen assays based on
VP40 detection.
All the reference materials listed above are available from the National
Institute for Biological Standards and Control, Potters Bar, the United Kingdom.
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For the latest list of appropriate WHO international standards and reference
materials, the WHO Catalogue of International Reference Preparations (79)
should be consulted.
A.2 Control of source materials

A.2.1 Viral vector
A.2.1.1 Virus master and working seeds
The use of any viral vector should be based on a master and working seed lot
system, analogous to the cell banking system used for production cells described
below in section A.2.2.
The rationale behind the development of the viral-vectored vaccine
should be described. The origin of all genetic components of the vaccine and
their function should be specified to allow for a clear overall understanding of
the functionality of the vaccine and of how it is attenuated, or made replication-
incompetent by genetic engineering. All intended and unintended genetic
modifications such as site-specific mutations, insertions, deletions and/or
rearrangements to any component should be detailed in comparison with their
natural counterparts. For a vaccine construct that incorporates genetic elements
to control the expression of a transgene – for example, in a tissue-specific manner
– evidence should be provided on product characterization and control to
demonstrate such specificity. RNA editing should be discussed if relevant.
All of the steps from the derivation of material that ultimately resulted
in the candidate vaccine to the virus master seed level should be described. A
diagrammatic description of the components used during vaccine development
should be provided and annotated. The method of construction of the viral-
vectored vaccine should be described and the final construct should be
genetically characterized according to the principles discussed in this section.
The cloning strategy should ensure that if any antibiotic resistance genes
are used during the development of the initial genetic construct, these are
absent from the viral vaccine seed.
The nucleotide sequence of the gene insert and of adjacent segments of
the vector should be provided, along with restriction-enzyme mapping of the
vector containing the gene insert. The genetic stability of the vector with the
recombinant construct should be demonstrated. The stability of a recombinant
vector should be assessed by comparing the sequence of the vector at the level
of a virus pre-master seed or virus master seed to its sequence at, or preferably
beyond, the anticipated maximum passage level. The comparison should
demonstrate that no changes occur in regions involved in attenuation (where
known) or replication deficiency. Any modifications to the sequence of the
heterologous insert should be investigated and demonstrated to have no impact
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on the resulting amino acid sequence (that is, it should be a conservative change)
or on the antigenic characteristics of the vaccine.
A.2.1.2 Tests on virus master seed and virus working seed

The virus master seed should be characterized as fully as possible. If this
characterization is limited (for example, because of limited quantities of material)
then the virus working seed should be fully characterized in addition to the
limited characterization of the virus master seed. It should be noted that it would
not be feasible to manufacture from the virus master seed in these circumstances.
Virus master seed characterization will include a description of the
genetic and phenotypic properties of the vaccine vector. This should include
a comparison with the parental vector – which is particularly important
where vector modification might affect attenuation or replication competency,
pathogenicity, and tissue tropism or species specificity of the vaccine vector
compared with the parental vector.
Genetic characterization will involve nucleotide sequence analysis of the
vaccine vector. Restriction mapping, southern blotting, PCR analysis or DNA
fingerprinting will also be useful adjuncts. Individual elements involved in the
expression of the heterologous gene(s) (including relevant junction regions)
should be described and delineated.
Genetic stability of the vaccine seed to a passage level comparable to
final virus bulk and preferably beyond the anticipated maximum passage level
should be demonstrated.
Phenotypic characterization should focus on the markers for attenuation/
modification and expression of the heterologous antigen(s), and should generally
be performed in vitro under conditions that allow for the detection of revertants
(including the emergence of replication-competent vectors from replication-
incompetent vectors during passage). However, other studies including antigenic
analysis, infectivity titre, ratio of genome copies to infectious units (for replicating
vectors) and in vitro yield should also form part of the characterization. For
replicating vectors, in vivo growth characteristics in a suitable animal model may
also be informative and should be performed if justified. For some vectors (for
example, adenoviral vectors), particle number should be measured in addition to
infectivity titre.
A subset of the above studies should be applied to the virus working seed
lot and justification for the chosen subset should be provided.
Information should be given on the testing carried out for adventitious
agents.
During a public health emergency it is anticipated that the majority
of the above information should be available and submitted in full for
evaluation since it is essential to demonstrate the suitability and safety of
the product.
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It may be justified to initiate clinical trials using a product which is

manufactured prior to establishment of the seed banking system. In such
a case, the suitability and safety of the product must be established prior
to its use – especially with regard to adventitious agents (24), replication
competence, attenuation and other phenotypic characteristics, stability
and suitable genetic sequence.
A.2.2 Cell substrates

The cell substrate for the manufacture of Ebola vaccine should be based on a cell
banking system or on controlled primary cells.
A.2.2.1 Cell banks and primary cells

A.2.2.1.1 Master and working cell banks (MCBs and WCBs)
The cell banks should conform to the WHO Recommendations for the evaluation
of animal cell cultures as substrates for the manufacture of biological medicinal
products and for the characterization of cell banks (24).
An appropriate history of the cell bank should be provided. This should
include information on its origin, identification, development manipulations and
characteristics for the purposes of the vaccine. Full details of the construction
of packaging cell lines should be given, including the nature and identity of
the helper viral nucleic acid and its encoded proteins/functions. If available,
information on the chromosomal location of the helper viral nucleic acid should
also be provided.
Genetic stability of the cell lines should be demonstrated. The stability
of a production cell line should be assessed by comparing the critical regions
of the cell line (and flanking regions) at the level of a pre-cell or master cell
to its sequence at or beyond the anticipated maximum passage level. Stability
studies should also be performed to confirm cell viability after retrieval from
storage, maintenance of the expression system, and so on. These studies may be
performed as part of routine use in production or may include samples taken
specifically for this purpose.
With regard to cell cultures, the maximum number of passages (or
population doublings) allowable from the MCB through to the WCB, and
through production in cells should be defined on the basis of the stability data
generated above, and should be approved by the NRA.
A.2.2.1.2 Primary cells

Primary cells are used within the first passage after establishment from the
original tissue, and so it is not possible to carry out extensive characterization
of the cells prior to their use. Therefore additional emphasis is placed on the
origin of the tissues from which the cell line is derived. Tissues should be derived
107
from healthy animals/embryonated eggs subjected to veterinary and laboratory

monitoring to certify the absence of pathogenic agents. Whenever possible,
donor animals/embryonated eggs should be obtained from closed, specific-
pathogen-free colonies or flocks. Animals used as tissue donors should not have
been used previously for experimental studies. Birds and other animals should
be adequately quarantined for an appropriate period of time prior to use for the
preparation of cells.
Information on the materials and components used for the preparation of
primary cell substrates should be provided, including the identity and source of
all reagents of human or animal origin. A description of the testing performed on
components of animal origin to certify the absence of detectable contaminants
and adventitious agents should be included.
The methods used for the isolation of cells from tissue, establishment of
primary cell cultures and maintenance of cultures should be described.
A.2.2.2 Testing of cell banks and primary cells

A.2.2.2.1 Tests on MCBs and WCBs
MCBs and WCBs should be tested for the absence of bacterial, fungal,
mycoplasmal and viral contamination by appropriate tests, as specified in the
WHO Recommendations for the evaluation of animal cell cultures as substrates
for the manufacture of biological medicinal products and for the characterization
of cell banks (24), or by a method approved by the NRA, to demonstrate that
they are not contaminated with adventitious agents.
Rapid sterility methods to demonstrate the absence of bacteria and fungi,
as well as NAT-based assays alone or in combination with cell culture, may be
used as an alternative to one or both of the compendial mycoplasmal detection
methods after suitable validation and agreement from the NRA (24).
The cell bank should be tested for tumorigenicity if it is of mammalian
origin, as described in Part B of the WHO Recommendations for the evaluation

of animal cell cultures as substrates for the manufacture of biological medicinal
products and for the characterization of cell banks (24). The tumorigenic potential
of the cell bank(s) should be described and strategies to mitigate risks that might
be associated with this biological property should be described and justified.
During a public health emergency, it is anticipated that the majority of

the above information should be available and submitted for evaluation
since it is essential to demonstrate the suitability and safety of the product.
However, it may be justified to initiate clinical trials using a product
which is manufactured prior to establishment of the cell banking system.
In such a case, the suitability and safety of the product must be established
prior to its use, especially with regard to adventitious agents (24).
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A.2.2.2.2 Tests on primary cells

The nature of primary cells precludes extensive testing and characterization
before use. Testing to demonstrate the absence of adventitious agents (bacteria,
fungi, mycoplasmas and viruses) is therefore conducted concurrently, and should
include, where relevant, the observation of control (uninfected) cultures during
parallel fermentations to the production runs. The inoculation of culture fluid
from production cultures and (where available) control cultures into various
susceptible indicator cell cultures capable of detecting a wide range of relevant
viruses (followed by examination for cytopathic changes and testing for the
presence of haemadsorbing viruses) should also be performed routinely for batch
release. In addition, pharmacopoeial testing for bacteria, fungi and mycoplasmas
in the production cultures and (if relevant) control cultures should be conducted.
Mycoplasmas and specific viruses of notable concern may also be tested for by
additional methods such as PCR.
In the specific case of chick embryo fibroblasts (CEFs), the tissue should
be sourced from specific-pathogen-free eggs. After preparation, the CEF cells
should be tested for: (a) bacterial, fungal and mycoplasmal contamination;
(b) viral adventitious agents by in vitro assay using three cell lines, including
avian and human cells (such as CEF, MRC-5 and Vero); (c) viral adventitious
agents by in vivo assay using mice and embryonated eggs; (d) avian leukosis
virus contamination; and (e) the presence of retroviruses by measuring reverse
transcriptase (RT) activity. Testing should take into consideration that CEF cells
are expected to be positive for RT activity due to the presence of endogenous
avian retroviral elements not associated with infectious retroviruses. It may
be necessary to use an amplification strategy (for example, co-culturing of
RT‑positive fluids on an RT-negative, retrovirus-sensitive cell line) to determine
whether a positive RT result can be attributed to the presence of an infectious
retroviral agent.
A.2.3 Source materials used for cell culture and virus propagation
If serum is used for the propagation of cells it should be tested to demonstrate
the absence of bacteria, fungi and mycoplasmas, as specified in the requirements
given in Part A – section 5.2 (80) and section 5.3 (81) – of the WHO General
requirements for the sterility of biological substances. Testing should also be
conducted to demonstrate freedom from adventitious viruses.
Detailed guidance on detecting bovine viruses in serum used to establish
MCBs and WCBs is provided in Appendix 1 of the WHO Recommendations
for the evaluation of animal cell cultures as substrates for the manufacture of
biological medicinal products and for the characterization of cell banks (24) and
should be applied as appropriate. This same guidance may also be applicable
to production cell cultures. As an additional monitor of quality, sera may be
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examined for endotoxin. Gamma irradiation may be used to inactivate potential

contaminant viruses, while recognizing that some viruses are relatively resistant
to gamma irradiation. Whichever viral inactivation process is used, a validation
study must be conducted to determine its consistency and effectiveness while
still maintaining serum performance. The use of non-inactivated serum should
be justified and is not advised without strong justification. Any non-inactivated
serum must meet the same criteria as inactivated serum when tested for sterility
and absence of mycoplasmal and viral contaminants.
The source(s) of animal components used in culture medium should
be approved by the NRA. These components should comply with the current
WHO guidelines on transmissible spongiform encephalopathies in relation to
biological and pharmaceutical products (82).
Bovine or porcine trypsin used to prepare cell cultures should be tested
and found free of bacteria, fungi, mycoplasmas and adventitious viruses, as
appropriate. The methods used to ensure this should be approved by the NRA.
The source(s) of trypsin of bovine origin (if used) should be approved by the
NRA and should comply with the current WHO guidelines on transmissible
spongiform encephalopathies in relation to biological and pharmaceutical
products (82).
In some countries, irradiation is used to inactivate potential contaminant
viruses in trypsin. If irradiation is used, it is important to ensure that a
reproducible dose is delivered to all batches and to the component units of each
batch. The irradiation dose must be low enough for the biological properties of
the reagents to be retained while being high enough to reduce virological risk.
Consequently, irradiation cannot be considered a sterilizing process (24). The
irradiation method should be validated and approved by the NRA.
Recombinant trypsin is available and should be considered – however, it
should not be assumed to be free of risk of contamination and should be subject
to the usual considerations for any reagent of biological origin (24).
Human serum should not be used.

If human serum albumin derived from human plasma is used at any
stage of product manufacture, the NRA should be consulted regarding the
requirements for this, as these may differ from country to country. At a minimum,
it should meet the WHO Requirements for the collection, processing and quality
control of blood, blood components and plasma derivatives (83). In addition,
human albumin and materials of animal origin should comply with the current
WHO guidelines on transmissible spongiform encephalopathies in relation to
biological and pharmaceutical products (82). Recombinant human serum
albumin is available and should be considered as a substitute for the plasma-
derived product.
Penicillin and other beta-lactams should not be used at any stage of
manufacture because they are highly sensitizing substances in humans. Other
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antibiotics may be used at any stage of manufacture, provided that the quantity
present in the final product is acceptable to the NRA.
Non-toxic pH indicators may be added (for example, phenol red at a
concentration of 0.002%). Only substances that have been approved by the NRA
may be added.
A.2.4 Special considerations for the development and testing

of the viral vector and production cell lines
Early-phase nonclinical and clinical studies are generally supplied with product
for which the level of knowledge of manufacture and control is expected to be
quite rudimentary since few batches will have been manufactured and analytical
methods will be in the early stages of development. The provision of material
is required for early safety and proof-of-concept studies, as well as to initiate
the dose-finding evaluation. Product will be tested initially in animals and
then in a small number of human subjects in a well-controlled environment.
This is the normal situation when there is no public health emergency and, in
these circumstances, guidance on the quality requirements for investigational
medicinal products in clinical trials is available (84).
Most data to be provided to the NRA before human studies can begin
will concern the derivation and safety of the viral vector and the production
cell line. The data will aim to show that the product and production system
are well designed, the function of each genetic element is known and its
inclusion in the product or cell line is justified. It should be confirmed that the
expected elements are present in the product and cell line and that the final
structure of the product is as predicted. A full description of the origin and
construction of the genetic components of the viral vector and cell line should
be provided, along with data on genetic stability up to (or preferably beyond)
the anticipated maximum passage level in manufacture. Ideally, a virus master
seed/virus working seed for the viral vector and MCB/WCB for the production
cell line should be prepared early in the development of the product – though
it is acknowledged that this may not be practical in the initial stages. Testing of
the seed lots and cell banks at the time of their establishment should confirm
comparability to the parental material. Any starting material (viral seeds and
production cell lines) used to manufacture product for clinical use must be fully
tested to ensure the absence of bacteria, fungi, mycoplasmas and adventitious
viruses (24, 80). Where applicable, freedom from TSEs must also be addressed
(82). The potential for tumorigenicity of the cell line should also be tested
and should meet current regulatory standards if it is of mammalian origin.
All reagents used in the manufacture of the virus seed or cell lines (including
cell culture solutions) should be tested and characterized as being of adequate
quality, particularly regarding freedom from adventitious agents.
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A.3 Control of Ebola vaccine production

A.3.1 Manufacture and purification
The manufacture of monovalent vaccine vectors starts with the amplification
of the vaccine vector seed stock in a suitable cell line. The number of passages
between the virus working seed lot and viral-vectored vaccine product should be
kept to a minimum and should not exceed the number used for production of
the vaccine shown in clinical studies to be satisfactory, unless otherwise justified
and authorized.
If applicable to the vector platform, a control cell culture should be
maintained simultaneously and in parallel with the production cell culture. Cells
should be derived from the same expansion series but no virus vector should
be added to the control cells. The growth medium and supplements used in
culturing should be identical for the production cell culture and control cell
culture. All other manipulations should be as similar as possible.
After harvesting of the culture product, the purification procedure can
be applied to a single harvest or to a pool of single monovalent harvests. The
maximum number of single harvests that may be pooled should be defined on
the basis of validation studies.
Multivalent vaccines are generally prepared by combining batches
of purified monovalent bulk that contain more than one EBOV strain and/or
MARV strain. However, if the vaccine consists of a single vector containing
genes encoding multiple antigens, then the recommendations for monovalent
bulk manufacturing should be followed, but testing should take into account the
multivalent identity and potency of the product.
By the time a marketing authorization application is submitted the
manufacturing process should be adequately validated by demonstrating that a
sufficient number of commercial-scale batches can be manufactured routinely
under a state of control by meeting predetermined in-process controls, critical
process parameters and lot release specifications. Any materials added during
the purification process should be documented and their removal should be
adequately validated or residual amounts tested for, as appropriate. Validation
should also demonstrate that the manufacturing facility and equipment have been
qualified, cleaning of product contact surfaces is adequate, and critical process
steps (such as sterile filtrations and aseptic operations) have been validated.
The purified viral vector bulk and intermediates should be maintained
under conditions shown by the manufacturer to ensure the retaining of the
desired biological activity. Hold times should be defined.
During early clinical trials it is unlikely that there will be data from
sufficient batches to validate/qualify product manufacture. However, as
development progresses, data should be obtained from subsequent manufacture
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and should be used in support of an eventual application for commercial supply

of the product.
During a public health emergency, on a case-by-case basis, some
requirements of process validation may be abbreviated provided it can be
demonstrated that the product will remain safe and well controlled. For
example, if platform-specific data have demonstrated that scale-up for a
vector is independent of the specific heterologous insert, this information
may be used to justify fewer full-scale batches with the EBOV gene insert
and a greater reliance on pre-validation and pilot-plant-scale batches.
Validation data from the manufacture of platform-related products may
provide useful supportive information, particularly in the identification
of critical parameters.
Since it is likely that there will initially be insufficient time to generate

full validation data during an emergency situation, as much information
as possible on the control of each batch should be presented to the NRA
as supporting evidence that batch manufacture is sufficiently controlled.
However, manufacturers should agree on the strategy with the NRA
before relying on platform-specific validation data.
In addition to control during manufacture, the products should be

adequately characterized by the stage of development. These attributes facilitate
understanding of the biology of the candidate vaccine and assessment of the
impact of any changes in manufacturing that are introduced as development
advances or following licensure. Assessing the immunogenicity of the product,
when relevant, should also be included in the characterization programme (for
example, as part of the nonclinical pharmacodynamic evaluation).
A.3.1.1 Tests on control cell cultures (if applicable)

When control cells are included in the manufacturing process due to limitations
on the testing of primary cells or viral harvests, or when their inclusion is
required by the NRA, the following procedures should be followed. From the
cells used to prepare cultures for vaccine production, a fraction equivalent to at
least 5% of the total or 500 ml of cell suspension or 100 million cells should be
used to prepare uninfected control cell cultures.
These control cultures should be observed microscopically for cytopathic
and morphological changes attributable to the presence of adventitious agents
for at least 14 days (at a temperature of 35–37 °C) after the day of inoculation
of the production cultures, or until the time of final virus harvest, whichever
comes last. At the end of the observation period, supernatant fluids collected
from the control culture should be tested for the presence of adventitious agents,
as described below. Samples that are not tested immediately should be stored at
−60 °C or lower until such tests can be conducted.
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If testing the control cultures for adventitious agents yields a positive

result, the harvest of virus from the parallel vaccine-virus-infected cultures
should not be used for production.
For the test to be valid, not more than 20% of the control culture flasks
should have been discarded for any reason by the end of the test period.
A.3.1.1.1 Tests for haemadsorbing viruses

At the end of the observation period a fraction of control cells comprising not
less than 25% of the total should be tested for the presence of haemadsorbing
viruses, using guinea-pig red blood cells. If the red blood cells have been stored
prior to use in the haemadsorption assay, the duration of storage should not
have exceeded 7 days and the temperature of storage should have been in the
range of 2–8 °C.
In some countries the NRA requires that additional tests for
haemadsorbing viruses are performed using other red blood cells, including
human (blood group O), monkey and/or chicken (or other avian species). All
haemadsorption tests should be read after incubation for 30 minutes at 0–4 °C,
and again after further incubation for 30 minutes at 20–25 °C. Tests using
monkey red blood cells should be read once more after additional incubation for
30 minutes at 34–37 °C.
For the tests to be valid, not more than 20% of the culture vessels should
have been discarded for any reason by the end of the test period.
A.3.1.1.2 Tests for other adventitious agents

At the end of the observation period, a sample of the pooled fluid and/or cell
lysate from each group of control cell cultures should be tested for adventitious
agents. For this purpose, an aliquot of each pool should be tested in cells of
the same species used for the production of virus, but not cultures derived
directly from the production cell expansion series for the batch which is subject
to the test. If primary cells are used for production then a different batch of
that primary cell type should be used for the test than was used for production.
Samples of each pool should also be tested in human cells and in a simian kidney
cell line. At least one culture vessel of each kind of cell culture should remain
uninoculated as a control.
The inoculated cultures should be incubated at the appropriate growth
temperature and should be observed for cytopathic effects for a period of at
least 14 days.
Some NRAs require that, at the end of this observation period, a
subculture is made in the same culture system and observed for at least an
additional 7 days. Furthermore, some NRAs require that these cells should be
tested for the presence of haemadsorbing viruses.
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For the tests to be valid, not more than 20% of the culture vessels should
have been discarded for any reason by the end of the test period.
A.3.2 Single virus harvest

The method of harvesting the vaccine vector should be described and the titre
of virus ascertained. A reference preparation should be included to validate the
titration assay. Minimum acceptable titres should be established for a single virus
harvest or pooled single harvests.
The integrity of the integrated heterologous gene should be confirmed.
An expression assay method should be described and should be performed on
production harvest material or downstream (for example, on purified final bulk).
A Western blot analysis or other method for confirming that the integrated gene
is present and expressed should be included in the testing of every batch.
A.3.2.1 Control tests on single virus harvest

Tests for adventitious agents should be performed on each single virus harvest
according to the relevant parts of section B.11 of the WHO Recommendations
for the evaluation of animal cells as substrates for the manufacture of
biological medicinal products and for the characterization of cell banks (24).
Additional testing for adventitious viruses may be performed using validated
NAT-based assays.
New molecular methods with broad detection capabilities are being
developed for adventitious agent detection. These methods include: (a) degenerate
NAT-based assays for whole virus families, with analysis of the amplicons by
hybridization, sequencing or mass spectrometry; (b) NAT-based assays using
random primers followed by analysis of the amplicons on large oligonucleotide
micro-arrays of conserved viral sequencing or by digital subtraction of expressed
sequences; and (c) high-throughput sequencing. These methods may be used
to supplement existing methods or as alternative methods to both in vivo and
in vitro tests after appropriate validation and agreement from the NRA.
Single or pooled virus harvests should be tested to demonstrate freedom
from bacteria, fungi and mycoplasmas, as specified in the requirements given in
Part A – section 5.2 (80) and section 5.3 (81) – of the WHO General requirements
for the sterility of biological substances.
For viral-vectored vaccines, due to the very high titres of the single
harvests, alternatives to the classical approaches to testing for adventitious agents
may be applied with the approval of the NRA.
Provided that the cell banks and viral seed stocks have been
comprehensively tested and released, demonstrating that they are free of
adventitious agents, the possibility of delaying in vitro testing for adventitious
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agents (viral pathogens and mycoplasmas) in the cell harvest or bulk substance,
or replacing it with validated PCR tests, could be evaluated subject to the
agreement of the NRA. The method of production should be taken into account
when deciding upon the nature of any specified viruses being sought.
Additional considerations for this approach are that no animal-derived
raw materials are used during manufacture, and that the manufacturing facility
operates under a GMP certificate (where applicable) with assurances that
prevention of cross-contamination is well controlled within the facility. Samples
should be retained for testing at a later date if required.
A.3.3 Pooled monovalent virus harvests

Single virus harvests may be pooled to form virus pools from which the final bulk
vaccine will be prepared. The strategy for pooling single virus harvests should
be described. All processing of the virus pool should be described in detail.
A.3.3.1 Control tests on pooled virus harvests

Virus pools should be tested to demonstrate freedom from bacteria, fungi and
mycoplasmas, as specified in the requirements given in Part A – section 5.2 (80)
and section 5.3 (81) – of the WHO General requirements for the sterility of
biological substances. Alternatively, if single virus harvests have been tested to
demonstrate freedom from bacteria, fungi and mycoplasmas then these tests
may be omitted on the pooled virus harvests.
A.3.4 Monovalent bulk vaccine

The monovalent bulk vaccine can be prepared from one or several virus pools
containing the same antigen, or it may be derived from a single virus harvest.
Substances such as diluents or stabilizers or any other excipients added
during preparation of the monovalent bulk or the final bulk vaccine should
have been shown not to impair the potency and safety of the vaccine in the
concentrations used.
A.3.4.1 Control tests on monovalent bulk

The monovalent bulk vaccine should be tested and consideration given to using
the tests listed below for the individual products as appropriate. Alternatively,
if the monovalent bulk will be held for only a short period of time, some of the
tests listed below could – if appropriate – be performed instead on the final
bulk or final lot. If sufficiently justified, some of the tests may be performed on
an earlier intermediate instead of on the monovalent bulk. All quality-control
release tests for monovalent bulk should be validated and shown to be suitable for
the intended purpose. Assay validation or qualification should be appropriate
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for the stage of the development life-cycle. Additional tests on intermediates

during the purification process may be used to monitor consistency and safety.
During an emergency situation it is anticipated that critical assays would
be fully validated. Specifications should also be given for each critical
parameter. Qualification or validation, as well as specifications for some
assays, may be based on related products (for example, products with the
same vector backbone but differing in heterologous gene from the Ebola
GP gene) where it can be justified that the specific heterologous gene
used is unlikely to have an impact on the result. An example of this would
be particle quantification by qPCR where the probe is demonstrated to be
a non-EBOV sequence in the vector.
With appropriate justification, validation for non-critical assays could

be completed after product approval, provided that assay verification
adequately demonstrates that the assay is fit for purpose and under
control.
Similarly, if adequately justified, not all of the proposed assays may need
to be completed for clinical trial batch release. If it can be justified that
product safety and potency are not compromised, that completion of
the test(s) would delay product availability for use in clinical trials, and/
or that the test(s) would use up an unacceptably large volume of the
product urgently required for clinical trials, it may be possible to omit or
delay the test, or replace it with one that is more acceptable in terms of
the overall aims of the clinical trials in an emergency situation.
However, all of the approaches discussed above should be agreed with

the NRA on a case-by-case basis.
A.3.4.1.1 Purity
The degree of purity of each monovalent bulk vaccine should be assessed using
suitable methods. This should include testing for the presence of fragments,
aggregates or empty particles of the product, as well as for contamination by
residual cellular proteins. Residual cellular DNA levels should also be assessed
when non-primary cell substrates are used for production. The content and
size of host cell DNA should not exceed the maximum levels agreed with the
NRA, taking into consideration issues such as those discussed in the WHO
Recommendations for the evaluation of animal cell cultures as substrates for the
manufacture of biological medicinal products and for the characterization of cell
banks (24).
Process additives should also be controlled. In particular, if any antibiotics
are added during vaccine production, the residual antibiotic content should be
determined and should be within limits approved by the NRA.
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In a public health emergency, theoretical calculations to determine

residual levels of process contaminants (except DNA and proteins)
may be acceptable at the time of licensure – data should however be
submitted as soon as possible post-licensure.
These tests may be omitted for routine lot release upon demonstration
that the process consistently clears the residuals from the monovalent bulk
vaccine, subject to the agreement of the NRA.
A.3.4.1.2 Potency
Each monovalent bulk vaccine should be tested for potency using a combination
of the following methods.
Particle number
For relevant vectors (for example, adenovirus vectors) the total number of
virus particles per millilitre, quantitated by techniques such as qPCR or high-
performance liquid chromatography, should be determined for each batch of
monovalent bulk.
Infectivity
The infectious virus titre for each batch of monovalent bulk should be
determined as a measure of active product. Direct methods such as a plaque-
forming assay or indirect methods such as qPCR (if suitably correlated with a
direct measure of infectivity) could be considered. The particle/infectivity ratio
should also be specified.
Expression of the heterologous antigen in vitro
The ability of the viral particles to express the heterologous gene should be
demonstrated (for example, by the generation of immunoblots using antigen-
specific antibodies) following amplification of the vector in a suitable cell line.
A.3.4.1.3 Identity
Tests used for assessing relevant properties of the viral vector – such as antigen
expression, restriction analysis, PCR with a specific probe or sequencing – will
generally be suitable for assessing the identity of the product.
A.3.4.1.4 Sterility or bioburden tests for bacteria and fungi

Each monovalent bulk should be tested for bacterial and fungal bioburden
or sterility. Bioburden testing should be justified in terms of product safety.
Sterility testing should be as specified in Part A, section 5.2 of the WHO General
requirements for the sterility of biological substances (80), or by methods
approved by the NRA.
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A.3.4.1.5 Bacterial endotoxins

Each monovalent bulk should be tested for bacterial endotoxins. At the
concentration of the final formulation of the vaccine, the total amount of residual
endotoxins should not exceed that found in vaccine lots shown to be safe in
clinical trials or the amount found in other lots used to support licensing. The
test may be omitted once production consistency has been demonstrated after
agreement from the NRA.
A.3.4.1.6 Reversion to replication competency or loss of attenuation

The viral-vectored Ebola vaccines under development are either replication-
incompetent in human cells or adequately attenuated to prevent disease
symptoms related to the viral vector backbone. Although manufacturers generally
provide theoretical justifications for why reversion to competency or virulence
is unlikely to occur, low levels of viral particles may emerge that have gained the
complementing gene from the production cell line by an unknown or poorly
characterized mechanism. These viral particles are considered to be an impurity
– it is not known whether they represent a safety concern. It should also be taken
into account that many individuals within the Ebola target population could be
immunocompromised. Consequently, it should be shown that the product is
still replication-incompetent or fully attenuated (whichever is relevant) in initial
batches of the product. After demonstrating this, it may be possible to omit such
tests in future batches provided a sufficient justification is made. Such justification
should include the demonstration of replication incompetence/attenuation, and
discussion of why reversion to competency or loss of attenuation will not occur
in future batches.
A.3.4.1.7 Preservative content (if applicable)

The monovalent bulk may be tested for the presence of preservative, if added. The
method used and the permitted concentration should be approved by the NRA.
A.3.5 Final bulk vaccine

To manufacture the final bulk vaccine, appropriate quantities of different
monovalent bulk vaccines should be pooled, mixed and formulated (if required)
to form an homogeneous solution. The final bulk can be made up of one or more
batches of a single monovalent vaccine, to give a monovalent vaccine product
or alternatively, batches of several different monovalent bulks may be mixed to
yield a multivalent vaccine.
For multi-dose preparations, the need for effective antimicrobial
preservation should be evaluated, taking into account possible contamination
during use and the maximum recommended period of use after opening the
container or after reconstitution of the vaccine. If an antimicrobial preservative
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is used, it should not impair the safety or potency of the vaccine; the intended
concentration of the preservative should be justified and its effectiveness should
be validated (85).
A.3.5.1 Control tests on final bulk vaccine

The following tests should be performed on the final bulk vaccine, unless
otherwise justified and agreed with the NRA.
A.3.5.1.1 Identity
See section A.3.4.1.3.
A.3.5.1.2 Antimicrobial preservative

Where applicable, the amount of antimicrobial preservative should be determined
by a suitable chemical method.
A.3.5.1.3 Sterility tests for bacteria and fungi

A.3.6 Special considerations for manufacture and validation

It is acknowledged that the fermentation and downstream processes might
undergo considerable optimization after the initial clinical batches are produced.
Where control cells are grown in parallel to production cells, their raw materials
and fermentation should be aligned with production cell manufacturing
procedures. Process and product characterization should ensure the comparability
of product throughout development. Some changes in product characteristics
can be anticipated (for example, intended improvements due to optimization
studies, or unintended changes due to a process change). All such changes should
be identified and presented in clinical trial submissions or during an application

for a product licence and the implications of the change should be discussed. It is
not expected that process consistency will be demonstrated during early clinical
development, partly because insufficient batches will have been produced to
allow for adequate process validation and also because the process is likely to be
undergoing optimization. However, all available batch data (including qualitative
and quantitative data) should be presented. The product must be demonstrated
to be free from contaminants and sufficiently characterized to allow bridging
to later clinical material and commercial product. Process validation should
address safety issues such as aseptic operations, sterile filtrations, cleaning
validations, environmental control of facilities and validation of process utilities
– such as heating, ventilation and air conditioning (HVAC) systems, and water
for injection systems.
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It is expected that during an emergency situation these validation criteria

would be adequately addressed.
During early development, validation of pooling of single viral harvests

may not have been completed and so the number of harvests pooled should be
defined based on other criteria such as production requirements.
During later clinical stages and at licensing submission, the manufacturing
process is normally firmly established and process-specific validation completed
by demonstrating that several consecutive full-scale commercial batches can be
made that conform to predetermined criteria.
Although the “Quality-by-Design” approach is not considered in these
WHO Guidelines, such an approach is not excluded provided that the principles
discussed throughout this document are adequately addressed.
A.3.7 Special considerations for Good Manufacturing Practice

The principles of GMP should be adhered to during the manufacture of product
for clinical studies – even during a public health emergency. This may be
particularly important if some normal elements of development or control have
been omitted because of the urgent need for product. For example, if certain
testing is to be omitted on the basis that the test is also conducted on an upstream
intermediate, it is essential that the process is operated under full control.
Validation and specifications are likely to be provisional during the manufacture
of product for clinical trials, and additionally the process is not likely to be well
understood since only a limited number of batches will have been produced.
Therefore, it becomes essential that the principles of GMP, as laid down for the
manufacture of investigational medicinal products, are followed (69, 70, 86).
A.3.8 Special considerations for analytical procedures and specifications

Testing of critical intermediates and of the final product, as well as in-process
control testing, should primarily confirm product safety for early clinical trial
batches. In this regard, tests for bioburden/sterility, endotoxin and freedom from
adventitious agents should be fully developed and validated and should be applied
to each batch (although some flexibility towards adventitious virus testing is also
discussed in these WHO Guidelines). Other tests may not be fully validated.
However, even from an early clinical phase, assay verification should have been
performed. This is likely to fall short of the full validation requirements detailed
in the International Council for Harmonisation of Technical Requirements
for Pharmaceuticals for Human Use (ICH) Guideline Q2(R1) (87), but should
nevertheless give an indication that each method is fit for purpose.
Tests for safety, quantity, potency, identity and purity are mandatory.
Upper limits should be set for quantity of impurities, taking safety considerations
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into account. For relevant virus vectors, reversion to competency should be

tested for. A justification should be provided for the quality attributes included in
the specification and for the acceptance criteria for purity, impurities, quantity,
potency and any other quality attributes which may be relevant to vaccine
performance. The justification should be based on relevant development data, the
batches used in nonclinical and/or clinical studies, and data from stability studies.
It is acknowledged that during early clinical development, the acceptance criteria
may be wider than the final specification for product intended for Phase III
studies and for commercial product. During the manufacture of products for
initial clinical trials, not all attributes tested may have established specification
ranges since insufficient batches may have been made to know what an acceptable
range is. Nor at this time is a clinically meaningful range always known. However,
as the clinical programme continues – and certainly by the time of initiation of
Phase III trials – specification ranges should be set for each attribute.
Product characteristics that are not completely defined in the early
stages of development, or for which the available data are too limited to establish
relevant acceptance criteria, should also be recorded. As a consequence, such
product characteristics could be included in the specification without predefined
acceptance limits. At the initial stages of development, testing may not be
required to determine residual levels of process contaminants (except DNA and
proteins) if sufficient justification can be provided by theoretical calculation.
However, data to confirm the calculations should be provided prior to the
licensing application.
For later-stage clinical trials, it is expected that all analytical procedures
would be validated according to the principles set out in ICH Q2(R1) (87).
Specifications for each parameter should be justified by process capability as
well as by clinical suitability. If justified, following the manufacture of additional
batches of product, the sponsor should commit to revise the specifications as
data on process capability are accumulated.
During a public health emergency, data on clinical suitability are likely

to be limited and should be taken into account to the extent that they
are available.
A.4 Filling and containers

The general requirements concerning filling and containers given in WHO good
manufacturing practices for biological products (70) should apply to vaccine
filled in the final form.
Care should be taken to ensure that the materials of which the
containers and closures (and, if applicable, the transference devices) are made
do not adversely affect the quality of the vaccine. To this end, a container
closure integrity test and assessment of extractables and/or leachables for the
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final container closure system are generally required for the qualification of
containers, and may be needed as part of stability assessments.
If multi-dose vaccine vials are used and these vaccines do not contain
preservative then their use should be time-restricted, as is the case for
reconstituted vaccines such as bacillus Calmette–Guérin (BCG) and measles-
containing vaccines (85). In addition, the multi-dose container should prevent
microbial contamination of the contents after opening. The extractable volume
of multi-dose vials should be validated.
The manufacturers should provide the NRA with adequate data to prove
the stability of the product under appropriate conditions of storage and shipping.
A.5 Control tests on final lot

Samples should be taken from each final vaccine lot – which may be monovalent
or multivalent. These samples must fulfil the requirements of this section. All
tests and specifications should be approved by the NRA. The specifications
should be defined on the basis of the results of tests on lots that have been shown
to have acceptable performance in clinical studies.
A.5.1 Inspection of containers

Every container in each final lot should be inspected visually or mechanically.
Those showing abnormalities should be discarded and each relevant abnormality
should be recorded. A limit should be established for the maximum number of
containers which can be discarded before investigation of the cause; potentially
resulting in batch failure.
A.5.2 Appearance
The appearance of the vaccine should be described with respect to its form
and colour.
A.5.3 Identity
See section A.3.4.1.3. For multivalent vaccine each antigen component should
be identified.
A.5.4 Sterility tests for bacteria and fungi

A.5.5 General safety test (innocuity)

The need to test the final lots of the Ebola vaccine for unexpected toxicity (also
known as abnormal toxicity) should be discussed and agreed with the NRA.
Some countries no longer require this test (88, 89).
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A.5.6 Purity
Testing for purity should be performed unless it is performed on the monovalent
bulk or final bulk vaccine. However, limited purity testing of the final lot may
be required even if purity is tested on the final bulk vaccine if, after taking
the manufacturing process and nature of the vector into consideration, it is
considered possible that the purity may have changed. This should be considered
on a case-by-case basis.
A.5.7 pH and osmolality

The pH and osmolality values of each final lot of containers should be tested.
Lyophilized products should be reconstituted with the appropriate diluent prior
to testing.
A.5.8 Test for pyrogenic substances

Each final lot should be tested for pyrogenic substances through intravenous
injection into rabbits. A Limulus amoebocyte lysate (LAL) test may be used in
lieu of the rabbit pyrogen test if it has been validated and the presence of non-
endotoxin pyrogens has been ruled out. A suitably validated monocyte-activation
test may also be considered as an alternative to the rabbit pyrogen test. The
endotoxin content or pyrogenic activity should be consistent with levels found
to be acceptable in vaccine lots used in clinical trials and should be approved by
the NRA.
A.5.9 Potency, particle number and infectivity

The potency specifications for live viral-vectored vaccines should be
set based on the minimum dose used to demonstrate efficacy or effectiveness
in human clinical trials and/or challenge studies with a suitable non-human

preclinical model plus human immunogenicity data. An upper limit should also
be defined based on available human safety data. For multivalent vaccines it may
be necessary to perform this test on the monovalent bulks instead if analytical
methods cannot distinguish between the different monovalent vaccines in the
final lot.
A.5.10 Extractable volume

It should be demonstrated that the nominal volume on the label can consistently
be extracted from the containers.
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A.5.11 Aggregates/particle size

Since virus particles are susceptible to aggregation, each final lot should be
examined for particle size/aggregate content at lot release and at end of shelf-life
unless it can be shown that the test is not necessary.
A.5.12 Preservatives (if applicable)

Each final lot should be tested for the presence of preservative, if added.
A.5.13 Residual moisture (if applicable)

For freeze-dried final product, the residual moisture should be shown to be
within acceptable limits.
A.5.14 Reconstitution time (if applicable)

For freeze-dried final product, the reconstitution time of the product should
conform to specification.
A.6 Records
The requirements given in section 17 of WHO good manufacturing practices for
biological products (70) should apply.
A.7 Retained samples

A.8 Labelling
The label on the carton, the container or the leaflet accompanying the
container should state:
■■ the name of the vaccine;

■■ the lot number;
■■ the nature of the cells used to grow the viral vector;
■■ the volume of one recommended human dose, the immunization
schedule and the recommended routes of administration;
■■ the amount of active substance(s) contained in one recommended
human dose;
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■■ the number of doses, if the product is issued in a multi-dose

container;
■■ the name and maximum quantity of any antibiotic present in the
vaccine;
■■ the name and concentration of any preservative added;
■■ the temperature recommended during storage and transport;
■■ the expiry/retest date;
■■ any special dosing schedules; and
■■ contraindications, warnings and precautions, concomitant vaccine
use advice, and potential adverse reactions.
Labelling should conform to the national requirements of the region in
which the vaccine will be used.
A.9 Distribution and transport

Further guidance is provided in the WHO Model guidance for the storage and
transport of time- and temperature-sensitive pharmaceutical products (90).
Efforts should be made to ensure that shipping conditions are such as
to maintain the vaccine in an appropriate environment. Temperature indicators
should be packaged with each vaccine shipment to monitor fluctuations in
temperature during transportation.
A.10 Stability testing, storage and expiry date

A.10.1 Stability testing
Adequate stability studies form an essential part of vaccine development.
Guidance on the evaluation of vaccine stability is provided in the WHO
Guidelines on stability evaluation of vaccines (91). Stability testing should be
performed at different stages of production, namely: on single harvests or single

harvest pools (if the process is held up for a period of time, which may affect
product attributes at these points); final monovalent bulk; final bulk; whenever
materials are stored for a period of time before further processing (which may
affect product attributes); and final lot. Stability-indicating parameters should be
defined or selected appropriately according to the stage of production. A shelf-
life should be established and assigned to all in-process materials during vaccine
production, and particularly to the vaccine intermediates.
Accelerated stability tests may be undertaken to give additional
information on the overall characteristics of a vaccine, and may also be useful
in assessing comparability when the manufacturer plans to make changes to
manufacturing.
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For vaccine licensure, the stability and expiry date of the vaccine in its
final container, when maintained at the recommended storage temperature,
should be demonstrated to the satisfaction of the NRA using final containers
from at least three final lots made from different vaccine bulks. During clinical
trials, fewer data are likely to be available. However, the stability of the vaccine
under the proposed storage conditions should be demonstrated for at least the
expected duration of the clinical trial.
Following licensure, ongoing monitoring of vaccine stability is
recommended to support shelf-life specifications and to refine the stability
profile (91). Data should be provided to the NRA according to local regulatory
requirements.
The final stability-testing programme should be approved by the NRA
and should include an agreed set of stability-indicating parameters, procedures
for the ongoing collection and sharing of stability data, and criteria for rejecting
vaccines(s).
In-use stability should also be specified and justified with adequate data
generated under real-time conditions.
In an emergency situation and during early clinical trials, limited stability
data on the monovalent or final bulk vaccine and finished product may
be acceptable to preserve scarce stocks of product for use in clinical
trials, or if there is insufficient time to generate real-time stability data.
Data from one batch of bulk and final product may be sufficient initially
but this should be supplemented with data from at least two more
batches of bulk and final product as material that is surplus to clinical
trial requirements becomes available.
Even if limited stability data are available, it is preferable to provide an

expiry or retest date on the immediate product label since this provides
important information to the user. If this goes beyond the available real-
time data, accelerated stability data should be available to help support
the proposed extrapolation to the shelf-life, and the clinical trial sites
should be able to demonstrate a robust system for recalling the product
if real-time data do not support the extrapolated shelf-life. In exceptional
circumstances, the rationale for omitting this information from the label
may be discussed with NRAs.
A.10.2 Storage conditions

Storage conditions should be fully validated. The vaccine should have been
shown to maintain its potency for a period equal to that from the date of release
to the expiry date. During clinical trials, this period should ideally be at least
equal to the expected duration of the clinical trial.
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A.10.3 Expiry date

The expiry date should be based on the shelf-life supported by stability studies
and should be approved by the NRA. The expiry date should be based on the date
of blending of final bulk, date of filling or the date of the first valid potency test
on the final lot.
Where an in vivo potency test is used, the date of the potency test is the
date on which the test animals are inoculated.
Part B. Nonclinical evaluation of Ebola vaccines

B.1 General remarks
The design, conduct and analysis of nonclinical studies should be based on the
WHO Guidelines on nonclinical evaluation of vaccines (20). Further guidance
can be found in WHO and national and regional documents on DNA vaccines
(14, 15) and live recombinant viral-vectored vaccines (16–18).
The nonclinical safety evaluation, whenever necessary, should yield
sufficient information to demonstrate that the candidate vaccine is reasonably
safe for use in humans.
The following sections describe the types of nonclinical information
that should be submitted to support the licensing of a new Ebola vaccine.
Wherever appropriate, recommendations are also made on the minimum
dataset required.
B.2 Product characterization and process development

It is vitally important that vaccine production processes are standardized and
appropriately controlled to ensure consistency in manufacturing. The extent of
process validation may vary with the stage of product development. The vaccine
lots produced for nonclinical good laboratory practice (GLP) safety studies
should be manufactured with production process, formulation and release
specifications similar to those of the lots intended for clinical use. Supporting
stability data generated under conditions of use should be provided.
For a live viral-vectored vaccine, the degree of attenuation and the
stability of the phenotype should be evaluated. The critical genetic and phenotypic
markers of stability of the vector genome should as far as is practical be defined.
Phenotypic markers are useful for the detection of reversion events and may
include, though are not restricted to, vector replication efficiency, induction of
viraemia and level of virulence, and neurovirulence. The need for neurovirulence
testing is discussed below in section B.4.
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B.3 Pharmacodynamic studies

B.3.1 Challenge-protection studies
In the past, rodents (mouse, guinea-pig) and non-human primates (cynomolgus
or rhesus macaques) have been used to study the pathogenesis of EBOV infection
and the mechanism of immune protection. Rodent models are frequently used to
provide initial evidence for the immunogenicity or efficacy of candidate vaccines.
However, non-human primates display natural susceptibility to EBOV infection
and similarity in genetics, morphology and immunology with humans, and more
closely mimic EVD observed in humans. As a consequence, the non-human
primate models are particularly useful for proof-of-concept challenge studies and
characterization of the mechanism of protection. It is expected that proof-of-
concept data be collected for each virus strain included in the candidate vaccines.
It should be noted that conducting proof-of-concept challenge studies
with wild-type EBOV requires a BSL-4 containment facility. The same requirement
may apply to running virus-neutralization assays when wild-type EBOV is used
to evaluate vaccine immunogenicity and to evaluate serology samples obtained
from animals after EBOV challenge. A BSL-2 facility is sufficient to contain
animals until the time of challenge, to run other immunological assays – such
as enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunospot
(ELISpot) and intracellular cytokine staining (ICS) – without involvement of a
wild-type EBOV, or to manufacture a genetically modified organism (GMO).
Due to limited availability of BSL-4 laboratories, the proof-of-concept
challenge studies will generally be small. Nonetheless, these studies are of
higher predictive value than immunogenicity studies for forecasting vaccine
performance in humans. The parallel assessment of vaccine immunogenicity
and efficacy (protection from EVD) in proof-of-concept challenge studies may
permit the establishment of an immune correlate of protection (ICP) and an
understanding of the underlying protective mechanism.
Either during a public health emergency or in a normal situation, the
challenge studies are not required prior to initiating Phase I clinical trials.
However, it is nevertheless desirable for proof-of-concept challenge
studies to be conducted early during product development since these
studies, in combination with immunogenicity assessment, could provide
important information regarding an ICP and protective mechanism,
which would assist in the selection of immunological end-points in
subsequent clinical trials.
The design of challenge-protection studies should take into account

the planned posology for a specific route of administration and valency of
candidate vaccines. For a multivalent candidate vaccine intended to induce
durable protective immunity, a heterologous prime-boost regimen may need to
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be considered. The protective activity of the vaccine with respect to each of the
Ebola strains targeted should be assessed.
As in any challenge-protection animal study, the end-points used to
define protection should normally correlate with the desired effect in humans
– typically a survival benefit or attenuation of severe disease indicators such as
viral shedding, body weight changes and other relevant clinical signs. Other
key characteristics of the experimental design include the use of appropriate
challenge virus strains, dose(s) and route of challenge. The challenge dose should
be sufficiently high to produce an appropriate degree of lethality in the control
group of animals so that the vaccine protective effect can be shown with adequate
statistical power. For example, doses of 100–1000 plaque-forming units (PFU)
have been used (92).
The collection of challenge-protection data should take account of
the proposed indication for use – that is, pre-exposure versus post-exposure
prophylaxis against EVD. Appropriate timing of the challenge is another
important consideration. For pre-exposure prophylaxis, animals are usually
challenged at the time when the peak level of vaccine response (for example, peak
antibody titres) has developed post-vaccination. Where feasible, it would also be
informative for various public health vaccine strategies to challenge animals at
other times (for example, before the peak response or after the immune responses
have waned). For post-exposure prophylaxis, challenge at various time points
should be considered.
B.3.1.1 Use of a challenge-protection animal study to support licensure

In some circumstances in which demonstrating vaccine efficacy in clinical trials
is not feasible – due to low rates of EVD or absence of an EVD outbreak, or
when a human ICP has not been established for a vaccine – manufacturers may
propose an alternative approach to estimating vaccine effectiveness to support
licensing (for example, by inferring animal challenge results to humans). If

this course is pursued – and agreed to by the relevant NRA – the study should
be adequately designed to generate reliable data for inferring effectiveness in
humans (see section C.2.5).
Beyond the key design elements discussed above, further considerations
may include the use of non-human primates, vaccinating animals with an
appropriate range of doses of the vaccine so that the level of immune response
developed in animals (for example, range of relevant antibody titres) can match
that in humans. Compliance with GLP also brings significant advantages
and is encouraged. However, it is acknowledged that compliance with GLP
may not be possible in BSL-4 laboratories. Consequently, well-controlled
and well-documented non-GLP studies are also acceptable. The use of good
documentation practices to ensure data integrity is required.
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The standardization of non-human primate challenge models is

important for generating reproducible and relevant data for the purpose of
supporting licensure, especially when different candidate Ebola vaccines are
compared. Relevant aspects here include species and age of animals, challenge
material (including virus strain/variant and passage number), challenge route,
challenge dose, criteria for animal euthanasia, and standardized data collection
and reporting. Further current thinking on this issue can be found elsewhere (4).
B.3.2 Immunogenicity studies

Immunogenicity studies in animal models can generate important information
on the immunological properties of the candidate vaccine. These studies should
evaluate immune responses both quantitatively and qualitatively as per intended
posology. The immune responses to each of the Ebola strains in a multivalent
vaccine should be assessed, including any potential immunological interference
between strains. Data on cross-neutralizing antibodies and cross-reactivity
should be obtained for monovalent and multivalent vaccines through the use of
heterologous viruses.
Such studies can provide evidence for the appropriateness of the vaccine
dose, the number of doses, dosing interval and dose–response relationship.
Either during a public health emergency or in a normal situation,
immunogenicity data derived from a relevant species responsive to
the vaccine antigen in terms of desired immune responses are an
expected minimum requirement prior to starting Phase I clinical trials.
Alternatively, strong supportive data generated from the same platform
technology (for example, the same vector and manufacturing process,
but expressing different vaccine antigens) may be considered sufficient
for Phase I trial initiation.
Immunogenicity should be measured as humoral, cellular or functional

immune responses, as appropriate to each of the intended protective antigens and
to the antigens of the vector used. For several leading candidate vaccines using
Ebola GP as a sole protective antigen, antigen-specific ELISA (which measures
the quantity of serum GP-specific IgG antibodies) has been routinely used to
characterize the humoral response. Evaluation of cellular responses should
include the phenotypic and functional characterization of CD8+ and CD4+
T cell responses using sensitive and highly specific assays such as ELISpot and
ICS by multiparameter flow cytometry. The functional activity of immune
responses may be measured in vitro in neutralization assays using either
wild-type virus or pseudovirion virus. More extensive analyses may include
examination of Th1 and Th2 responses, the kinetics and duration of CD8+ and
CD4+ T cells and antibody responses, as well as assessment of the quality or fine
specificity of the antibody response.
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As discussed in section B.3.1, the assessment of immunogenicity

parameters in proof-of-concept challenge studies may allow for the establishment
of a correlation between an antibody or other immune response (such as cellular
immunity or cytokine response) and the level of protection from disease or
death, or for understanding the underlying protective mechanisms. These key
data may be expected to be generated during the development of the product.
Assessment of immunogenicity against multiple EBOV types should
be performed for multivalent vaccines and should also be considered for
monovalent vaccines.
B.4 Nonclinical safety studies (toxicity testing)

A safety assessment, including repeat-dose toxicity and local-tolerance studies,
is generally required for all new candidate vaccines, unless otherwise adequately
justified (20). In general, these studies will have been completed and analysed
prior to the initiation of Phase I clinical trials. Additional safety testing may be
necessary depending on the properties of the candidate vaccines. For a replicating
recombinant vaccine vector with neurovirulent potential, neurovirulence testing
in an animal species acceptable to the relevant NRA is an important consideration
and should be conducted before proceeding to trials in humans.
During a public health emergency, interim data from ongoing toxicity
studies (including on the immediate effect on survival and vital
physiological functions) and the submission of draft unaudited toxicity
study reports may be sufficient to support proceeding to Phase I clinical
trials with a novel platform/candidate vaccine.
As in a normal non-emergency situation, the omission of toxicity studies

may be possible if there are adequate platform toxicology data and
clinical safety experience. For example, for the viral-vectored vaccines
that this document focuses on, toxicity studies were not required during
the 2014–2016 EVD epidemic.
Such a limited dataset should be of good quality – that is, it should

be generated from a relevant animal species and should follow GLP
principles.
Since the use of a reduced toxicity dataset during a public health

emergency provides less certainty about the safety of the product,
additional data should be submitted once they become available,
including data on any delayed effect observed at later time points in
repeat-dose toxicity studies, histopathological data and the final signed
audited reports. Early discussion with NRAs in the countries where the
Phase I clinical trials are to be conducted is encouraged.
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Since Ebola vaccines are also beneficial for women of childbearing

potential, a reproductive-toxicity study will need to be conducted at an
appropriate point during product development. Serious consideration should be
given to vaccine administration that results in the exposure of pregnant animals
to a vaccine response during the early phase of implantation/organogenesis.
For a replicating recombinant vaccine vector that may have a direct effect on
the embryo/fetus, the dosing regimen should ensure a sufficient level of vaccine
vector in the blood of exposed pregnant animals.
The requirement for a developmental toxicity study is an important
issue for consideration, depending on the level of threat or control of
the disease. During the 2014–2016 EVD epidemic, large-scale Phase III
efficacy trials were approved in endemic countries without intentionally
enrolling pregnant women. With decreasing numbers of cases as the
2014–2016 epidemic was brought under control, the local NRAs required
that developmental toxicity data be made available to support the
enrolment of pregnant women.
B.5 Pharmacokinetic (biodistribution) studies

Classic pharmacokinetic studies with live viral-vectored vaccines are normally
not required. However, a biodistribution study in a relevant species should
generally be considered if the recombinant viral vector has any of the following
characteristics: (a) it is a novel viral vector or a known vector with a novel
envelope and there are no existing biodistribution data for the platform; (b) there
is a likelihood of altered infectivity and tissue tropism due to recombination; or
(c) a novel route of administration and formulation is to be used.
B.6 Environmental risk

The use of Ebola vaccines based on recombinant viral vectors could result in the
release of recombinant microorganisms into the environment. Some countries
have legislation covering environmental and other concerns related to the use
of live vaccines derived by recombinant DNA technology since they may be
considered as GMOs, and an environmental risk assessment (ERA) must be
submitted with any application to market these products. The specifics of the ERA
assessment within each country/region vary. Manufacturers are encouraged to
start a dialogue with the responsible authorities, including regulatory authorities
in countries where clinical trials are planned, early in the development of this
class of product.
The WHO Guidelines on the quality, safety and efficacy of dengue
tetravalent vaccines (live, attenuated) (93) provide advice in this respect that may
also be useful in the case of Ebola vaccines.
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The primary environmental risk of a replicating recombinant vaccine

vector relates to vaccine vector shedding and shedding-based transmission to
third parties – that is, to unvaccinated humans or domestic animals following
human administration. In the case of a replication-incompetent recombinant
viral vector, no shedding experiment is required. For future candidate novel
live recombinant vaccines based on a GMO, an ERA of the possible shedding
of the vaccine organisms following administration is required as part of the
preclinical evaluation.
Part C. Clinical evaluation of Ebola vaccines

C.1 General considerations
Clinical development programmes for Ebola vaccines must take into account the
epidemiology of the disease, the infrastructure for conducting clinical trials in
affected areas and the regulatory frameworks of particular NRAs. However, key
points that should be common to all such programmes are: (a) the standards for
demonstrating Ebola vaccine safety and effectiveness are the same as for other
vaccines; and (b) clinical studies are to be conducted in accordance with the
principles described in the WHO Guidelines for good clinical practice (GCP)
for trials on pharmaceutical products (94) and the WHO Guidelines on clinical
evaluation of vaccines: regulatory expectations (22).
As for all vaccines, close monitoring of studies by an independent data
monitoring committee (if warranted), the ethics committee(s) and the sponsor
should help to ensure study integrity. Meetings between sponsors and the relevant
NRA at critical time points during clinical development should be encouraged,
as well as meetings to discuss scientific and medical questions that may arise at
any time during an investigation.
C.1.1 Study population

Study population characteristics (for example, demographics, location, underlying
medical conditions and Ebola immune status) may vary by phase of clinical
development, as further discussed in section C.1.2. Specific considerations for
the evaluation of Ebola vaccines in the paediatric population are discussed in
section C.7.2.
Inclusion and exclusion criteria for participants should be defined for
each study planned. Exclusion criteria may include previous receipt of an Ebola
vaccine and possible previous contact with a person with EVD. Consideration
should be given to excluding subjects at risk of loss to follow-up (for example,
individuals not planning to live in the area for the duration of safety follow-up),
as well as immunodeficient or immunosuppressed subjects, particularly in the
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case of live vaccines based on replication-competent viral vectors. Additional

exclusion criteria should be based on clinical experience with the particular
vaccine, with the aim of excluding individuals who may have an increased risk
of significant adverse reactions, and individuals whose underlying conditions
may make it difficult to interpret safety data. For example, an investigational
recombinant VSV-vectored Ebola vaccine has been associated with arthritis in
one study. Consideration should be given to excluding individuals with arthritis
or related conditions (active or in past medical history) from participating in
initial studies of this vaccine, taking into account their risk of contracting Ebola,
and pending subsequent determination of the frequency, duration and severity
of this adverse event. Thus, considerations for exclusion would likely differ for
studies of healthy volunteers with a low risk of exposure to EBOV and for studies
conducted in the setting of an active outbreak.
The phase of clinical development and circumstances of the study should
also be considered when developing inclusion and exclusion criteria. For
example, a later-phase study being conducted in an emergency situation
in a population at high risk of EVD would probably have fewer exclusion
criteria than a Phase I study of healthy volunteers not at risk of EVD. The
phases of clinical development are described below in section C.1.2.
Pre-vaccination sera should be collected, at least in early-phase trials,

to assess pre-existing antibodies to EBOV and vaccine vector viruses, as well
as to assess aspects of baseline health status. The laboratory values expected for
the study population and any exclusion criteria should be specified in the study
protocol. Stored pre-vaccination serum may also be useful in the assessment of
certain post-vaccination adverse events that may occur. Assessment of possible
causal associations between vaccination and adverse events can also be facilitated
by knowledge of the background rates of events in the relevant general population.
C.1.2 Phases of clinical development

The phases of vaccine clinical development are typically a continuum from
Phase I, which often includes the first-in-human clinical trials carried out
primarily to assess safety and preliminary immunogenicity, to Phase II to further
describe safety and dose relationship to immunogenicity, and then to Phase III
pivotal studies to demonstrate the safety and effectiveness of a product in support
of licensure.
As for all vaccines, Phase I and Phase II studies of investigational Ebola
vaccines are expected to provide initial safety and immunogenicity data,
and to assess the optimal dose. The epidemiology of the disease is likely
to have a major impact on the timing and design of Phase III studies. In
the face of an outbreak, without available preventive vaccines, vaccine
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evaluation should adhere to the principles of this phased approach

but intervals between phases of evaluation may be compressed and
overlapping. For example, compressed timelines for clinical development
may be achieved by initiating Phase III studies based on interim safety
and immunogenicity data from earlier-phase studies rather than on
data from final study reports. Clinical development of an Ebola vaccine
in the setting of an outbreak is complex. Close collaboration between
public health authorities, NRAs, the community, clinical investigators
and the vaccine developer is essential to ensure that studies will meet
licensure requirements, including requirements for ethical conduct.
Phase II and Phase III clinical trials may be designed with prospectively
planned adaptive features that allow for changes in design or analyses
based on examination of the accumulated data at pre-specified interim
points in the trial. Such adaptive features may make trials more efficient.
For detailed considerations regarding approaches and the designing of
studies to demonstrate vaccine effectiveness see section C.2.
C.1.2.1 Phase I studies

The primary purpose of Phase I vaccine studies is to obtain preliminary safety
and immunogenicity data. For Ebola vaccines, these studies would generally be
first conducted in a small number (for example, < 100) of healthy adult volunteers
previously unexposed to EBOV and at low risk of EVD.
However, in the face of an outbreak, NRAs may consider larger Phase
I clinical studies (for example, by enrolling more sites) to increase the
early safety and immunogenicity database, as well as the use of study
populations similar to the eventual target population, thus facilitating
timely initiation of Phase II clinical studies.
The design of Phase I studies can be uncontrolled and open label or

may include a placebo control. When possible, the concomitant use of other
vaccines should be avoided to optimize the safety evaluation. The study design
may include sequential dose-escalation whereby subjects enrolled in lower-dose
cohorts are closely monitored for safety for a defined period (for example, 1–2
weeks or as appropriate for the characteristics of the vaccine) and the resulting
data are reviewed before subsequent enrolment of additional subjects in
successively higher-dose cohorts. All study participants should be actively and
closely monitored for safety.
C.1.2.2 Phase II studies

Phase II studies are initiated once satisfactory safety and immunogenicity data
from Phase I studies are available. In the absence of safety concerns from short-
term post-vaccination follow-up in Phase I studies (for example, 7 days or as
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appropriate for the specific vaccine), development may in some cases proceed to
Phase II studies in parallel with the continued collection of longer-term safety
data from Phase I studies. Phase II studies provide further information on safety
and immunogenicity to determine the optimal dose and dosing regimen, and
to support initiation of Phase III studies. Phase II studies typically involve up
to several hundred subjects and are frequently randomized, double-blind and
controlled. The comparator is usually an inert placebo or a control vaccine that
provides protection against disease unrelated to EVD. Phase II trials should be
of sufficient size to test hypotheses on dose and dosing regimen. Phase II studies
should be conducted in the proposed target population or in a population
similar to the target population in terms of demographic and ethnic factors, and
other factors that might impact on vaccine effectiveness or safety (for example,
concomitant infections). Detailed safety and immunogenicity data should be
obtained in Phase II studies.
C.1.2.3 Phase III studies

Large-scale Phase III clinical studies involve more-extensive testing to provide a
rigorous assessment of vaccine effectiveness that may include direct evaluation
of efficacy in protecting against clinical disease, expanded safety evaluation and
opportunities to potentially identify an ICP. Definitions of vaccine effectiveness
and vaccine efficacy are provided in section C.2.1. Phase III clinical trials may
also permit clinical evaluations of lot-to-lot manufacturing consistency. The
target population for Phase III clinical trials with candidate Ebola vaccines should
consist of individuals at high risk for the disease (that is, populations residing in
EVD outbreak areas, relevant health-care providers, laboratory personnel or first
responders). The design of Phase III effectiveness studies must be of adequate
scientific rigour to support effectiveness claims, while adhering to ethical
standards. Ideally, effectiveness is evaluated in randomized, double-blind, well-
controlled trials with a parallel control group receiving an inert placebo such
as saline injection or a vaccine that provides protection against another disease.
In some settings, the balance between scientific rigour and ethical standards
may preclude the use of a placebo group – for example, if there is an existing
efficacious Ebola vaccine that those in the trial might be eligible to receive.
Ethical considerations for the use of placebos in vaccine research, including in
circumstances in which an efficacious vaccine is already available, are discussed
in the WHO meeting report Expert consultation on the use of placebos in vaccine
trials (95). As discussed in section C.2 below, other study designs for obtaining
effectiveness data for candidate Ebola vaccines may be considered if a placebo-
controlled trial is not considered ethical or is not feasible.
To demonstrate vaccine effectiveness, Phase III trials may be based
on a disease end-point or, as described in section C.2, they may be based on
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the attainment of a level of an immune marker predictive of protection. The

incidence of EVD and ethical considerations will be primary determinants of
the approach used to evaluate vaccine effectiveness and the design of clinical
end-point efficacy studies, as also discussed in more detail in section C.2. For
many disease end-point clinical efficacy study designs, large sample sizes may
be needed, particularly if the incidence of the disease in the study population
is expected to be low or to decline during the study period. Adequate statistical
justification of the size and duration of the trial should be provided, and trial
end‑points and criteria for trial success specified prior to initiation of the
study. Plans should be included to monitor the conduct of the trial, taking
into consideration the potential for changes in disease incidence which may
necessitate trial design modification. It is important that some attempt should
be made to define an ICP as part of efficacy studies. For such an evaluation to be
clinically meaningful, validated standardized assays are essential.
Clear and definite evidence that the vaccine is safe and effective is required
for regulatory decision-making. Discussions should be held with relevant NRAs
on the study design and on plans for conducting the study and analysing its
results at the early conceptual stage of the Phase III study, and agreement reached
with the NRAs prior to trial initiation. Close consultation with local community
leaders, health policy-makers and ethics committee(s) in EVD outbreak regions
where efficacy studies are planned is also crucial.
C.2 Demonstration of effectiveness of candidate Ebola vaccines

C.2.1 Definitions of effectiveness and efficacy
It is important to distinguish vaccine effectiveness from vaccine efficacy. Vaccine
efficacy is an estimate of the reduction in the incidence of clinical disease
observed in a vaccinated group relative to the incidence of disease in a group
not vaccinated against the disease to be prevented. Vaccine efficacy measures
direct protection (that is, protection induced by vaccination in the vaccinated

population sample). The best estimates of vaccine efficacy come from randomized
controlled clinical trials.
Vaccine effectiveness is an estimate of the protection conferred by
vaccination. It is usually obtained by monitoring the disease to be prevented by
the vaccine during routine use in a specific population. It may measure both
direct and indirect protection (for example, the estimate may reflect in part the
protection of non-vaccinated people secondary to the effect of the vaccine in
the vaccinated population). Thus, the term vaccine effectiveness may be used
broadly to encompass vaccine efficacy (direct protection) as well as indirect
protection. Evidence for vaccine effectiveness may be derived from challenge-
protection studies conducted in animal models or from a vaccine-induced
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immune response (for example, pre-specified antibody threshold induced by the

vaccine in vaccinated people).
For any preventive vaccine, the most direct approach for demonstrating
effectiveness is based on clinical end-point efficacy trials showing protection
against disease, or alternatively, based on clinical trials evaluating a scientifically
well-established ICP (for example, antibody response).
C.2.2 Immunological evaluation of Ebola vaccines

Clinical disease end-point efficacy trials provide an opportunity to identify an
ICP. The derivation of an ICP is facilitated by the availability of post-vaccination
serum samples from a relatively large number of protected trial participants as
well as from vaccinated participants who develop disease. Thus, for all Ebola
vaccine clinical disease end-point efficacy trials, post-vaccination serum samples
(and preferably also pre-vaccination serum samples) would ideally be collected
from all subjects, with post-vaccination sampling at regular predefined intervals
throughout the study period. If this is not feasible, pre- and post-vaccination
serum samples should be collected from as many subjects as possible. Ebola
prevalence studies in various African countries have revealed unexpectedly high
rates of baseline Ebola seropositivity in some regions, as measured by serum IgG
antibodies, underscoring the importance of collecting baseline serum samples
in studies conducted in these countries (96–101). Consideration should also
be given to the collection of blood samples for the evaluation of cell-mediated
immunity which may play a role in protection for some vaccines.
Even if it is not possible to identify an ICP from a clinical end-point
efficacy trial, immunogenicity data from Phase II and Phase III studies are
crucially important for the use of alternative approaches to assess vaccine
effectiveness based on surrogate immune response end-points likely to predict
protection and/or for challenge-protection studies conducted in animal models
(see sections C.2.4 and C.2.5 respectively).
Potentially important immunogenicity end-points include EBOV IgG
ELISA antibody titre and presence/levels of EBOV neutralizing antibody. End-
points evaluating T cell mediated responses following vaccination may also be
considered. Specific considerations regarding immunological assays are discussed
below in section C.6.
In evaluating antibody response to vaccination, it is important to
stratify analyses by baseline serostatus and to pre-specify the definition of
seroresponse, and seroconversion. Seroresponse is typically based on an x-fold
rise in antibody level from pre-vaccination to post-vaccination in initially
seropositive individuals. Seroconversion is typically based on achieving a
measurable antibody level post-vaccination in individuals who were initially
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seronegative. A detailed justification for the definition of each term should be

provided. The definition of seroresponse may differ for different Ebola vaccines
and assays. Serological end-points and evaluation criteria should be determined
following input from, and agreement by, the NRA before study un-blinding and
serological analysis.
As an ICP (including potential antibody thresholds associated with
protection) or a surrogate immune marker may differ for different vaccines, it
is important to obtain vaccine-specific human serological data. Ideally, vaccine-
specific human cellular immune response data would also be obtained (102).
Applicability of an ICP or a surrogate immune marker will depend on specific
vaccine characteristics such as antigen structure, mode of delivery, antigen
processing in the vaccinee and virus serotype. For example, an ICP established
for an adenovirus-vectored Ebola vaccine cannot be presumed to be applicable
to a VSV-vectored Ebola vaccine given that the two vaccines present antigen
differently and engender different types of protective immune responses.
Similarly, Ebola vaccines that are, for example, based on VSV and adenovirus
vectors and administered using a prime-boost regimen may induce different
protective immune responses than Ebola vaccines based on different platforms
or technologies and administered using a different regimen. As another example,
an ICP or a surrogate immune marker identified for a vaccine containing a
particular EBOV (for example, ZEBOV) cannot be assumed to be applicable to
another vaccine containing a different EBOV (for example, SUDV).
C.2.3 Clinical disease end-point studies

C.2.3.1 General principles of clinical disease end-point studies
In general, the crucially important aspects of clinical disease end-point efficacy
studies include: (a) an appropriate control group; (b) appropriate methods for
randomization, as applicable; (c) masking procedures, as applicable; (d) a pre-
specified primary end-point (for example, EVD confirmed by PCR); (e) pre-
specified important secondary end-points (for example, EVD not laboratory
confirmed); (f) pre-specified, detailed clinical case definitions for the primary
end-point; (g) validated diagnostic assays to support the pivotal efficacy analyses;
(h) unbiased case-ascertainment methods; and (i) adherence to relevant
statistical principles. Measures to reduce potential bias are important in all trials,
but particularly so for designs other than randomized, double-blind, controlled
trials with a parallel control group. Specific considerations regarding the design
of clinical end-point efficacy studies and diagnostic tests for EVD are discussed
below in sections C.2.3.2 and C.6.1, respectively. Consideration should be given
to the establishment of an independent data-monitoring committee for clinical
end-point efficacy studies of Ebola vaccines in order to advise the sponsor on the
continuing validity and scientific merit of the study.
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C.2.3.2 Design of clinical disease end-point studies

C.2.3.2.1 Randomized controlled trials
The prospective randomized, double-blind, placebo-controlled trial with
an EVD end-point is the gold standard for demonstrating the efficacy of
any investigational Ebola vaccine(s) when no licensed efficacious vaccine is
available. This design avoids potential bias in the assessment of end-points and
maximizes the chance that a difference in disease incidence observed between
the vaccinated and unvaccinated groups is due to a true effect of the vaccine
being evaluated. The unit of randomization is usually the individual subject
enrolled in the trial, although other units of randomization may be considered.
While direct assessment of vaccine efficacy in randomized controlled trials
provides the most definitive evidence of effectiveness, it requires a sufficiently
high disease incidence and a correspondingly adequate sample size.
C.2.3.2.2 Ring vaccination design

In settings with relatively low disease incidence, vaccine efficacy clinical trial
designs – such as ring vaccination in which people at highest risk of infection are
recruited – may be considered in order to maximize statistical power (64, 103).
A novel cluster randomized controlled trial design to evaluate vaccine
efficacy and effectiveness during outbreaks, the ring vaccination trial
design was developed with special reference to Ebola (101). The approach
taken to increase statistical power is to recruit those at highest risk of
infection (for example, individuals who are socially or geographically
connected to an index case). An important consequence of this increase
in power is that this trial design has the potential to yield an estimate
of vaccine efficacy within a shorter period of time and possibly with a
smaller sample size, compared to more-common trial designs.
A ring is a socio-geographical population group made up of the contacts

and contacts of contacts of the index case. Rings are randomly assigned
to immediate or delayed vaccination, with the delayed vaccination rings
serving as controls. Vaccine efficacy is calculated on the basis of the
relative rates of disease in the immediate and the delayed vaccination
rings. An efficacy trial using ring vaccination with an investigational
Ebola vaccine was conducted in Guinea in 2015 (64, 103).
C.2.3.2.3 Stepped wedge randomized cluster trial

After licensure of an Ebola vaccine – and in some settings even before licensure
– the high case-fatality rate of EVD may raise ethical concerns about non-
vaccination in a parallel control group. To mitigate these concerns, a stepped
wedge randomized cluster trial (SWRCT) design in which clusters of participants
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are sequentially vaccinated over a number of time periods, may be considered.

In this design, all participants start in the control group and, at predefined time
points, a cluster of participants is vaccinated in a random order (known as
‘‘steps’’). Vaccine efficacy is calculated on the basis of the relative rate of disease
in the vaccinated population compared to the unvaccinated population. This
design, in which all participants are vaccinated by the end of the study, may
be ethically acceptable in settings where the candidate vaccine is not available
simultaneously for all participants and where the use of a placebo group is
considered unacceptable.
Disadvantages of the SWRCT design include difficulty in blinding,
attrition in the later vaccinated clusters and the more complex analysis required.
In addition, an underlying requirement for validity of an SWRCT design is that
disease incidence rates must remain fairly stable throughout the trial. If disease
incidence rates are not expected to remain reasonably constant during the course
of the trial, data analyses may be performed separately within narrow windows of
time (for example, by day or week) within which it can be assumed that disease
incidence rates are stable. This time stratification will necessitate more careful
recording of disease incidence rate with time. The impact of misclassification
of disease incidence rate with time will need to be considered. Another issue is
that SWRCT designs randomize the timing of vaccination of the clusters, which
unlike most designs disallows the flexibility to move vaccination to high-risk
areas that evolve while the trial is ongoing, and which could also potentially cause
the SWRCT to take longer to complete compared to other trial designs (104).
C.2.3.2.4 Test-negative case control design

Once a vaccine has been deployed in a population, it may be possible to estimate
vaccine effectiveness using a test-negative case control design (105–107). In the
test-negative case control design, patients seeking health care for symptoms
compatible with EVD are recruited into the study and tested for the disease.
Vaccine effectiveness is estimated by comparing the odds of vaccination in
subjects testing positive for Ebola (cases) to the odds of vaccination in subjects
testing negative (controls).
Test-negative case control studies are relatively low cost and easy to
conduct. However, controlling for potential bias in this non-randomized design
is particularly challenging because vaccinated and unvaccinated individuals
may have different risk factors for disease.
Test-negative case control studies are also subject to the same sources of
bias and measurement error as other non-randomized studies – some of which
may not be recognized or adequately adjusted for in the statistical analyses.
Furthermore, it may be difficult to assure comparable disease severity across
participants at study entry or to achieve complete ascertainment of vaccination
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status. Potential sources of bias and limitations inherent in this design need to
be carefully considered in planning study procedures and statistical analysis, as
well as in interpreting the results.
C.2.4 Surrogate end-points for demonstration of effectiveness

For diseases like EVD, for which there is no well-established ICP, if disease
incidence is too low to feasibly conduct clinical end-point efficacy studies then
effectiveness may be based on controlled clinical studies which establish an
effect on a surrogate end-point (for example, immune response) considered
likely to predict clinical benefit. The surrogate end-point used to evaluate
effectiveness could be derived from human studies – for example, immune
responses in vaccinated individuals from Phase II and Phase III studies and/
or from a comparison of antibody responses post-vaccination in protected
vaccinees with those of vaccinees who contract EVD. In this scenario, immune
responses such as antibody titres achieved in vaccinated non-human primates
that correlate with protection from challenge may also help in determining an
immunogenicity end-point likely to predict protection in humans. Some NRAs
may have provisions that would allow for the licensing of an Ebola vaccine
based on such an approach for demonstrating effectiveness. Specific regulatory
requirements associated with such provisions (for example, post-licensure
studies to verify clinical benefit, and requirements for pre-licensure clinical
safety studies in humans) must be adhered to.
As discussed above in section C.2.2, a surrogate immune marker
identified for a particular vaccine may not be applicable to another vaccine.
C.2.5 Animal efficacy data for demonstration of effectiveness

If clinical end-point efficacy studies in humans are not ethical or feasible and
there is no well-established ICP or surrogate immune marker likely to predict
protection then evidence for effectiveness may be based on controlled challenge-
protection studies conducted in an appropriate animal model (see section B.3.1)
and clinical immunogenicity data. A central principle of approaches based on
animal efficacy data is that the results of the animal studies establish that the
vaccine is likely to produce clinical benefit in humans. Some NRAs may have
provisions that would allow for the licensing of an Ebola vaccine based on such
an approach for demonstrating effectiveness. Specific regulatory requirements
associated with such provisions (for example, meeting certain criteria for the
animal model(s), accrual of information in animals and humans to allow for
selection of an effective dose in humans, pre-licensure safety studies in humans,
and post-licensure studies to verify clinical benefit when such studies are feasible
and ethical) must be adhered to.
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C.2.6 Special considerations

C.2.6.1 Evaluation of effectiveness of candidate vaccines
after initial licensure of an Ebola vaccine
Licensure of an Ebola vaccine may facilitate the evaluation of effectiveness of
a new candidate Ebola vaccine if an ICP is established or a surrogate immune
marker likely to predict clinical benefit is identified during development of the
licensed vaccine and is considered to be applicable to new candidate vaccines.
In such cases, an adequately conducted, randomized, controlled clinical trial(s)
comparing the immune response, as measured by the relevant immunological
parameter(s), in recipients of the candidate vaccine to that of recipients of the
already licensed vaccine, using pre-specified statistical criteria, appropriate
statistical methods and validated assays, could provide sufficient evidence of
effectiveness to support licensure. As previously described, if the estimate of
effectiveness is based on a surrogate marker likely to predict clinical benefit,
approval may be subject to post-marketing requirements to verify the clinical
benefit of the vaccine.
Alternatively, an Ebola vaccine may be licensed without an ICP or
surrogate immune marker likely to predict protection considered to be applicable
to other candidate Ebola vaccines. It may therefore be necessary to demonstrate
vaccine effectiveness using other approaches (for example, animal challenge-
protection studies combined with clinical immunogenicity studies). For this
purpose, the animal challenge-protection studies should be adequately designed
to provide reliable data, as discussed in B.3.1.
Licensure of an Ebola vaccine may make it infeasible and unethical to
conduct pre-licensure clinical end-point efficacy trials with new candidate
Ebola vaccines. Even conducting a comparative efficacy trial to demonstrate
non-inferiority of a new candidate vaccine to the licensed vaccine would be
challenging.
C.2.6.2 Evaluation of effectiveness of multivalent vaccines

For multivalent vaccines (for example, containing more than one EBOV strain,
or an EBOV strain(s) and MARV) effectiveness (that is, based on clinical end-
point efficacy studies, animal efficacy data and/or human immune response
data) will need to be demonstrated for each strain contained in the vaccine.
C.2.6.3 Duration of immune response and protection,

and need for booster vaccinations
The long duration of the 2014–2016 EVD epidemic and the potential for future
exposures highlight the need to consider the durability of vaccine-induced
protection and the potential need for booster doses in the evaluation of Ebola
vaccines. This evaluation could be facilitated by the identification of an ICP.
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Importantly, Phase II and Phase III clinical trials should attempt to identify
ICPs and should evaluate the kinetics of the immune response and induction of
immunological memory.
C.3 Safety evaluation of candidate Ebola vaccines

C.3.1 General considerations
Sponsors must comply with the adverse event reporting requirements of the
relevant NRA and the independent ethics committee(s). Templates of the
forms used to monitor and document adverse events should be provided with
each protocol. Sponsors are encouraged to initiate early dialogue with the
appropriate NRAs to reach agreement on the size of the safety database needed
to support licensure of a particular vaccine. As with all vaccines, the size of
the safety database depends in part on the characteristics of the candidate
vaccine as well as on available preclinical and clinical safety data. Safety data
from previous preclinical and clinical experience with related vaccines using
the same platform may also be considered when determining the size of the
safety database.
Safety-monitoring methods should be tailored to the specific study
population (for example, children, adults, pregnant women or people living
in areas where EVD is endemic), with consideration given to adverse events
known to be associated with a particular vaccine – for example, in some Ebola
vaccine studies, fever, arthralgia and arthritis have been observed. Study
protocols should specify methods for monitoring and documenting adverse
events, including: (a) use of standardized subject diaries and case report forms;
(b) procedures for inquiring about adverse events at study visits; (c) severity
grading scales; (d) definitions for adverse event categories – for example,
serious, new-onset chronic medical condition, and adverse event of special
interest (AESI); and (e) requirements for prompt reporting of serious adverse
events (SAEs) to the sponsor.
In early-phase clinical studies (and at later phases if warranted),
consideration may be given to pre- and post-vaccination assessment of
safety laboratory parameters, including haematological and clinical chemistry
evaluations. If such parameters are monitored, grading scales appropriate for
the study population should be utilized.
It is also important to establish stopping rules for subsequent doses for
individual study participants who experience an SAE, as well as study pausing/
stopping rules for SAEs overall. Consideration should also be given to the
establishment of an independent data-monitoring committee to advise the
sponsor with regard to the continuing safety of trial participants and those to
be recruited into the trial, particularly for any trials involving children and any
large-scale later-phase trials.
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Other aspects of safety that should be addressed in the study protocol

include assessment of virus shedding and the potential for secondary
transmission of replicating or potentially replication-competent live vaccine
virus vectors, at least in early-phase studies, as well as procedures to minimize
the risk of EBOV transmission to study personnel involved in clinical end-point
efficacy studies.
C.3.2 Monitoring for common, solicited adverse reactions

In Phase I and Phase II studies, all participants should be monitored for pre-
specified, solicited local and systemic adverse reactions at specified time points,
for a specified period following vaccination (for example, daily for at least
7 days, or longer if warranted based on vaccine characteristics and available
preclinical and clinical data). In Phase III studies, it may be acceptable to
actively monitor only a subset of participants (for example, several hundred per
group) for common, non-serious local and systemic adverse reactions. Data-
collection methods may include the use of memory aids in literate populations
and telephone interviews.
C.3.3 Monitoring for unsolicited adverse events

All study participants should also be monitored for unsolicited adverse events,
including new-onset chronic medical conditions and exacerbation of medical
conditions that may not necessarily meet the NRA’s definition of serious.
Whereas monitoring for all unsolicited adverse events may be conducted for
relatively short periods post-vaccination (for example, 21 days, or 42 days for
replicating live viral vaccines), monitoring for new-onset chronic medical
conditions for a longer period (for example, 6–12 months) may be useful in
detecting unexpected safety signals.
C.3.4 Monitoring for serious adverse events

While the exact definition of an SAE can vary across different NRAs, the ICH
Guideline E2A defines an SAE as any untoward medical occurrence that results
in death, is life-threatening, requires inpatient hospitalization or prolongation of
existing hospitalization, results in persistent or significant disability/incapacity
or is a congenital anomaly/birth defect (108). WHO considers an adverse event
following immunization as “serious” if it meets any of the above criteria or if it
requires intervention to prevent permanent impairment or damage (109).
All participants in pre-licensure clinical trials of Ebola vaccines should
be closely and actively monitored (for example, with diary cards or follow-up
visits) for SAEs for at least 21 days (or 42 days for replicating live viral vaccines)
after each vaccination. A method to further query for SAEs over a minimum
of 6 months following the last vaccination should also be incorporated into
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the study protocol. A longer-term safety follow-up period for the assessment
of SAEs (for example, through the 12 months following the last vaccination)
may be warranted for some vaccines (for example, vaccines containing
novel adjuvants). Long-term safety follow-up (that is, for 6–12 months post-
vaccination) may be accomplished by telephone follow-up or other methods
appropriate for the setting.
C.3.5 Monitoring for adverse events of special interest

All study participants should be monitored for any AESIs for a particular
vaccine for a specified period post-vaccination (for example, 6–12 months). The
period of follow-up may vary for different AESIs, depending on the anticipated
window of risk.
C.4 Ethical considerations

Compliance with good clinical practice standards (22, 94) provides assurance
that the rights, safety and well-being of study participants are protected and
study integrity is preserved. For any clinical study, a review by an independent
ethics committee is mandatory and the approval of this committee must be
obtained prior to study initiation. Informed consent must be given freely by
every study participant and should be documented. For children participating
in clinical studies, consent must be given by their parent or legal guardian. The
informed consent process may need to be more specifically tailored to take into
account local cultural views or practices. Child participants should be informed
about the study to the extent compatible with their understanding and, if
capable, should provide their assent. Participants in vaccine studies should not
be exposed to unreasonable or serious risks of illness or injury. A study should
be initiated and continued only if the anticipated benefits justify the risks. Low-
resource communities, which are often those at greatest risk of EVD, should not
be exploited in conducting research (for example, where there will be no long-
term benefit to the community because the developer does not intend to seek
licensure in the country where the vaccine is studied).
See section C.7.2 for considerations regarding initiation of clinical
studies in the paediatric population.
C.5 Statistical considerations

C.5.1 General statistical principles
General statistical principles for clinical trials should be based on the relevant
WHO document (21), where available, and other guidelines such as ICH E9
(110). Phase I studies are generally exploratory and may lack statistical power
for hypothesis testing.
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Phase II studies are for selecting the final optimal dose and dosing
regimen and should be rigorously designed and analysed. The potential role of
immunogenicity data should be taken into consideration to ensure the adequacy
of data to support licensure if necessary.
Phase III studies are designed to provide robust data on vaccine
effectiveness and more-extensive data on safety. The study protocols should
clearly describe the procedures for randomization and blinding, primary and
secondary objectives, end-points to be analysed, null and alternative hypotheses
to be tested, level of type I error, sample size calculations, statistical methods for
assessing each end-point, and analysis populations (per-protocol and intent-to-
treat). If interim analyses for efficacy are planned, detailed information should
be included in the protocol regarding the timing of interim analyses, type I
error allocated to each analysis, and stopping rules. The study reports should
include detailed information on subject disposition. Statistical estimates should
be presented along with confidence intervals.
C.5.2 Statistical considerations for evaluating vaccine effectiveness

The effectiveness of a new Ebola vaccine is most convincingly demonstrated in a
randomized, double-blind, placebo-controlled study based on an EVD end-point
– though circumstances may dictate that alternative trial designs be considered.
Vaccine efficacy and the corresponding confidence interval (usually 95%) should
be estimated. Sample size for these trials depends on disease incidence rates in
the study population, the level of vaccine efficacy considered to be clinically
relevant and the chosen trial design.
Rapidly changing and/or declining incidence rates during an
outbreak may need to be considered when choosing a study design. In some
circumstances, designs such as cluster randomization may need to be used. For
cluster-randomized trials, data should be analysed using statistical methods
appropriate for the study design and study objectives. If inference will be at
the usual individual level rather than the cluster level, sample size calculations
and statistical analysis methods should appropriately address the within-cluster
correlation, as feasible. Randomization should be carefully planned to avoid
imbalance in disease risk or incidence rate between clusters randomized to be
vaccinated or to serve as controls. As mentioned in section C.2.3.2.2, seeking
to confine a trial to individuals at relatively high risk of EVD (as with the ring
vaccination trial design) may have higher statistical power to detect vaccine
efficacy than a trial in a population at lower risk of disease and, as a consequence,
can potentially require a smaller sample size and achieve faster completion time
compared to other study designs.
When ICPs established in animal challenge studies are being used to
define immune response end-points for effectiveness evaluation or to infer
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clinical benefit under other alternative licensure pathways, these studies (for
example, in non-human primates) should be conducted using an appropriate
dose range and an adequate number of animals such that the relationship
between immune response and protection, and the protective threshold, can be
estimated with satisfactory precision (see Part B).
C.5.3 Statistical considerations for evaluating vaccine safety

Safety evaluation is inherently exploratory and typically uses descriptive
statistics. The calculation of p-values is sometimes useful as a flagging device
applied to a large number of outcomes to detect differences that may need
further evaluation. Multiplicity adjustment is not performed in order to increase
the ability to detect potential signals. However, the potential for false-positive
signals resulting from multiple tests must be considered prior to drawing firm
conclusions.
If detection of several pre-specified SAEs is the primary focus of a large
pre-licensure safety trial then multiplicity adjustment for testing a small number
of hypotheses can be considered. When specific safety issues are identified
during preclinical studies or early clinical trials (for example, cases of post-
vaccination arthritis in clinical studies with certain viral-vectored vaccines) then
prospective monitoring for related events as well as formal statistical testing
should be considered.
C.6 Serological and diagnostic assays

The incubation period for EVD is 2–21 days. While patients are infectious by
the time symptoms are evident, levels of virus in saliva or blood may not reach
detectable levels until two or three days later. At this point in the course of
infection, viral antigen can be detected by immunoassay and viral nucleic acid
by a NAT-based assay. For both antigen and nucleic-acid-based tests the use of
blood is preferred due to lower sensitivity of these assays with saliva. While serum
IgM may also be detectable at this time, there is a risk of obtaining false-negative
results so early in the course of infection. Serological testing should therefore
be reserved for confirming prior infection or for evaluating vaccine responses.
Isolation of EBOV in tissue culture must be performed in a high-containment
laboratory, of which there are few, and this is therefore not routinely performed.
C.6.1 Diagnostic tests

All currently available EBOV NAT-based assays are based on the same principle
– detection of an EBOV nucleic acid target sequence after extraction of viral
nucleic acids from clinical samples, reverse transcription of RNA and in vitro
amplification. The primers used in different NAT-based assays target different
viral genome regions, which should be considered, particularly when used
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in vaccine trials, so that infection can be distinguished from vaccination. For

example, if the EBOV gene targeted by the NAT-based assay is also expressed by
the vaccine, a positive result on a blood sample could mean that the subject may
have EVD or it could mean that the subject is shedding vaccine virus.
Although many EBOV diagnostic kits have received approval for
emergency use, this should not be taken to mean that they have been validated
for non-emergency purposes, such as establishing vaccine efficacy in field trials.
Assay performance parameters investigated as part of emergency-use approval
often do not include more rigorous assessments, such as repeatability over the
operating range, inter-assay precision or performance in the field. Appropriate
RNA process controls and international reference standards became available for
these assays in 2015 (see section A.1.1), which should now enable assessment of
assay performance and comparison of results across different assay platforms.
Rapid diagnostic tests (RDTs) designed for EBOV antigen detection
provide results more rapidly (sometimes within minutes), are easier to perform
compared to NAT-based assays and do not require complex equipment (or
electricity). However such tests are less sensitive than NAT-based assays and
results should be confirmed by NAT-based assay where possible. As with NAT-
based assays, care should be exercised when interpreting the results of RDTs
using samples obtained from vaccinees, given that the antigen targeted by the kit
may share homology with vaccine antigen.
C.6.2 Immunological tests

Although an ICP against EVD has not been established, myriad immunological
tests have been developed. Of these, the EBOV IgG ELISA has gained the
greatest acceptance based mostly on studies of experimentally vaccinated non-
human primates in which high IgG levels have been linked to protection
against subsequent challenge. Whether protection was via antibody detected by
ELISA, or whether the presence of high levels of ELISA antibody is a marker
of some other more meaningful form of immune response, is not known. In

the absence of available data from humans defining an ICP (for example, data
from a successful vaccine efficacy trial), an ICP may have to be established in
an animal model. On the basis of data available to date, non-human primates
appear to be an acceptable animal model for such an exercise, with inadequate
information to support the use of other animal species.
Few EBOV immunoassays are commercially available – most reside in
research laboratories where they were developed for use in preclinical or clinical
trials of investigational vaccines. For this reason, most ELISAs are designed
to detect antibodies against the EBOV GP – that is, the protein expressed by
most investigational vaccines. There are numerous concerns about these tests
and care should be taken in interpreting the data they produce. ELISA plates
coated with lysates of cells expressing non-EBOV antigen that is also contained
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in, or expressed by, the vaccine may be prone to yielding false-positive results.
Other issues for consideration are the source of virus antigen used in the ELISA
(reduced cross-recognition between virus strains), conformational changes of
the antigen upon binding to the plate and antigen stability over time.
Since ELISAs are not necessarily informative of functional immunity,
assays that measure virus neutralization and cell-mediated responses have been
developed. The neutralization assays generally employ pseudovirions (such as
VSV in which the GP gene has been replaced with that of EBOV) or lentivirus
packaging systems. Consideration should be given as to whether virus-
neutralizing activity detected in these in vitro assays is predictive of EBOV-
neutralizing activity in vivo. It is also important to consider false positivity
through the use of ELISA plates coated with non-EBOV vaccine components.
For example, non-EBOV antibodies generated in response to receipt of a
VSV-vectored Ebola vaccine may have an impact on the performance of VSV-
based neutralization assays. This is less of a problem for neutralization assays
using wild-type EBOV as the target virus but it highlights the need for careful
evaluation of assay specificity as part of assay validation.
Although not well established, there is evidence supporting the
importance of T cell-mediated responses in preventing EVD. In a study of
an Ad5-vectored Ebola vaccine in non-human primates, depletion of CD8+
T cells in vaccinated animals before challenge abrogated protection (111).
Several different types of tests for cell-mediated immunity have been developed,
including the ELISpot and ICS tests. These tests present additional challenges,
including determination of the appropriate peptide pools to be used and
logistical and safety issues concerning the collection and storage of peripheral
blood mononuclear cells, as well as assay validation issues.
In general, there are few published data on the performance of assays to
detect immunological responses to EBOV infection or to Ebola vaccines. Where
available, international standards or reference reagents (see section A.1.1) should
be used to standardize assay performance, and improve comparison of results
across vaccines, across studies and across different assay platforms.
C.7 Special populations

Ideally, developers of candidate Ebola vaccines will perform studies to gather
data in at least some, if not all, of the relevant populations discussed below.
C.7.1 Pregnant women

Evidence from the 2014–2016 EVD epidemic suggests that EVD is associated
with high rates of maternal and neonatal mortality (112). The use of Ebola
vaccine in pregnant women may have potential benefits in: (a) preventing EVD
in the mother and reducing maternal morbidity; (b) preventing EVD in the
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early neonatal period; and (c) limiting the spread of EVD from pregnant women
during labour and delivery to health-care workers in an outbreak setting (113).
The following concepts should be considered when planning clinical
trials in pregnant women. Details regarding such trials should be discussed
with the respective NRA(s) and can also be found in the WHO Guidelines on
clinical evaluation of vaccines: regulatory expectations (22). Prior to enrolling
pregnant women in clinical trials, developmental toxicity studies in animal
models are needed to address the potential reproductive risk of the product
(see section B.4). In addition, supportive safety data from completed Phase
I and Phase II clinical trials in healthy men and non-pregnant women should
be available. The consent form should include information on what is known
and unknown regarding the potential risks and benefits of the investigational
product to both mother and infant, and should reflect available data from non-
pregnant adults and nonclinical studies. A reasonable effort should be made to
accurately calculate gestational age for pregnant participants prior to enrolment,
taking into consideration the standard of care in the region where the clinical
trial is being conducted. For studies of preventive vaccines in general (including
Ebola vaccines), consideration should be given, as part of a cautious approach, to
excluding women in the first trimester of pregnancy.
Safety data specific to both the pregnant mother and her fetus should
be collected. Information on pregnancy-related outcomes (such as spontaneous
abortion or intrauterine growth restriction) and on pregnancy-related
complications (such as new-onset gestational diabetes or placenta previa)
should be collected. In addition, severity scales used for the grading of adverse
outcomes should be based on pregnancy-specific physiological and laboratory
values, if available. Efforts should be made to monitor infants for developmental
abnormalities.
Paediatric populations
C.7.2
A paediatric clinical development plan for a vaccine to protect against EVD
should be considered early (prior to Phase III) and should take into account
the incidence and prevalence of EVD, as well as existing therapies, in the
paediatric population, including neonates. In general, enrolment of children in
Ebola vaccine studies should be considered when there is sufficient evidence
to support the safety of studies in the paediatric population and there is
a reasonable demonstration of a sufficient prospect of direct benefit from
animal and/or human adult studies to justify the risks. Scientific and ethical
considerations regarding the initiation of paediatric studies of Ebola vaccines
should be discussed with the relevant NRA early in clinical development.
Available preclinical data and clinical data in older age groups should support
the paediatric dose and regimen to be evaluated, and should guide decisions on
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the potential need for incremental evaluation in older paediatric groups first,
followed by younger children and possibly infants. Safety considerations will be
critical when deciding upon the potential study of Ebola vaccines based on live,
replication-competent viral vectors in infants younger than 1 year of age.
Whether evidence of effectiveness can be extrapolated from adults to
specific paediatric age groups or from older to younger paediatric age groups
will depend on the similarities between the relevant age groups with respect to
factors such as the course of the disease and the immune response to vaccination.
Consideration may also be given to bridging effectiveness from older to younger
populations on the basis of a comparison of immune responses, as measured
by a validated assay using an immune marker that is thought to predict clinical
benefit. In some cases, immunological markers that are thought to contribute
to protection may be used to bridge across age groups even if they are not
scientifically well-established correlates of protection.
If the adult formulation of a vaccine is not suitable for certain paediatric
age groups (for example, due to the large dose volume), sponsors should plan for
the development of an age-appropriate paediatric formulation.
In paediatric studies, grading scales for adverse events and normal ranges
for laboratory tests should be specifically tailored to the age group studied.
C.7.3 Immunocompromised individuals and

individuals with underlying disease
Countries that have experienced prior Ebola outbreaks frequently have a
relatively high prevalence of concomitant illnesses or conditions such as HIV/
AIDS, tuberculosis, malaria and malnutrition. This prompts a number of unique
considerations with respect to clinical development programmes for Ebola
vaccines. Information on underlying medical conditions that may have an impact
on the safety and effectiveness evaluations of a vaccine should be collected for
participants in clinical trials.
The safety evaluation of investigational vaccines in immunocompromised
individuals should include assessment of exacerbation of the underlying disease
post-vaccination. For example, plasma HIV viral load has been shown in some
studies, but not in others, to transiently increase following vaccination with
influenza and pneumococcal vaccines – though without established clinical
consequence. Product-specific considerations may preclude the use of some
vaccines in certain populations due to unacceptable risks (for example, risk of
disseminated disease following immunization of HIV-infected individuals with
BCG vaccine).
The effectiveness of an Ebola vaccine may differ in countries according
to the prevalence of certain underlying medical conditions. Thus, effectiveness
data should be obtained in the region where the vaccine is most likely to be used.
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C.8 Post-marketing surveillance

As part of preparing for marketing approval of any new Ebola vaccine,
pharmacovigilance plans specific to each vaccine should be developed.
Depending on the situation, these plans could be prepared/implemented by
vaccine manufacturers and public health authorities in the countries where
the vaccine will be used, or through cooperative efforts that could also include
participation by regulators, WHO and other institutions.
According to the ICH, a pharmacovigilance plan should be prepared
for any new vaccine (114). A first step towards the preparation of such a plan
is the “safety specification” which summarizes: (a) the important identified and
potential risks of the vaccine; and (b) the important missing information. The
safety specification should also describe the populations that are potentially at
risk for EVD (that is, the populations in which the vaccine will most likely be
used) and any outstanding safety questions which warrant further investigation.
The safety specification is intended to help industry, regulators and other
institutions involved in the process to identify any need for specific data
collection and to facilitate preparation of the pharmacovigilance plan (114). The
safety specification is usually prepared by the sponsor (the institution submitting
the vaccine for marketing authorization, which is usually, but not always, the
manufacturer) during the pre-marketing phase. For products of international
public health importance, such as Ebola vaccines, pharmacovigilance planning
would benefit from dialogue not only with regulators but also with public health
authorities, WHO and other institutions involved in the process.
In the case of vaccines for which no specific concerns have arisen, routine
pharmacovigilance should be sufficient for post-approval safety monitoring.
Nevertheless, for products with important identified risks, important potential
risks or important missing information (which may be the case with new
Ebola vaccines) the pharmacovigilance plan should consider appropriate risk-
management and risk-minimization activities to address these concerns (114).
The strategies proposed for the identification and investigation of vaccine

safety signals should be specified in the pharmacovigilance plan. These may
depend, in part, on decisions made regarding the use of the vaccine(s) during
epidemic and inter-epidemic periods. Specifically, pharmacovigilance activities
may need to be adapted to situations in which the vaccine is recommended
for: (a) well-defined and relatively small groups (for example, first responders,
health-care workers and/or specific groups at high risk such as the close contacts
of suspected cases); (b) large demographic groups (for example, all individuals
in a certain age range or the inhabitants of a specific geographical region); or
(c) the overall population of a country or region.
Ideally, the pharmacovigilance plan should permit the detection of new
safety signals (a role performed mainly by spontaneous or passive reporting
154
Annex 2
systems) and confirmation of the association between the suspected event(s)

and the vaccine being investigated (115, 116). Currently, no effective post-
marketing surveillance systems with clear protocols, tools and a mandate exist
in countries affected by the 2014–2016 EVD epidemic. Thus, enhanced capacity
for vaccine pharmacovigilance may be needed, in accordance with the WHO
Global vaccine safety blueprint (23). This blueprint defines the need for enhanced
capacity as follows:
Enhanced vaccine pharmacovigilance, at a minimum level, includes
improved data collection, in passive surveillance, towards higher data quality
and more complete data sets, but also improved collation, verification,
analysis and communication by building capacity for stimulated and active
surveillance. It also includes the ability to perform population-based studies
and appropriate epidemiologic studies testing hypotheses by assessing
relative and absolute risk ratios, when appropriate.
The document goes on to state that:
Spontaneous reporting systems are insufficient to enable rapid assessment
and adequate public health response to vaccine safety signals. Rapid
response to vaccine safety signals is required to identify those rare instances
where real adverse reactions occur, so that their impact can be minimized
as they emerge. Countries where an increased level of vaccine safety activity
is judged to be necessary are those where newly developed vaccines are
being introduced and in countries that manufacture and use prequalified
vaccines (23).
The WHO Global Advisory Committee on Vaccine Safety (GACVS)
has reviewed safety data from Phase I studies of two investigational Ebola
vaccines (117). The adverse event profiles from these studies provide useful
information for planning safety evaluations in further studies of these vaccines.
Pharmacovigilance plans for the introduction of Ebola vaccines should take
into account the observed safety profiles from clinical studies and should be
aligned with WHO guidance.
In summary, the implementation of an adequate pharmacovigilance plan
for the post-marketing evaluation of adverse events following the introduction
of Ebola vaccines requires a functioning spontaneous reporting system, active
surveillance systems and the ability to perform appropriate epidemiological
studies to further investigate any possible association between suspected event(s)
and the vaccine. Given existing limitations in countries that were affected by the
2014–2016 EVD epidemic, an enhanced capacity for pharmacovigilance may be
needed in some countries, and more than one active surveillance approach may
need to be implemented to achieve effective pharmacovigilance.
155
Part D. Guidelines for NRAs

D.1 General
The general recommendations for control laboratories given in the WHO
Guidelines for national authorities on quality assurance for biological products
(118) and WHO Guidelines for independent lot release of vaccines by regulatory
authorities (119) should apply after the vaccine product has been granted a
marketing authorization. These recommendations specify that no new biological
substance should be released until consistency of batch manufacturing and
quality has been established and demonstrated. The recommendations do not
apply to material for clinical trials.
The detailed production and control procedures, as well as any significant
changes in them that may affect the quality, safety and efficacy of viral-vectored
vaccines, should be discussed with and approved by the NRA.
The NRA may obtain the product-specific working reference from
the manufacturer to be used for lot release until the international or national
standard preparation is established.
Consistency of production has been recognized as an essential
component in the quality assurance of vaccines. In particular, during review
of the marketing authorization dossier, the NRA should carefully monitor
production records and quality control test results for clinical lots, as well as for
a series of consecutive lots of the vaccine, produced using the procedures and
control methods that will be used for the marketed vaccine.
D.2 Release and certification

A vaccine lot should be released to the market only if it fulfils all national
requirements and/or satisfies Part A of these WHO Guidelines (119). A protocol
for the manufacturing and control of Ebola vaccines, based on the model
protocol provided in Appendix 1 and signed by the responsible official of the

manufacturing establishment, should be prepared and submitted to the NRA in
support of a request for the release of a vaccine for use.
A Lot Release Certificate signed by the appropriate NRA official
should then be provided if requested by a manufacturing establishment, and
should certify whether or not the lot of vaccine in question meets all national
requirements, as well as Part A of these WHO Guidelines. The purpose of
this official national release certificate is to facilitate the exchange of vaccines
between countries, and should be provided to importers of the vaccines. A model
NRA Lot Release Certificate is provided below in Appendix 2.
156
Annex 2
Authors and acknowledgements

The first and second drafts of these WHO Guidelines were prepared by a WHO
Drafting Group comprising Dr E. Griffiths, Consultant, Kingston-upon-Thames,
the United Kingdom; Dr M. Gruber, United States Food and Drug Administration,
Center for Biologics Evaluation and Research, the USA; Dr Y. Sun, Paul-Ehrlich-
Institut, Germany; Dr M. Udell, Medicines and Healthcare Products Regulatory
Agency, the United Kingdom; and Dr I. Knezevic, Dr D. Wood and Dr T.Q. Zhou,
World Health Organization, Switzerland. Acknowledgment is also due to Dr R.
Sheets, Consultant, Silver Spring (MD), the USA for conducting a review of the
current state of development of Ebola vaccines.
The third draft was reviewed at a WHO informal consultation on
regulatory considerations for evaluation of Ebola vaccines intended for
emergency use held in Geneva, Switzerland, 18–19 March 2015 and attended
by: Dr E. Abwao, Pharmacy and Poisons Board, Kenya; Dr M. Cavaleri, European
Medicines Agency, the United Kingdom; Dr G. Coleman, Health Canada,
Canada; Dr J. Djonova, Swissmedic, Switzerland; Mr D. Etuko, National Drug
Authority, Uganda; Dr E. Griffiths, Consultant, Kingston-upon-Thames, the
United Kingdom; Dr J.L. Goodman, Georgetown University, the USA; Dr M.
Gruber, United States Food and Drug Administration, Center for Biologics
Evaluation and Research, the USA; Mrs T. Jivapaisarnpong, Ministry of Public
Health, Thailand; Dr W. Johnson and Dr O. Abiri, Pharmacy Board of Sierra
Leone, Sierra Leone; Dr O. Kiselev, Russian Academy of Medical Sciences,
Russian Federation; Dr A. Marti, Swissmedic, Switzerland; Ms M. Malhame,
GAVI, the Vaccine Alliance, Switzerland; Dr P. Minor, National Institute for
Biological Standards and Control, the United Kingdom; Ms. K. Mwamwita and
Ms S. Mziray, Tanzania Food and Drugs Authority, United Republic of Tanzania;
Mrs P. Nkambule, Medicines Control Council, South Africa; Dr G. Osanjo,
University of Nairobi, Kenya; Dr M. Pfleiderer, Paul-Ehrlich-Institut, Germany;
Dr C. Perrin, Médecins Sans Frontières, France; Dr A. Rinfret, Health Canada,
Canada; Dr E. Sadove, United States Food and Drug Administration, the USA;
Dr T. Scharton-Kersten, International AIDS Vaccine Initiative, the USA; Dr R.
Sheets, Grimalkin Partners, the USA; Dr Y. Sun, Paul-Ehrlich-Institut, Germany;
Dr M. Udell, Medicines and Healthcare Products Regulatory Agency, the United
Kingdom; Ms W. Wei, Chinese Food and Drug Administration, China; Dr J.
Zhang, Military Academy of Medical Sciences, China; Dr X. Zhang, General
Logistic Ministry, China; Dr K. Zoon, National Institutes of Health, the USA;
and Dr C. Davis, Mr K. De Joncheere, Dr I. Knezevic, Dr O.C. Lapujade,
Mrs J. McMahon, Dr D. Meek, Ms C.A. Rodriguez-Hernandez, Dr D. Wood
and Dr T.Q. Zhou, World Health Organization, Switzerland; representatives of
the International Federation of Pharmaceutical Manufacturers & Associations
(IFPMA): Dr M.L. Dekleva, Merck, the USA; Mr A. Hacker, Janssen-Cilag Ltd,
157
the United Kingdom; Dr P. Peeters, Dr C. Saillez and Mr G. Liebaut, GSK

Vaccines, Belgium; Dr G. Preuveneers, Merck, Belgium; and Dr J. Sadoff, Johnson
& Johnson Crucell Holland B.V., Netherlands; representative of the Developing
Countries Vaccine Manufacturers Network (DCVMN): Dr N.R. Hegde (on behalf
of Bharat Biotech International Limited, India), India; representatives of other
companies: Ms A. Fix and Mr G. Glenn, Novavax, the USA; and Dr X. Yu, Tianjin
CanSino Biotechnology Inc., China;
On the basis of the comments received during the above WHO informal
consultation, the fourth and fifth drafts (the latter corresponding to document
WHO/BS/2015.2276) were prepared by the WHO Drafting Group comprising
Dr E. Griffiths, Consultant, Kingston-upon-Thames, the United Kingdom; Dr M.
Gruber, United States Food and Drug Administration, Center for Biologics
Evaluation and Research, the USA; Dr Y. Sun, Paul-Ehrlich-Institut, Germany;
Dr M. Udell, Medicines and Healthcare Products Regulatory Agency, the United
Kingdom; and Dr I. Knezevic, Dr D. Wood and Dr T.Q. Zhou, World Health
Organization, Switzerland. Acknowledgement is also due to Dr P. Minor, National
Institute for Biological Standards and Control, the United Kingdom and Dr M.
Nübling, World Health Organization, Switzerland for their inputs to the section
on international standards.
Acknowledgment is also given to the following experts who provided
written comments in response to the posting of document WHO/BS/2015.2276
on the WHO Biologicals website for the first round of public consultation
during August and September 2015: Dr A. Boyd, Dr D. Greene, Dr M. Gruber,
Dr S. Rubin, Dr C-L. Sun, Dr W. Sun and Dr D. Volokhov, United States Food
and Drug Administration, Center for Biologics Evaluation and Research,
the USA (comments coordinated by Dr G. Raychaudhuri); Dr G. Coleman,
Health Canada, Canada; Dr A. Hacker, Janssen-Cilag Ltd, the United Kingdom;
Dr N.R. Hegde (on behalf of Bharat Biotech International Limited, India), India;
Dr P. Minor and Dr D. Wilkinson, National Institute for Biological Standards
and Control, the United Kingdom; Dr C.V. Munoz, European Centre for
Disease Prevention and Control, Sweden; Dr J. Russell, National Institutes of
Health, the USA; Dr R. Sheets, Grimalkin Partners, the USA; Dr A.K. Tahlan
and Mr S. Kumar, Central Research Institute, India; M. Xydia-Charmanta and
Dr J. Wolf, Merck & Co. Inc., the USA (respectively coordinated and provided
the consolidated comments of the IFPMA); Dr K. Zoon, National Institutes of
Health, the USA; and Dr M.R. Balakrishnan, Dr C. Maure and Dr P.L. Zuber,
World Health Organization, Switzerland.
The document WHO/BS/2015.2276 was subsequently reviewed at the
meeting of the WHO Expert Committee on Biological Standardization held in
Geneva, Switzerland, 12–16 October 2015. A sixth draft document was then
prepared by the above WHO Drafting Group, taking into account comments
received during both the previous public consultation and the meeting of the
158
Annex 2
WHO Expert Committee. The resulting document was then posted on the WHO
Biologicals website for a second round of public consultation from January to
March 2016. Acknowledgments are due to the following experts who provided
written comments: Dr G. Coleman, Health Canada, Canada; Dr A.G. Fula
Argüello, Dr M.A. Alarcón Mora and Dr J.A. García Cortés, Instituto Nacional
de Vigilancia de Medicamentos y Alimentos; Dr A. Hacker, Janssen-Cilag
Ltd, the United Kingdom; Dr G. Raychaudhuri, United States Food and Drug
Administration, Center for Biologics Evaluation and Research, the USA; Dr A.
Rinfret, Health Canada, Canada; Dr J. Wang, National Institutes for Food and
Drug Control, China; Dr J. Wolf, Merck & Co., Inc., the USA (provided the
consolidated comments of the IFPMA); and Dr M. Nübling and Dr P.L. Zuber,
Acknowledgments are also due to the following participants in a WHO
working group meeting on guidelines on the quality, safety and efficacy of Ebola
vaccines held in Geneva, Switzerland, 4–5 May 2016: Dr E. Griffiths, Consultant,
Kingston-upon-Thames, the United Kingdom; Dr M. Gruber, United States
Food and Drug Administration, Center for Biologics Evaluation and Research,
the USA; Dr Y. Sun, Paul-Ehrlich-Institut, Germany; Dr M. Udell, Medicines
and Healthcare Products Regulatory Agency, the United Kingdom; Dr G.
Coleman, Health Canada, Canada; Dr M. Darko, Food and Drugs Authority,
Ghana; Mr D. Etuko, National Drug Authority, Uganda; Dr S. Kennedy,
University of Liberia, Liberia; Dr J. McEwen, Canberra, Australia; Dr P. Minor,
National Institute for Biological Standards and Control, the United Kingdom;
Dr R. Sheets, Consultant, Grimalkin Partners, Silver Spring (MD), the USA;
Dr J. Southern, Medicines Control Council of South Africa, South Africa; Dr K.
Zoon, National Institutes of Health, the USA; and Dr M. Friede, Dr I. Knezevic,
Dr O.C. Lapujade, Dr D. Wood, Dr T.Q. Zhou and Dr P.L. Zuber, World Health
Organization, Switzerland.
A further revised version (WHO/BS/2016.2279) was then prepared by
the above WHO Drafting Group, taking into account comments received from
the above consultations.
Acknowledgments are due to the following experts who provided written
comments in response to the posting of document WHO/BS/2016.2279 on the
WHO Biologicals website between July and September 2016 for a third round of
public consultation: Prof. S. Efstathiou, National Institute for Biological Standards
and Control, the United Kingdom; Dr A. Hacker, Janssen-Cilag Ltd, the United
Kingdom; Dr H. Mao and Dr X. Yu, CanSino Biotechnology Inc., China; Dr G.
Raychaudhuri, United States Food and Drug Administration, Center for Biologics
Evaluation and Research, the USA (provided the consolidated comments of the
Center for Biologics Evaluation and Research); Dr A. Rinfret, Health Canada,
Canada; and Dr J. Wolf, Merck & Co., Inc., the USA (provided the consolidated
comments of the IFPMA).
159
The document WHO/BS/2016.2279 was subsequently reviewed at the

meeting of the WHO Expert Committee on Biological Standardization held in
Geneva, Switzerland, 17–21 October 2016. A further revised version (WHO/
BS/2017.2327) was then prepared by the WHO Drafting Group, taking into
account comments received during the meeting of the WHO Expert Committee
and from: Prof. P. Smith, London School of Hygiene & Tropical Medicine, the
United Kingdom; and Dr M-P. Kieny, Dr M. Friede and Dr A.M. Henao Restrepo,
Acknowledgments are also due to the following experts who provided
written comments in response to the posting of document WHO/BS/2017.2327
on the WHO Biologicals website during August and September 2017 for a fourth
round of public consultation: Dr G. Coleman, Health Canada, Canada; Dr I.K.
Damon, Dr R. Carter and Dr B. Mahon, United States Centers for Disease
Control and Prevention, the USA; Dr G. Enwere, World Health Organization,
Switzerland; Dr A. Rinfret, Health Canada, Canada; Prof. P. Smith, London
School of Hygiene & Tropical Medicine, the United Kingdom; Dr J. Southern,
Advisor, Simon’s Town, South Africa; and Dr J. Wolf, Merck & Co., Inc., the USA
(on behalf of the IFPMA).
Acknowledgements are also due to Dr K. Farizo, Dr D. Pratt, Dr H.
Izurietta and Dr S. Rubin, Center for Biologics Evaluation and Research, the
USA who provided direct inputs during the development of the series of draft
guidelines.
Further changes were subsequently made to document WHO/BS/
2017.2327 by the WHO Expert Committee on Biological Standardization.
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Appendix 1
Model protocol for the manufacturing and control of
viral-vectored Ebola vaccines
The following provisional protocol is intended for guidance. It indicates the
information that should be provided as a minimum by the manufacturer to
the NRA after the vaccine product has been granted a marketing authorization.
The protocol is not intended to apply to material intended for clinical trials.
Since the development of these vaccines is incomplete at the time of
writing this document, detailed requirements are not yet finalized. Consequently
only the essential requirements are provided in this appendix. Information and
tests may be added or omitted (if adequate justification is provided) as necessary
to be in line with the marketing authorization approved by the NRA. It is therefore
possible that a protocol for a specific product will differ from the model provided
here. The essential point is that all relevant details demonstrating compliance
with the licence and with the relevant WHO Guidelines on a particular product
should be given in the protocol submitted.
The section concerning the final product should be accompanied by
a sample of the label and a copy of the leaflet that accompanies the vaccine
container. If the protocol is submitted in support of a request to permit
importation, it should also be accompanied by a Lot Release Certificate from the
NRA of the country in which the vaccine was produced and/or released stating
that the product meets national requirements as well as Part A of these WHO
Guidelines.
1. Summary information on finished product (final vaccine lot)

International name:
Commercial name:
Product licence (marketing authorization) number:
Country:
Name and address of manufacturer:
Name and address of product licence-holder if different:
Viral vector(s):
Ebola virus strain(s):
Batch number(s):
Type of container:
Number of filled containers in this final lot:
Number of doses per container:
Composition (viral vector concentration)/volume of single human dose:
171
Target group:
Expiry date:
Storage conditions:
2. Control of source material

2.1 Virus seeds (repeat for each monovalent vaccine component)
2.1.1 Seed banking system
■■ Name and identification of viral vector:
■■ Origin of all genetic components:
■■ Construction of viral vector:
■■ Nucleotide sequence of the transgene and flanking regions:
■■ Antigenic analysis, infectivity titre, in vitro yield:
■■ Comparison of genetic and phenotypic properties with parental
vector:
■■ Seed bank genealogy with dates of preparation, passage number and
date of coming into operation:
■■ Tests performed for detection of adventitious agents at all stages of
development:
■■ Freedom from TSE agents:
■■ Details of animal or human components of any reagents used in the
manufacture of seed banks, including culture medium:
■■ Genetic stability at the level of a virus pre-master seed or virus
master seed to its sequence at, or preferably beyond, the anticipated
maximum passage level:
■■ Confirmation of approval for use by manufacturer, and the basis for
that approval:
2.2 Cell cultures (if applicable) (repeat for each

monovalent vaccine component)
2.2.1 Cell banking system
■■ Name and identification of cell substrate:
■■ Origin and history of cell substrate:
■■ Details of any manipulations (including genetic manipulations)
performed on the parental cell line in the preparation of the
production cell line:
■■ Cell bank genealogy with dates of preparation, passage number and
date of coming into operation:
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■■ Confirmation of approval for use by manufacturer, and the basis for

that approval:
■■ Tests performed for detection of adventitious agents at all stages of
development:
■■ Test for tumorigenic potential (if of mammalian origin):
■■ Details of animal or human components of any reagents used in
manufacture of cell banks, including culture medium:
■■ Genetic stability (if genetically manipulated):
2.2.2 Primary cells (if generated)

■■ Source of animals and veterinary control (for example, specify if
animals or eggs are sourced from closed, pathogen-free colonies):
■■ Name, species and identification of primary cell batches:
■■ Details of animal or human components of any reagents used in
manufacture of cells:
■■ Methods of isolation of the cells:
■■ Tests performed for detection of adventitious agents during
manufacture (may be performed on control cells if necessary):
3. Control of vaccine production (repeat for each

monovalent vaccine component)
3.1 Control of production cell cultures/control cells
3.1.1 Information on preparation
■■ Lot number of master cell bank:
■■ Lot number of working cell bank:
■■ Date of thawing ampoule of working cell bank:
■■ Passage number of production cells:
■■ Date of preparation of control cell cultures:
■■ Result of microscopic examination:
3.1.2 Tests on cell cultures or control cells

■■ Adventitious agents:
■■ Sterility (bacteria, fungi, mycoplasmas):
173
3.2 Viral vector harvests or pooled viral vector harvests

3.2.1 Information on manufacture
■■ Batch number(s):
■■ Date of inoculation:
■■ Date of harvesting:
■■ Lot number of virus master seed lot:
■■ Lot number of virus working seed lot:
■■ Passage level from virus working seed lot:
■■ Methods, date of purification if relevant:
■■ Volume(s), storage temperature, storage time and approved storage
period:
3.2.2 Tests
■■ Adventitious virus tests:
■■ Bacteria/fungi/mycoplasmas:
■■ Virus titre:
3.3 Monovalent viral vector bulk

■■ Date of formulation:
■■ Total volume of monovalent bulk formulated:
■■ Virus pools used for formulation:
■■ Lot number/volume added:
■■ Virus concentration:
■■ Name and concentration of added substances (for example, diluent,
stabilizer if relevant):
period:
3.3.2 Tests
■■ Identity:
■■ Purity:
■■ Residual HCP:
■■ Residual HC DNA (if non-primary cell lines):
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■■ Potency:
–– Particle number (for adenovirus):
–– Infectious virus titre:
–– Particle-to-infectivity ratio (for adenovirus):
–– Expression of heterologous antigen in vitro:
■■ Replication competence (for adenovirus):
■■ pH:
■■ Preservative content (if applicable):
■■ Endotoxin:
■■ Sterility or bioburden:
3.4 Final viral vector bulk

■■ Date of formulation:
■■ Total volume of final bulk formulated:
■■ Monovalent virus pools used for formulation:
■■ Lot number/volume added:
■■ Virus concentration:
■■ Name and concentration of added substances (for example, diluent,
stabilizer if relevant):
period:
3.4.2 Tests
■■ Identity:
■■ Sterility or bioburden:
■■ Concentration of antimicrobial agent, if relevant:
4. Filling and containers

Lot number:
Date of filling:
Type of container:
Volume of final bulk filled:
Filling volume per container:
Number of containers filled (gross):
175
Number of containers rejected during inspection:

Number of containers sampled:
Total number of containers (net):
Maximum period of storage approved:
Storage temperature and period:
5. Control tests on final vaccine lot

Inspection of containers (that is, inspection container integrity):
Appearance (that is, appearance of container content):
Identity:
pH and osmolality:
Potency (if feasible to measure in a multivalent system):
■■ Particle number (adenovirus):
■■ Infectious virus titre:
■■ Particle-to-infectivity ratio (for adenovirus):
■■ Expression of heterologous antigen in vitro:
General safety tests (initial batches only):
Endotoxin:
Sterility:
Extractable volume:
Aggregate/particle size:
Presence of preservative (if relevant):
Residual moisture content (for freeze-dried product):
Reconstitution time (for freeze-dried product):
6. Certification by the manufacturer

Name of Head of Production (typed)

Certification by the person from the control laboratory of the manufacturing
company taking overall responsibility for the production and control of the vaccine.
I certify that lot no. of Ebola vaccine, whose number
appears on the label of the final containers, meets all national requirements and
satisfies Part A1 of the WHO Guidelines on the quality, safety and efficacy of
Ebola vaccines 2 (if applicable)
1
With the exception of provisions on distribution and shipping, which the NRA may not be in a position
to assess.
2
WHO Technical Report Series, No. 1011, Annex 4.
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Annex 2
Name (typed)
Signature
Date
7. Certification by the NRA

If the vaccine is to be exported, attach the NRA Lot Release Certificate (as shown
in Appendix 2), a label from a final container and an instruction leaflet for users.
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Appendix 2
Model NRA Lot Release Certificate for viral-vectored
Ebola vaccines
This certificate is to be provided by the NRA of the country where the vaccine
has been manufactured, on request by the manufacturer.
Certificate no.
The following lot(s) of Ebola vaccine produced by 1
in 2
whose lot numbers appear on the labels of the
final containers, complies with the relevant specification in the marketing
authorization and provisions for the release of biological products 3 and Part A4
of the WHO Guidelines on the quality, safety and efficacy of Ebola vaccines 5 and
comply with WHO good manufacturing practices for pharmaceutical products:
main principles,6 WHO good manufacturing practices for biological products,7
and Guidelines for independent lot release of vaccines by regulatory authorities.8
The release decision is based on 9
The certificate may include the following information:

■■ name and address of manufacturer;
■■ site(s) of manufacturing;
■■ trade name and common name of product;
■■ marketing authorization number;
■■ lot number(s) (including sub-lot numbers and packaging lot
numbers if necessary);
1
Name of manufacturer.
2
Country of origin.
3
If any national requirements are not met, specify which one(s) and indicate why release of the lot(s) has
nevertheless been authorized by the NRA.
4
With the exception of provisions on distribution and shipping, which the NRA may not be in a position
to assess.
5
6
7
8
9
Evaluation of product-specific summary protocol, independent laboratory testing, and/or specific
procedures laid down in a defined document, etc., as appropriate.
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Annex 2
■■ type of container used;

■■ number of doses per container;
■■ number of containers or lot size;
■■ date of start of period of validity (for example, manufacturing date)
and expiry date;
■■ storage conditions;
■■ signature and function of the person authorized to issue the
certificate;
■■ date of issue of certificate;
■■ certificate number.
The Director of the NRA (or other authority as appropriate):
Name (typed)
Signature
Date
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Guidelines on procedures and data requirements for
changes to approved biotherapeutic products
1. Introduction 184
2. Purpose and scope 185
3. Terminology 186
4. General considerations 191
5. Special considerations 193
5.1 Comparability exercise 193
5.2 Bridging studies 194
5.3 Similar biotherapeutic products 194
6. Reporting categories for quality changes 195
6.1 Major quality changes 196
6.2 Moderate quality changes 196
6.3 Minor quality changes 197
6.4 Quality changes with no impact 197
7. Reporting categories for safety, efficacy and/or product labelling
information changes 198
7.1 Safety and efficacy changes 199
7.2 Product labelling information changes 200
7.3 Urgent product labelling information changes 201
7.4 Administrative product labelling information changes 201
8. Procedures 202
8.1 Procedures for prior approval supplements 206
8.2 Procedures for minor quality changes and quality changes with no impact 209
8.3 Procedures for urgent product labelling
information changes 210
8.4 Procedures for administrative product labelling information changes 211
9. Authors and acknowledgements 211
10. References 214
Appendix 1 Reporting categories and suggested review timelines 216
Appendix 2 Changes to the drug substance 219
Appendix 3 Changes to the drug product 247
Appendix 4 Safety, efficacy and product labelling information changes 276
181
Guidelines published by the World Health Organization (WHO) are

intended to be scientific and advisory in nature. Each of the following
sections constitutes guidance for national regulatory authorities
(NRAs) and for manufacturers of biological products. If an NRA so
desires, these WHO Guidelines may be adopted as definitive national
requirements, or modifications may be justified and made by the NRA.
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Abbreviations
ALIFAR Asociación Latinoamericana de Industrias Farmacéuticas
BSE bovine spongiform encephalopathy
GCP good clinical practice
HPLC high-performance liquid chromatography
HSA Health Sciences Authority
ICH International Conference on Harmonisation of Technical
Requirements for Registration of Pharmaceuticals for
Human Use
& Associations
IGBA International Generic and Biosimilar Medicines Association
IQ installation qualification
OQ operational qualification
PAS prior approval supplement
PDA Parenteral Drug Association
PK/PD pharmacokinetic/pharmacodynamic
PPTA Plasma Protein Therapeutics Association
PQ performance qualification
SBP similar biotherapeutic product
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1. Introduction
Biotherapeutic products are an increasingly important component of global
health care. Several WHO guidelines on the evaluation of biotherapeutic
products have been produced (1–3) that provide a set of principles on the
regulatory evaluation of such products. During international consultations on
the development of these guidelines, and their subsequent implementation, it
became clear that there was a need for WHO guidance on making post-approval
changes to biotherapeutic products in order to help address the complexity
and other challenges associated with the global life-cycle management of such
products. In May 2014, the Sixty-seventh World Health Assembly adopted
two relevant resolutions: one on promoting access to biotherapeutic products
and ensuring their quality, safety and efficacy (4) and the other on regulatory
systems strengthening (5). In support of these resolutions, WHO was requested
to provide guidance on how to deal with increasingly complex biotherapeutic
products, including similar biotherapeutic products (SBPs). In addition, the 16th
International Conference of Drug Regulatory Authorities recommended that
WHO assist Member States in ensuring regulatory oversight throughout the life-
cycle of biotherapeutic products (6).
This document is intended to provide guidance to national regulatory
authorities (NRAs) and manufacturers on regulating changes to already licensed
biotherapeutic products in order to assure their continued quality, safety and
efficacy, as well as continuity in supply and access. The term “biotherapeutic
products” as used in this document collectively includes the originator products
and SBPs (also called “biosimilars”).
Changes are essential for the continual improvement of the manufacturing
process and for maintaining state-of-the-art control of biotherapeutic products,
and often need to be implemented after the product has been approved (that is,
when it has been licensed or when marketing authorization has been received).
Changes may be made for a variety of reasons, including: (a) to maintain routine
production (for example, replenishment of reference standards, or change of raw
materials); (b) to improve product quality, or the efficiency and consistency of
manufacture (for example, changes in the manufacturing process, equipment or
facility, or adding a new manufacturing site); (c) to make safety or efficacy changes
(for example, adding a new indication, changing the dosage regimen, or adding
information on co-administration with other medicines); (d) to update product
labelling information (for example, improvement of the management of risk by
addition of a warning statement for a particular target population, or limiting the
target population); or (e) to address administrative changes (for example, change
in the proper/nonproprietary or trade name of a biotherapeutic product).
NRAs and marketing authorization holders should recognize that:
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■■ any change to a biotherapeutic product has a potential impact on

the quality, safety and/or efficacy of that product;
■■ any change to the information associated with the product (that is,
product labelling information) may have an impact on its safe and
effective use.
The regulation of changes to approved biotherapeutic products is key
to ensuring that products of consistent quality, safety and efficacy are marketed
after they receive authorization or licensure. Many NRAs of Member States
have requested guidance on the data needed to support changes to approved
biotherapeutic products in order to ensure comparability of the pre-change
and post-change products with respect to quality, safety and efficacy. Although
it is difficult to provide a set of guidelines that apply to all national situations,
an attempt has been made to cover a range of possible changes in manufacture,
quality control, safety, efficacy and product labelling information.
This document is intended to serve as a guide for establishing national
requirements for the regulation of post-approval changes to biotherapeutic
products. The categories of changes and reporting procedures are provided in the
main body of the document and the data requirements to support the proposed
changes are provided in the appendices. If an NRA so desires, these WHO
Guidelines may be adopted as definitive national requirements. It is possible that
modifications to this document may be justified due to risk–benefit and legal
considerations specific to each NRA. In such cases, it is recommended that any
modifications should not depart from the principles outlined in this document.
NRAs are encouraged to apply the concepts of reliance or work-sharing or to use
collaborative approaches when reviewing post-approval changes, as indicated in
section 8 below.
2. Purpose and scope

These WHO Guidelines provide guidance for NRAs and marketing authorization
holders on the regulation of changes to the original marketing authorization
dossier or product licence for an approved biotherapeutic product in terms
of: (a) the procedures and criteria for the appropriate categorization and
reporting of changes; and (b) the data required to enable NRAs to evaluate
the potential impact of the change on the quality, safety and efficacy of the
product. Additionally, the purpose of these WHO Guidelines is to assist NRAs in
establishing regulatory procedures for post-approval changes to such products.
The guidance applies in principle to all biologically active protein
products used in the treatment of human diseases (for example, plasma-
fractionated products) and those intentionally modified by, for example, fusion
185
proteins, PEGylation, conjugation with a cytotoxic drug or modification of

rDNA sequences. The guidance also applies to protein products used for in vivo
diagnosis (for example, monoclonal antibody products used for imaging).
While these WHO Guidelines apply to products that have received a
licence or a marketing authorization, the principles described herein may also
apply to quality changes that occur during development of a product and where
comparability needs to be demonstrated. However, the amount and type of data
submitted for such products will be limited and will vary according to the nature
of each product and its stage of development. In addition, the legal status of
investigational products varies from country to country and should therefore be
discussed with the NRA.
Prophylactic vaccines against infectious diseases, and gene and cell
therapy products, are not covered by these WHO Guidelines. Detailed and
specific guidance for prophylactic vaccines are available in a separate WHO
Guidelines document (7). However, the principles set out in this document may
apply to low molecular weight heparins. Other WHO guidelines with relevance
to this area include those covering good manufacturing practices (GMP) for
biological and pharmaceutical products (8, 9).
3. Terminology
The definitions given below apply to the terms used in these WHO Guidelines.
They may have different meanings in other contexts.
Acceptance criteria: criteria, expressed by numerical limits, ranges or
other suitable measures, which should be met to release the drug substance or
drug product or materials at different stages of their manufacture.
Biotherapeutic product: a biological medicinal product with the
indication of treating human disease. For the purpose of these WHO Guidelines,
biotherapeutic products include all biologically active protein products

(including plasma-fractionated products) which are used in the treatment
of human diseases, and those intentionally modified by, for example, fusion
proteins, PEGylation, conjugation with a cytotoxic drug or modification of
rDNA sequences. They also include protein products used for in vivo diagnosis
(for example, monoclonal antibody products used for imaging).
Change: refers to a change that includes, but is not limited to, the
product composition, manufacturing process, quality controls, analytical
methods, equipment, facilities or product labelling information made to an
approved marketing authorization or licence by the marketing authorization
holder. Also referred to as “variations” or “post-notice of compliance changes”
in other documents (10–14).
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Comparability exercise: the activities – including study design,

conducting of studies and evaluation of data – that are designed to investigate
whether a pre-change product and a post-change product are highly similar (1).
Comparability protocol: a well-defined plan for future implementation
of quality change(s) (for example, manufacturing-related changes, change of
analytical method or site transfer). Also referred to as “post-approval change
management protocol” in other documents (15). A comparability protocol
establishes the tests to be performed and acceptable limits to be achieved to
demonstrate the comparability of pre-change and post-change products following
specific quality change(s).
Container closure system: refers to the following components:
■■ A primary container closure system is a packaging component
that is in, or may come into, direct contact with the drug product
dosage form (for example, vial or pre-filled syringe) or components
that contribute to the container/closure integrity of the primary
packaging material for a sterile product.
■■ A secondary container closure system is a packaging component
that is not, and will not be, in direct contact with the dosage form
(for example, carton or tray).
■■ A functional secondary container closure system is a packaging
material that is not in direct contact with the product and that
provides additional protection or serves to deliver the product.
Control strategy: a planned set of controls derived from current product
and process understanding that ensures process performance and product
quality. The controls can include parameters and attributes related to drug
substance and drug product materials and components, facility and equipment
operating conditions, in-process controls, finished product specifications, and
the associated methods and frequency of monitoring and control (16).
Critical quality attribute: a physical, chemical, biological or
microbiological property or characteristic that is selected for its ability to
indicate the consistent quality of the product within an appropriate limit, range
or distribution to ensure the desired product quality (1).
Design space: the multidimensional combination and interaction of
input variables (for example, material attributes) and process parameters that
have been demonstrated to provide assurance of quality (16).
Dosage form: the physical form in which a pharmaceutical product is
presented by the manufacturer (form of presentation) and the form in which it
is administered (form of administration). Also referred to as “pharmaceutical
form” in other documents.
187
Drug product: a pharmaceutical product type in a defined container

closure system that contains a drug substance, generally in association with
excipients.
Drug substance: the active pharmaceutical ingredient and associated
molecules that may be subsequently formulated to produce the drug product.
Excipient: any component of the drug product, other than the active
component/drug substance and the packaging material, generally added during
formulation. Also referred to as “inactive ingredient” in other documents.
Final batch: a collection of sealed final containers that is homogeneous
with respect to the composition of the product. A final batch must have been
filled in one continuous working session.
Formulated bulk: an intermediate in the drug product manufacturing
process, consisting of the final formulation of drug substance and excipients at
the concentration to be filled into primary containers.
In-process control: checks performed during manufacture to monitor
or to adjust the process in order to ensure that the intermediate or final product
conforms to its specifications. The control of the production environment or
equipment may also be regarded as part of in-process control.
Intermediate: a material produced during steps in the manufacture of a
biotherapeutic product that undergoes further processing before it becomes the
drug product. See also the definition for Drug substance.
Manufacturer: any person or legal entity engaged in the manufacture
of a product subject to marketing authorization or licensure. In other
documents, “manufacturer” may also refer to any person or legal entity that
is an applicant or holder of a marketing authorization or product licence
where the applicant assumes responsibility for compliance with the applicable
product and establishment standards. See also the definition for Marketing
authorization holder.
Marketing authorization: a formal authorization for a medicine to be

marketed. Once an NRA approves a marketing authorization application for
a new medicine, the medicine may be marketed and may be available to be
prescribed by physicians. Also referred to as “product licence” or “licence” in
this and other documents.
Marketing authorization application: a formal application to the
NRA for approval to market a new medicine. The purpose of the marketing
authorization application is to determine whether the medicine meets the
statutory standards for safety, efficacy, product labelling information and
manufacturing. Also referred to as “product licence application” or “licence
application” in this and other documents.
Marketing authorization holder: any person or legal entity that has
received a marketing authorization or licence to manufacture and/or distribute
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a medicine. It also refers to a person or legal entity allowed to apply for a change
to the marketing authorization or licence.
Master cell bank (MCB): an aliquot of a single pool of cells which
generally has been prepared from the selected cell clone under defined conditions,
dispensed into multiple containers and stored under defined conditions.
Primary packaging site: site involved in the activity of putting a drug in
its primary container which is, or may be, in direct contact with the dosage form.
Process validation: documented evidence which provides a high degree
of assurance that a specific process will consistently result in a product that
meets its predetermined specifications and quality characteristics.
Product labelling information: refers to printed materials that
accompany a prescription medicine and all labelling items, namely:
■■ prescribing information (an instruction circular that provides
product information on indication, dosage and administration, safety
and efficacy, contraindications, warnings and a description of the
product for health-care providers (also referred to as “summary of
product characteristics” or “package insert” in various countries);
■■ patient labelling or consumer information;
■■ inner label or container label;
■■ outer label or carton.
Quality attribute: a physical, chemical, biological or microbiological
property or characteristic.
Quality change: a change in the manufacturing process, product
composition, quality control testing, equipment or facility. Also referred to as
“chemistry manufacturing and control (CMC) change” in other documents.
Raw materials: a general term used to denote the culture media
components, reagents or solvents intended for use in the production of starting
material, drug substance, intermediates or drug products.
Real-time release testing: testing that provides the ability to evaluate
and ensure the quality of in-process and/or final product based on process data,
which typically include a valid combination of measured material attributes and
process controls (16, 17).
Reference standards/materials: well-characterized materials used as
references against which batches of biological products are assessed. These
materials remain fundamental to ensuring the quality of biological products as
well as the consistency of production, and are essential for the establishment of
appropriate clinical dosing.
Safety and efficacy change: a change that has an impact on the clinical
use of the biotherapeutic product in relation to safety, efficacy, dosage and
189
administration, and that requires data from clinical or post-marketing studies,

and in some instances clinically relevant nonclinical studies, to support the
change.
Secondary packaging facility: site involved in packaging activities using
a packaging component that is not, and will not be, in direct contact with the
dosage form (for example, putting the primary container in the outer container
or affixing labels).
Shelf-life: the period of time during which a drug substance or drug
product, if stored under the conditions defined on the container label, is
expected to comply with the specification, as determined by stability studies on
a number of batches of the product. The expiry date is assigned to each batch by
adding the shelf-life period to the date of manufacture.
Similar biotherapeutic product (SBP): a biotherapeutic product that is
similar in terms of quality, safety and efficacy to an already licensed reference
biotherapeutic product, and which was developed and approved on the basis of
the principles outlined in relevant WHO guidelines (2, 3).
Source material/starting material: material from a biological source
that marks the beginning of the manufacturing process of a drug as described
in a marketing authorization or licence application and from which the active
ingredient is derived either directly (for example, plasma derivatives, ascitic fluid
or bovine lung) or indirectly (for example, cell substrates, host/vector production
cells, eggs or viral strains).
Specification: a list of tests, references to analytical procedures and
appropriate acceptance criteria which are numerical limits, ranges or other
criteria for the tests described. Specifications are critical quality standards that
are proposed and justified by the manufacturer and approved by the regulatory
authorities.
Supplement: a written request submitted to the NRA to approve a change
in the original application for the marketing authorization (or product licence)
or any other notification to add to (that is, to supplement) the information in
the original marketing authorization or product licence file. A prior approval
supplement (PAS) is a supplement requiring approval from the NRA prior to
implementation of the change. Also referred to as “change application dossier” in
other documents.
Validation: the demonstration, with documentary evidence, that any
procedure, process, equipment, material, activity or system will consistently
produce a result meeting predetermined acceptance criteria.
Working cell bank (WCB): the working cell bank is prepared from
aliquots of a homogeneous suspension of cells obtained from culturing the
master cell bank under defined culture conditions.
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4. General considerations
Changes to approved biotherapeutic products or SBPs are categorized on the
basis of a risk analysis which takes into consideration the complexity of the
production process and product, the patient population and the proposed
changes. When a change affects the manufacturing or the control strategy, the
assessment should include evaluation of the impact of the change on quality (that
is, identity, strength, purity and potency) as it may relate to the safety and/or
efficacy of the product. When a change affects the clinical use of a product or of
product labelling information, this assessment should include evaluation of the
effect of the change on the safety and efficacy of the product.
Prior to implementing a change with a potential impact on quality, the
marketing authorization holder should demonstrate through appropriate studies
(analytical testing, functional assays and, if needed, clinical and/or nonclinical
studies) that the pre-change and post-change products are comparable in terms
of quality, safety and efficacy.
For each change, the marketing authorization holder should decide if the
information in the original marketing authorization or product licence needs
to be supplemented (that is, requires an official submission of a supplement to
the NRA) based on the recommendations provided in these WHO Guidelines.
Supplements requiring approval by the NRA prior to the implementation of a
change – that is, for changes that potentially have a major or moderate impact
– are referred to as prior approval supplements (PASs) and must be submitted
in advance to the NRA. For supplements that do not require approval prior
to implementation – that is, for changes that potentially have a minor impact
on product quality – the NRA should be notified following implementation of
the change.
For each change, the supplement should contain information developed
by the marketing authorization holder to allow the NRA to assess the effects
of the change. All changes, regardless of their impact on quality, safety and
efficacy, should be recorded and retained by the manufacturer or marketing
authorization holder in accordance with the applicable regulatory requirements
for document retention (8, 9).
For manufacturing changes not specifically described in these WHO
Guidelines, the marketing authorization holder is encouraged to use scientific
judgement, leverage competent regulatory authority guidance or to contact the
NRA to determine the potential impact of the change on quality, safety and
efficacy in order to discuss the appropriate reporting category.
Assessment of the extent to which a quality change (also referred to as a
manufacturing change) affects the quality attributes of the product is generally
191
accomplished by comparing manufacturing steps and test results from in-

process, release, and characterization testing of the pre-change product (for
example, using historical data) with those of the post-change product. It can then
be determined if the test results are comparable – that is, if the drug substance,
intermediate or drug product made after the change is comparable to, and/or
meets the predefined acceptance criteria of, the drug substance or drug product
made before the change. Where minor differences in quality are identified,
these may be considered acceptable provided that they are shown not to have an
adverse impact on the quality, safety or efficacy of the product (see sections 5.1
and 5.2). In some cases, additional supporting data may be required, as noted in
Appendices 2, 3 and 4 below.
A marketing authorization holder or manufacturer making a change to
an approved biotherapeutic product should also conform to other applicable laws
and regulations, including good manufacturing practices (GMP), good laboratory
practices (GLP) and good clinical practices (GCPs). Marketing authorization
holders and drug substance/product manufacturers should also comply with
relevant GMP validation and record-keeping requirements and should ensure
that relevant records are readily available for examination by authorized NRA
personnel during inspections. For example, changes in equipment used in
the manufacturing process generally require installation qualifications (IQs),
operational qualifications (OQs) and performance qualifications (PQs). This
information does not need to be included in a PAS for equipment changes
but is part of GMP requirements and should be available during inspections.
Inspections (on-site or paper-based) may occur routinely or may be required
during submission review of a PAS for a major manufacturing change such as a
move to a new facility.
Certain major changes, such as changes to the molecule (for example,
changing amino acid sequence or conjugating to PEG moieties) will lead to a
new molecular entity and are not considered as post-approval changes. For
these changes, submission of a product licence application for a new marketing

authorization may be required. In some countries, a change in the quantity of
drug substance per dose of biotherapeutic product also requires a product
licence application for a new marketing authorization.
The implementation of new regulations for post-approval changes
should take product supply into consideration. Any negative impact on access
to approved products should be minimized. Therefore, NRAs are strongly
encouraged to establish requirements that are commensurate with their own
regulatory capacity, experience and resources. NRAs of countries procuring
products are encouraged to consider establishing procedures for the expedited
approval of changes based on previous expert review and approval of the same
changes by the NRAs of the countries where these products are licensed, or based
on the decision of a recognized regional regulatory authority. If a change has been
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approved by another competent NRA, the NRA receiving the submission may
choose to recognize this approval decision or may make an independent decision
based on its own assessment. Foreign approval documentation may accompany
the required information and may be used as supporting evidence for the post-
approval change, as outlined in this document. The responsibility for the final
regulatory decision on the approval of the change still lies with the receiving
NRA (see section 8 and Appendix 1).
To ensure product supply and encourage adequate reporting of changes
by manufacturers, NRAs should consider establishing procedures for the
concurrent (that is, parallel) review of changes to the product. The manufacturing
of biotherapeutic products requires, for example, the replenishment of biological
starting materials such as WCBs and secondary/working reference standards
which are considered as routine changes. Consequently, these changes often
need to be reviewed concurrently with other manufacturing or safety and efficacy
changes. Conversely, clinical safety and efficacy changes, such as the addition of a
new indication or new age group for the use of a biotherapeutic product, require
considerable supporting data including clinical studies; thus, review time should
not impact the review of unrelated manufacturing changes or the immediate
implementation of urgent changes to product labelling information. However,
multiple related changes, or those supported by the same information, may be
submitted in the same supplement (see “Multiple changes” in section 8).
In these WHO Guidelines, descriptions of the reporting categories
for quality changes are provided in section 6, and the reporting categories for
information changes on safety, efficacy and product labelling are provided in
section 7. Proposed regulatory procedures for the reporting of changes to NRAs
are described in section 8. Examples of suggested review timelines for changes in
the various categories are given in Appendix 1. A comprehensive list of quality
changes and the type of information that should be included in a supplement
application are provided in Appendix 2 (for the drug substance and intermediates)
and in Appendix 3 (for the drug product). Examples of changes that affect clinical
use of a product and product labelling information (on safety, efficacy, dosage,
administration and product components) are provided in Appendix 4.
5. Special considerations
5.1 Comparability exercise
The need for – and extent of – a comparability exercise depends upon the
potential impact of the change(s) on the quality, safety and efficacy of the
product. Comparability exercises can range from analytical testing alone (for
example, where process changes have no impact on any quality attribute) to a
comprehensive exercise requiring nonclinical and clinical bridging studies. For
example, a change in the culture conditions or in the purification process may
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cause the alteration of the glycosylation profile of the product, including site-
directed glycosylation. Alteration of glycosylation profiles may cause a change
in the pharmacokinetic/pharmacodynamic (PK/PD) profile of the product (see
also section 5.2 on “Bridging studies”). If comparability can be demonstrated
through analytical studies alone, nonclinical or clinical studies with the post-
change product are not necessary. However, where the relationship between
specific quality attributes and safety and efficacy has not been established, and/
or differences are observed between some critical quality attributes of the pre-
change and post-change product, it may be necessary to include a combination
of quality, nonclinical and/or clinical studies in the comparability exercise (1, 11).
5.2 Bridging studies

Nonclinical and clinical bridging studies are studies in which a parameter of
interest (such as a manufacturing process or formulation) is directly compared
with a changed version of that parameter with respect to the effect of the change
on the product’s clinical performance. If the physicochemical properties,
biological activity, purity and/or level of impurities of the pre-change and post-
change product are comparable, the safety and efficacy of the biotherapeutic
product can be inferred. However, nonclinical and/or clinical bridging studies
may be required when analytical data alone either do not establish comparability
or are insufficient to do so. The comparison of efficacy responses and safety
outcomes (for example, PK/PD profile, or rates of common adverse events and
serious adverse events) is often the primary objective. For ethical reasons, it is
desirable to apply the 3R principles (Replacement, Reduction, Refinement) to
the use of animals where scientifically appropriate. The following are examples
of changes that are likely to require nonclinical and/or clinical bridging studies:
(a) generation of a new MCB derived from a different host cell line; (b) a new
dosage form; (c) a new formulation (for example, a new excipient); (d) a new
presentation (for example, addition of pre-filled pens to vials); (e) a new route
of administration; and (f) a new dosing schedule. For these and comparable
changes, any proposed use of alternative approaches to a bridging study must be
justified and discussed with the NRA.
5.3 Similar biotherapeutic products

Following approval, an SBP is considered to be independent from the reference
product and has its own life-cycle (3). The manufacturer is not required to
re‑establish similarity to the reference product when comparability exercises
are conducted.
A major change in clinical use for an SBP that relies on the previously
demonstrated similarity provided in the original approval of the SBP may be
considered by the NRA on a case-by-case basis. For example, a new indication
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given to the reference product after approval of an SBP should not automatically
be given to the SBP. However, when new safety information on the reference
product is added after the original approval of the SBP, the labelling information
changes of the SBP should follow the changes made for the reference product
unless it can be demonstrated that the new information on the reference product
is not relevant to the SBP.
6. Reporting categories for quality changes

On the basis of the potential effect of the quality change (for example,
manufacturing change) on the quality attributes (that is, identity, strength, purity
and potency) of the biotherapeutic product, and on the potential impacts of this
on the safety or efficacy of the product, a change should be categorized as:
■■ a major quality change
■■ a moderate quality change
■■ a minor quality change, or
■■ a quality change with no impact.
The implementation of changes in the major or moderate categories
must be reported to the NRA in order to supplement the information in the
original marketing authorization or product licence. Major and moderate quality
changes should be reviewed and approved by the NRA prior to implementation
of the change (that is, prior to distribution of the post-change product).
Quality changes that are expected to have minimal potential to have an
impact, or to have no impact on the quality, safety or efficacy of the biotherapeutic
product, do not require submission of a PAS. The changes included in these
categories may be implemented by the marketing authorization holder without
prior review and approval by the NRA. However, quality changes with minimal
potential to have an impact should be notified to the NRA within established
timelines following implementation.
For each approved product, the marketing authorization holder or
manufacturer should maintain a comprehensive chronological list of all quality
changes, including minor quality changes. Additionally, this list should include a
description of the quality changes, including the manufacturing site(s) or area(s)
involved, the date each change was made, and references to relevant validations
and standard operating procedures. All data supporting minor quality changes,
as listed in Appendices 2 and 3 below, should be available on request to the NRA
or during inspections in accordance with local regulations.
Further information on each category of change is given below in
sections 6.1–6.4, with Appendices 2 and 3 providing a comprehensive list of
major, moderate and minor quality changes, and the information required to
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support each change. The quality changes listed in Appendices 2 and 3 should
be reported or recorded in the appropriate categories, as recommended in
this section and in the appendices. If a quality change may potentially have an
impact on the quality, safety and efficacy of the biotherapeutic product, but is
not included in Appendix 2 or 3, the NRA may be consulted for the correct
classification. When procedures and timelines for such consultations are not in
place, manufacturers should determine the classification of the change on the
basis of a change-specific risk assessment using the principles and examples
provided in these WHO Guidelines. The NRA should consider establishing a
mechanism that allows for its guidelines to be updated to address technological
changes requiring regulatory category classifications.
6.1 Major quality changes

Major quality changes are changes to the product composition, manufacturing
process, quality controls, facilities or equipment that have significant potential
to have an impact on the quality, safety or efficacy of the biotherapeutic product
or SBP. The marketing authorization holder should submit a PAS and receive a
notification of approval from the NRA before implementing the change. NRAs
should consider establishing a mechanism that allows for clear review timelines
and a consistent means of ensuring that those timelines are met (see section 8
and Appendix 1).
For a change in this category, the PAS should specify the products
concerned and should include a detailed description of the proposed change.
Additional supporting information is needed for the drug substance (as
noted in Appendix 2) and for the drug product (as noted in Appendix 3) and
could include: (a) information on the methods used and studies performed to
evaluate the effect of the change on the product’s quality attributes; (b) the data
derived from those studies; (c) relevant validation protocols and results; and
(d) updated product labelling information. In some cases, major quality changes
may also require nonclinical and/or clinical data. Relevant considerations on
the data required can be found in the WHO Guidelines on the quality, safety
and efficacy of biotherapeutic protein products prepared by recombinant DNA
technology (1).
6.2 Moderate quality changes

Moderate quality changes are changes to the product composition, manufacturing
process, quality controls, facilities or equipment that have a moderate potential
to have an impact on the quality, safety or efficacy of the biotherapeutic product
or SBP. The marketing authorization holder should submit a PAS and receive
a notification of approval from the NRA before implementing the change. The
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requirements for the PAS for moderate quality changes are the same as those
for major quality changes (see section 6.1); however, the amount of supporting
data required will generally be less than that required for major changes and the
review timeline should be shorter.
6.3 Minor quality changes

Minor quality changes are changes to the product composition, manufacturing
process, quality controls, facilities or equipment that have a minimal potential to
have an impact on the quality, safety or efficacy of the biotherapeutic product or
SBP. Changes in this category may be implemented by the marketing authorization
holder without prior review by the NRA. However, the NRA should be notified
of the changes within a specified timeline (see Appendix 1). The justification and
supporting documentation for minor quality changes are not needed for such
notification but should be made available by the marketing authorization holder
upon request from the NRA.
When a minor quality change affects the lot release specifications (for
example, narrowing of a specification, or compliance with pharmacopoeial
changes) and affects the quality control testing as summarized in the lot release
protocol, the marketing authorization holder should inform the institution
responsible for reviewing the release of lots (see introductory sections in
Appendices 2 and 3).
Minor quality changes that are related to a major or moderate change
should be described in the supplement for the major or moderate quality
change (see section 8.2 for additional details).
6.4 Quality changes with no impact

Quality changes that have no impact on product quality, safety or efficacy
may be implemented by the marketing authorization holder without prior
review by the NRA. Information on such changes must be retained as part
of the manufacturer’s GMP records or marketing authorization holder’s
product records, as applicable. These changes must comply with the applicable
GMP requirements and must be available for review during GMP inspections.
Examples of such changes include, but are not limited to:
■■ non-critical changes to the licensed application, including spelling
corrections and editorial clarifications made to documents (such as
validation summaries and/or reports, analytical procedures, standard
operating procedures or production documentation summaries) that
have no impact on the quality, safety and efficacy of the product;
■■ replacement of equipment with identical equipment;
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■■ change in specifications for a compendial raw material, a compendial

excipient or a compendial container closure component to comply
with an updated pharmacopoeial standard/monograph;
■■ transfer of quality control testing activities to a different facility
within a GMP-compliant site;
■■ with the exception of a potency assay or a bioassay, transfer of the
quality control testing activities for a pharmacopoeial assay to a
different facility within the same company;
■■ change in the in-process controls performed at non-critical
manufacturing steps;
■■ addition of a new GMP-compliant storage warehouse for raw
materials, master and working cell banks, and drug substance;
■■ installation of non-process-related equipment or rooms to improve
the facility, such as warehousing refrigerators or freezers;
■■ addition of time point(s) into the post-approval stability protocol;
■■ deletion of time point(s) from the post-approval stability protocol
beyond the approved shelf-life.
7. Reporting categories for safety, efficacy and/

or product labelling information changes
After assessing the effect of a change related to the clinical use of a product or to
product labelling information on the safe and effective use of a biotherapeutic
product, marketing authorization holders should classify this change as one of
the following reporting categories:
■■ safety and efficacy change;
■■ product labelling information change;

■■ urgent product labelling information change; or
■■ administrative product labelling information change (in cases where
prior approval before implementation is needed).
The product labelling information includes prescribing information
(or package insert) for health-care providers or patients, outer label (that is,
carton) and inner label (that is, container label). After approval, the marketing
authorization holder should promptly revise all promotional and advertising
items relating to the biotherapeutic product to make them consistent with
implementation of the product labelling information change.
Further information on each category is provided below in sections
7.1–7.4. In addition, examples of efficacy, safety and product labelling
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information changes considered to be appropriate for each category are provided

in Appendix 4.
7.1 Safety and efficacy changes

Safety and efficacy changes are changes that have an impact on the clinical
use of the biotherapeutic product in relation to safety, efficacy, dosage and
administration. To support such changes, data are required from clinical
studies and, in some cases, from clinically relevant nonclinical studies. Safety
and efficacy changes also require supplement submission and approval prior to
implementation of the change.
In general, safety and efficacy changes affect the product labelling
information and have the potential to increase or decrease the exposure levels of
the biotherapeutic product either by expanding the population that is exposed
or by changing dosage or dosing. These changes may be related to clinical use of
the biotherapeutic product, and can include:
■■ addition or expansion of a safety claim or efficacy claim, including
expansion of the population that is exposed;
■■ change in the strength or route of administration; 1
■■ change in the recommended dose and/or dosing schedule;
■■ co-administration with other biotherapeutic products or medicines;
■■ deletion or reduction of existing risk-management measures (for
example, contraindications, adverse events, warnings or cautionary
text/statements in the product labelling information).
The type and scope of the required nonclinical and/or clinical safety
and efficacy data are determined case by case on the basis of risk–benefit
considerations related to the impact of the changes, the biotherapeutic product
attributes and the disease that the biotherapeutic product is designed to prevent.
Other considerations include:
■■ the nature of the disease treated (that is, morbidity and mortality,
acute or chronic disease, current availability of disease therapy, and
size and nature of patient population);
■■ safety considerations (for example, adverse drug reactions observed,
adverse events in specific patient populations, management of adverse
reactions and change in rates of adverse reactions);
■■ the availability of animal models.
1
Some NRAs consider that changes in the route of administration or strength may require a new marketing
authorization.
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Marketing authorization holders are encouraged to consult with the NRA

on the adequacy of the clinical and/or nonclinical data needed to support a safety
and efficacy change, if deemed necessary. Additionally, some changes such as
dosage form, content of excipients or residual components, or delivery device
may require clinical data as well as revision of the product labelling information.
The NRA should be consulted on the data required to support such changes.
For nonclinical and clinical studies, the recommendations given in the
WHO Guidelines on the quality, safety and efficacy of biotherapeutic protein
products prepared by recombinant DNA technology (1) should apply. Guidance
on approaches to the nonclinical and clinical comparability exercise can also be
found in WHO guidelines on the evaluation of SBPs (2, 3).
For a change under this category, the marketing authorization holder
should submit a supplement to the NRA that includes the following where
applicable:
■■ a detailed description of – and rationale for – the proposed change;
■■ a summary of the methods used and studies performed to evaluate
the effect of the change on the safety or efficacy of the biotherapeutic
product;
■■ amended product labelling information;
■■ information on clinical studies (protocol, statistical analysis plan
and clinical study report);
■■ information on clinical assay methods (standard operating
procedures) and validations; and
■■ the pharmacovigilance plan.
7.2 Product labelling information changes

Product labelling information changes are changes to the labelling items that
have the potential to improve the management of risk to the population for
which use of the biotherapeutic product is currently approved through:
■■ the identification or characterization of any adverse event resulting
in the addition or strengthening of risk-management measures
for an adverse event considered to be consistent with a causal
association with the biotherapeutic product concerned;
■■ the identification of subgroups for which the benefit-to-risk profile
of the biotherapeutic product has the potential to be less favourable;
and
■■ the addition or strengthening of risk-management measures,
including instructions on dosing or any other conditions of use.
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Product labelling information changes require the filing of a PAS and

a notification of approval from the NRA prior to distribution of the product.
Supplements for product labelling information changes related to the clinical use
of a product often require data from pharmacovigilance reports (that is, periodic
safety update reports). Changes supported by large clinical or nonclinical studies
are usually not considered as product labelling information changes but as safety
and efficacy changes.
For a change under this category, the marketing authorization holder
should submit to the NRA a PAS that includes the following where applicable:
■■ a detailed description of – and rationale for – the proposed change;
■■ pharmacovigilance reports and statistical analysis of results; and
■■ amended product labelling information.
7.3 Urgent product labelling information changes

Urgent product labelling information changes are changes to the labelling items
that need to be implemented in an expedited manner in order to mitigate a
potential risk to the population in which the biotherapeutic product is currently
approved for use. Marketing authorization holders should consult with the NRA
and agree on the required supporting documentation and time frames for the
labelling changes or the need for a Dear Health-Care Professional Letter (that is,
a formal letter from a manufacturer to health-care professionals) to convey the
information prior to the submission of the supplement(s).
7.4 Administrative product labelling information changes

Administrative product labelling information changes are changes that are not
expected to affect the safe and efficacious use of the biotherapeutic product.
In some cases these changes may require reporting to the NRA and receipt
of approval prior to implementation, while in other cases reporting may not
be required.
■■ Examples of product labelling information changes that require
approval by the NRA prior to implementation are changes in the
proper/nonproprietary name or trade name of the biotherapeutic
product. Changes in this category are considered important for
reasons of liability and monitoring.
■■ Examples of product labelling information changes that do
not require approval by the NRA prior to implementation are
administrative changes such as those related to labelling (for
example, minor changes in format without any negative effect on
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readability). These changes should be reported to the NRA as part

of a subsequent PAS for safety and efficacy changes or product
labelling information changes when updated product labelling
information is included.
Manufacturers are encouraged to consult with the NRA regarding the
appropriate reporting category for labelling changes to approved products.
8. Procedures
The establishment of procedures and criteria for the adequate oversight of
changes to approved biotherapeutic products is the responsibility of the
regulator. Therefore, NRAs should establish written instructions regarding
submission procedures and timelines (with action dates) for consultation by
marketing authorization holders as they prepare to submit a supplement for
a change. These instructions should cover: (a) the identification of emergency
use; (b) expanded access; and (c) expedited and/or priority review, timelines
and procedures for life‑saving medications to address an unmet need. As
supplements for a major quality change or an efficacy and safety change require
extensive documentation and data, the review times should be longer than those
for supplements for moderate quality changes or product labelling information
changes. Furthermore, NRAs may establish different timelines for the review of
major quality changes that do not require clinical data as compared with safety
and efficacy changes that do require clinical data. Appendix 1 provides examples
of different regulatory categories and their suggested review timelines.
If a change is not included in Appendices 2, 3 or 4, marketing
authorization holders are encouraged to use scientific judgement, leverage
competent regulatory authority guidance or to contact the NRA to determine the
appropriate category of a supplement prior to submission of the information in
support of a change. Similarly, marketing authorization holders should consult

NRAs for major changes that require the inclusion of a GMP certificate and
which may trigger a pre-submission inspection, or that may require clinical
and/or nonclinical data to support a change in safety and efficacy or in product
labelling information. Marketing authorization holders are encouraged to
contact the NRA regarding plans for future changes and proposed filing dates for
changes to existing products in order to assist NRAs in planning the allocation of
review resources. NRAs should establish procedures with appropriate timelines
for the conducting and recording of communications between themselves and
marketing authorization holders.
To assist in the acceptance of submissions for review, the covering
letter or the Module 1 documentation of the Common Technical Document
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accompanying a supplement for a quality change should clearly specify the

selected category by labelling the submission as either a major quality change or
a moderate quality change.
The covering letter accompanying a supplement for a safety, efficacy or
product labelling information change should specify that the change is being
reported in the selected category by labelling the submission as:
■■ a safety and efficacy change;
■■ a product labelling information change;
■■ an urgent product labelling information change; or
■■ an administrative product labelling information change (in cases
where prior approval is needed before implementation).
Major quality change supplements that contain both quality data and
revised product labelling information but no clinical and/or nonclinical data
should be labelled “Major quality change and Product labelling information
change” and the covering letter should specify that the submission includes both
quality changes and revised product labelling information items.
Major quality change supplements that contain quality, safety and efficacy
data (from clinical studies and/or clinically relevant nonclinical studies) and
revised product labelling information, should be labelled “Major quality change
and Safety and efficacy change” and the covering letter should specify that the
submission includes quality changes, results from clinical and/or nonclinical
studies, and revised product labelling information items.
Each supplement should include a list of all the changes contained in
the submission. The list should describe each change in sufficient detail to allow
the NRA to determine quickly whether the appropriate reporting category has
been used. If the submission has been inappropriately classified, the marketing
authorization holder should be notified. Minor quality changes that are related/
consequential to moderate or major quality changes should be described in
the PAS. In addition, any minor changes that have been implemented should
be annotated in the affected documents (for example, Common Technical
Document sections) and reported in any future filing to the NRA. For example,
a minor change such as narrowing of a specification should be included in a
supplement for a moderate or major change which includes updated quality
control release information.
The regulation of post-approval changes is part of the entire regulatory
framework which includes marketing authorization, GMP inspection and post-
marketing surveillance. These activities are often performed by different units
of the NRA. It is essential that these different units – especially the marketing
authorization (or regulatory affairs) and GMP inspection units – interact and
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exchange information effectively, and that the roles and responsibilities of each
unit are clearly defined, particularly when they operate as separate entities. When
multiple units are involved in the evaluation of a supplement, a formal decision-
making process should be in place to discuss, for example, whether a change may
require a GMP inspection or may be reviewed during the next routine inspection.
Procedures should also be established so that the outcomes of inspections are
verified or taken into account prior to the approval of supplements. Good
coordination and communication between different units of the NRA are pivotal
in ensuring continuity of supply and access to products of assured quality, safety
and efficacy. Some regulatory authorities may be willing to cooperate more
closely and to share information on GMP inspections under a mutual agreement
(for example, the Pharmaceutical Inspection Cooperation Scheme – PIC/S).
Expedited review procedures

NRAs of product-procuring countries that decide to recognize or rely on the
decisions of other NRAs should establish alternative regulatory procedures for
the expedited approval of changes based on previous expert review and approval
by the NRA of the country where the biotherapeutic products are licensed (see
Appendix 1). Accordingly, the product-procuring NRAs should also create a
list of the NRA approvals they will recognize. On the basis of regulatory and
regional considerations, procedures for recognition of the decisions of other
NRAs on the approval of changes could include the following pathways:
■■ The NRA recognizes the decision of other regulatory authorities
and does not perform a review of supporting data, but is notified
of the change. The submission consists of a covering letter from
the marketing authorization holder informing the procuring NRA
about the change and including as an attachment a copy of the
approval letter from the NRA of the licensing country stating the
relevant changes.
■■ The NRA performs an assessment of the decision of the NRA of the
licensing country to determine whether recognition of that NRA’s
decision is appropriate. The submission consists of:
–– the covering letter from the marketing authorization holder
informing the procuring NRA of the change;
–– a copy of the approval letter issued by the NRA of the licensing
country;
–– assessment reports and relevant correspondence from the NRA
of the licensing country (if made available by the NRA);
–– a detailed description of the change; and
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–– supporting data submitted as necessary if assessment reports are

not available.
■■ The NRA performs a partial review and evaluation of a complete
package of supporting data, as originally submitted in the product-
licensing country.
Similarly, recognition of inspection activities conducted by the authorities
that license the product may be considered as part of the expedited review
process and may be included in the regulatory pathways listed above.
Additionally, for previously approved changes addressing urgent safety
issues in the product labelling information, procedures should be in place to
allow for the expedited implementation of such changes (see section 8.3 and
Appendix 1).
In special or urgent circumstances, a marketing authorization holder may
ask the NRA to expedite the review of a supplement for public health reasons
(for example, a product shortage or safety update) or if a delay in making the
change would impose extraordinary hardship on the marketing authorization
holder or manufacturer.
Multiple changes
Multiple related changes, involving various combinations of individual changes,
may be submitted in the same supplement. For example, a manufacturing site
change may also involve changes to the equipment and manufacturing process.
For submissions that include multiple changes, the marketing authorization
holder should clearly specify which data support each change.
Multiple major or moderate quality changes for the same product may be
filed in a single submission provided that the changes are related and/or supported
by the same information. Minor quality changes that were implemented previously
and that are related and/or consequential to a moderate or major quality change
should be described in the PAS for the moderate or major quality change. If
the proposed changes are related, the marketing authorization holder should
indicate the association between them. The marketing authorization holder
should also clearly specify which supporting data support which change. Such
changes could affect both the drug substance and the drug product. If too many
changes are filed within the same submission, or if major issues are identified
with a change and extensive time would be required to review them, the NRA
may ask the marketing authorization holder to divide the changes into separate
submissions and to resubmit the file. If the recommended reporting categories
for the individual changes differ, the submission should be in accordance with
the most restrictive of the categories recommended for the individual changes.
In the case of numerous changes of the same category, the NRA may reclassify
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the submission to the next higher level on the basis of the potential impact of the
totality of the changes on the quality, safety and efficacy of the biotherapeutic
product or SBP. This reclassification should be communicated to the marketing
authorization holder at the start of the assessment.
8.1 Procedures for prior approval supplements

The procedures in this section apply to all changes requiring approval prior
to implementation: namely, major and moderate quality changes, safety and
efficacy changes, product labelling information changes, urgent product labelling
information changes and selected administrative product labelling information
changes.
The following items should be included, where applicable, in the
supplement submission for post-approval changes:
■■ a covering letter that includes:
–– the type of submission (for example, major quality change,
moderate quality change or safety and efficacy change),
–– a list of the change(s) and a rationale for the change(s) with
sufficient detail (including a justification for the selected reporting
category) to allow for processing and reviewer assignments by
NRAs,
–– an indication of the general type of supporting data, and
–– cross-referenced information (including product name, marketing
authorization holder’s name, submission type and date of
submission/approval);
■■ completed documents or forms based on NRA requirements, such
as a medicine submission application form, signed and dated;
■■ the anticipated date for implementation of the change (recognizing
that in some cases the implementation of the change may be delayed

after approval to allow for depletion of the previously approved
biotherapeutic or to allow for global staggered approval depending
on supply/demand);
■■ GMP information (for example, inspection history and/or evidence
of GMP compliance rating by experienced NRAs), as applicable;
■■ when relevant, a side-by-side comparison showing the differences
between the approved manufacturing process (including quality
control tests) and the proposed one(s) (see section 5);
■■ when relevant, clinical and/or nonclinical study reports,
pharmacovigilance reports, and annotated and clean drafts of product
labelling information (see section 7).
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In addition to the above general information, the specific information

required to support the various quality changes is outlined in Appendices 2 and
3. It should be noted that the general information is not repeated under each
of the various changes outlined in the appendices. All data recommended to
support a change should be provided with the submission, in addition to the
general information as appropriate. If recommended supporting data are not
submitted, a detailed rationale should be provided to explain why.
If the same change is applicable to multiple products, a separate
submission is generally required for each product – though the data may be
cross-referenced. NRAs may in some cases allow a common change to be
bundled into one submission for multiple products. When cross-references are
made to information that has been submitted previously, details of the cross-
referenced information should be provided in the covering letter.
Submissions filed in electronic or paper format should be based on the
requirements of the NRA. The data submitted should be well organized and
should be provided in the format defined by the NRA.
After the NRA completes the review of the supporting data in a
supplement, the following outcomes are possible:
■■ If the NRA determines that the information submitted in a
supplement supports the quality, safety and efficacy of the product
manufactured with the change, the NRA will issue a written
notification of approval stating that the change can be implemented
and the product manufactured with the change can be distributed.
■■ If the NRA determines that the information submitted in a
supplement fails to support the quality, safety or efficacy of the
product manufactured with the change, the NRA will issue a written
request notification for additional documentation, information and
clarification to be submitted by the marketing authorization holder.
If the identified deficiencies are minor, they may be addressed
without stopping the review process. If the deficiencies are major
or are not resolved during the allotted review period following
rounds of questions and requests for more information, the NRA
may decide to issue a written notification of noncompliance, as a
result of which the review process is stopped, the change may not be
implemented and the product manufactured with the change may
not be distributed. In the case of a notification of noncompliance
being issued, the following outcomes are possible:
–– If the marketing authorization holder’s response document to
the notification of noncompliance is adequate and all identified
deficiencies are resolved in a satisfactory manner, the NRA will
207
issue a written notification of approval stating that the change

can be implemented and the product manufactured with the
change can be distributed.
–– If the information in the marketing authorization holder’s
response document to the notification of noncompliance is not
adequate and not all identified deficiencies are resolved in a
satisfactory manner, the NRA will issue a written notification of
rejection stating that the change cannot be implemented and the
product manufactured with the change cannot be distributed.
The NRA should establish procedures and timelines for the review of
marketing authorization holders’ responses to the notification of noncompliance
in cases where the review has been stopped. Documentation subsequent to
the original supplement submission (in response to information requests or
notifications of noncompliance) should be submitted and filed as amendments
to the original supplement, and all communications with sponsors should be
properly recorded.
Appeal procedures should be established for resolving disagreements
and disputes between the NRA and the marketing authorization holder. Such
procedures should allow the marketing authorization holder to request a
re‑evaluation of the submitted application in case the application is initially
rejected by the NRA.
NRAs may consider the use of a “comparability protocol” when a
marketing authorization holder submits changes:
Comparability protocol
A comparability protocol (also referred to as “post-approval change management
protocol” in other documents) establishes a framework for a well-defined plan
for future implementation of a quality change. This will include the tests to be
done and acceptable limits to be achieved when assessing the effect of specific
changes on the quality, safety or efficacy of a biotherapeutic product or SBP. For
some changes, the routine quality tests performed to release the drug substance
or drug product are not considered sufficient for assessing the impact of the
change, and additional in-process tests and characterization tests may be needed.
Comparability protocols are often used for the routine replenishment of WCBs
and reference standards used in quality control tests when the remaining aliquots
of reference standards expire or diminish.
The purpose of a comparability protocol is to provide transparency in
the data requirements for changes and increase the predictability of the effects
of changes. This allows for the more expedient distribution of a product by
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Annex 3
permitting the marketing authorization holder to submit a protocol for a change

which, if approved, may justify a reduced reporting category for the change when
the comparability data are obtained and the change is implemented. It is for the
NRA to decide whether or not to include the review and approval of comparability
protocols in its approach to regulating changes to approved biotherapeutic
products or SBPs; however, the concept of using comparability protocols is
encouraged. For NRAs currently taking this approach, a comparability protocol
can be provided in the original submission. Otherwise, a new comparability
protocol, or a change to an existing one, requires submission of a supplement
and approval prior to implementation because it may result in a lower reporting
category for the changes covered in the comparability protocol once the actual
comparability data are submitted. The change in reporting category for a change
covered by a comparability protocol and the supporting data to be generated
should be established by the NRA at the time the comparability protocol is
approved. For a minor quality change that results from the execution of a
comparability protocol, the change should be notified to the NRA immediately
after implementation. For some marketing authorization holders with multiple
related products and facilities, an expanded change protocol can be proposed.
The scope of an expanded change protocol may cover multiple related products
or manufacturing changes (for example, facility changes) (15).
Production documents
Production documents (that is, executed batch records) are not generally
required to support changes to the marketing authorization dossier or product
licence. However, such documents may be requested during review and should
be made available to the NRA on request. These documents should be retained
in accordance with GMP and should be available in their local official language
during inspections. If English translations are required, NRAs are encouraged
to establish a mechanism to make this requirement known to marketing
authorization holders accordingly.
8.2 Procedures for minor quality changes and

quality changes with no impact
Implementation of minor quality changes does not require prior approval
from the NRA but should be notified to the NRA. Each NRA is responsible for
determining the timelines for reporting the notification (for example, annually).
Supporting data should not be provided with the notification unless it may help
in justifying the reporting category. However, as recommended in Appendices
2 and 3 below, the minor quality changes should be recorded or compiled with
related supporting data generated by the manufacturer in a document or file
209
dedicated to minor changes. The documents or files for all minor quality changes
should be available to the NRA on request or during inspection.
NRAs may audit minor quality changes by requesting and reviewing the
supporting data, as deemed appropriate during an inspection or review of related
changes. If the classification of a change or the supporting data are not considered
to be acceptable then the marketing authorization holder may be requested to file
a supplement for a major or moderate quality change.
Minor quality changes that have previously been implemented and are
related and/or consequential to a major or moderate quality change should be
described in the relevant parts of the documentation when submitting a PAS for
the major or moderate change. As for all minor quality changes, the supporting
data for these changes do not need to be included in the supplement but should
be retained by the manufacturer.
Changes that have no impact on the quality, safety and efficacy of the
product are not reported, but if the NRA determines (during an inspection or a
review of related changes) that the information for the change fails to demonstrate
the continued safety or efficacy of the product manufactured using the changes,
the NRA may work to resolve the problem with the marketing authorization
holder. If the NRA finds that the product in distribution poses a danger to public
health, or if it determines that there are unresolved issues, it may require the
marketing authorization holder to cease distribution of the product manufactured
using the changes or to remove the product from distribution pending resolution
of the issues related to the changes.
8.3 Procedures for urgent product labelling

information changes
For urgent changes to product labelling information which address safety updates
and have the potential to have an impact on public health (for example, addition
of a contraindication or a warning), NRAs should establish a specific mechanism

to allow for the immediate or expedited approval and implementation of such
changes on a case-by-case basis after previous agreement between the NRAs and
Since product labelling safety updates invariably need to be implemented
and are generally approved, NRAs in procuring countries should establish a
mechanism by which urgent product labelling changes that have been approved
in the country where the biotherapeutic products in question are produced and/
or licensed may be implemented immediately upon receipt of the supplement
from marketing authorization holders or manufacturers. Such accelerated
procedures would contribute to the dissemination of the most current
information to health-care providers and would help to mitigate discrepancies
between the labels used in the various countries and posted on websites.
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8.4 Procedures for administrative product

labelling information changes
Depending on the scope of the change, administrative product labelling
information changes may require approval prior to implementation. For example,
changes in the proper/nonproprietary name or trade name of the biotherapeutic
product require approval before implementation, while minor formatting
changes do not (see section 7.4 for further details).
For an administrative product labelling information change that requires
approval prior to implementation the marketing authorization holder should
submit a supplement containing background information on the change and
annotated and clean drafts of the product labelling information.
Administrative product labelling information changes that do not need
prior approval and that have been implemented since the last approved product
labelling information should be included when submitting a subsequent PAS for
safety and efficacy changes or for product labelling information changes. In these
cases, the product labelling information should be annotated when filing the next
PAS to indicate the new changes and those administrative changes that have been
implemented since the last approval.
9. Authors and acknowledgements

The first draft of these WHO Guidelines was prepared by Dr E. Griffiths,
Consultant, Kingston-upon-Thames, the United Kingdom; Mr H. Hamel, Health
Canada, Canada; Mrs T. Jivapaisarnpong, Ministry of Public Health, Thailand;
Dr H-N. Kang, World Health Organization, Switzerland; Dr E. Lacana, Food and
Drug Administration, the USA; Dr I. Oh, Ministry of Food and Drug Safety,
Republic of Korea; Dr R. Thorpe, Consultant, Welwyn, the United Kingdom;
Dr T. Yamaguchi, Pharmaceuticals and Medical Devices Agency, Japan; and
Dr M. Wadhwa, National Institute for Biological Standards and Control, the
United Kingdom, following a meeting held in Geneva, Switzerland, 30–31
August 2016, and taking into consideration the WHO Guidelines on procedures
and data requirements for changes to approved vaccines (7) and comments
received from the following experts: Dr A. Abdelaziz, Jordan Food and Drug
Administration, Jordan; Mrs J. Bernat (provided the consolidated comments of
the International Federation of Pharmaceutical Manufacturers & Associations
(IFPMA)), Switzerland; Dr H-K. Heim, Federal Institute for Drugs and Medical
Devices, Germany; Dr D. Khokal (provided the consolidated comments of
the Health Sciences Authority (HSA)), Singapore; Dr B. Kim, Dr K. Kim and
Dr I. Oh, Ministry of Food and Drug Safety, Republic of Korea; Dr Y. Kishioka
and Dr T. Yamaguchi, Pharmaceuticals and Medical Devices Agency, Japan;
211
Dr H. Meyer, Paul-Ehrlich-Institut, Germany; and Dr M. Welin, Medical

Products Agency, Sweden.
The resulting draft document was posted on the WHO Biologicals
website for a first round of public consultation from 11 October to 16 December
2016 and comments were received from the following reviewers: Mr D. Baker
(provided the consolidated comments of the Parenteral Drug Association (PDA)),
the USA; Mrs J. Bernat (provided the consolidated comments of the IFPMA),
Switzerland; Dr D. Goryachev, Ministry of Health, Russian Federation; Dr H-K.
Heim, Federal Institute for Drugs and Medical Devices, Germany; Dr S. Jadhav
(provided the consolidated comments of the Serum Institute of India), India;
Dr D. Khokal (provided the consolidated comments of the HSA), Singapore;
Dr Y. Kishioka, Pharmaceuticals and Medical Devices Agency, Japan; Mrs S.
Kox (provided the consolidated comments of the Biosimilars Committee of the
International Generic and Biosimilar Medicines Association (IGBA)), Belgium;
Dr C. Liang, National Institutes for Food and Drug Control, China; Mr M.
Maito (provided the consolidated comments of the Asociación Latinoamericana
de Industrias Farmacéuticas (ALIFAR)), Argentina; Dr D. Misztela (provided
the consolidated comments of the Plasma Protein Therapeutics Association
(PPTA)), Brussels, Belgium; Mrs J. Rodgers, Food and Drugs Authority, Ghana;
Dr G.R. Soni, National Institute of Biologicals, India; Dr P. Swann (provided the
consolidated comments of Biogen), the USA; Dr R. Thorpe, Consultant, Welwyn,
the United Kingdom; and Dr M. Welin, Medical Products Agency, Sweden.
The document WHO/BS/2017.2311 was prepared by Ms J. Dahlan,
National Agency of Drug and Food Control, Indonesia; Dr E. Griffiths,
Consultant, Kingston-upon-Thames, the United Kingdom; Mr H. Hamel, Health
Canada, Canada; Mrs T. Jivapaisarnpong, Ministry of Public Health, Thailand;
Dr H-N. Kang, World Health Organization, Switzerland; Dr E. Lacana, Food
and Drug Administration, the USA; and Dr M. Wadhwa, National Institute
for Biological Standards and Control, the United Kingdom, taking into
consideration comments received from the first round of public consultation as

well as from a WHO informal consultation on the development of guidelines
on procedures and data requirements for changes to approved biotherapeutic
products including biosimilars held in Seoul, Republic of Korea, 27–28 April
2017 and attended by: Dr M. Allam, National Organization for Research and
Control of Biologicals, Egypt; Mrs B. Always (IFPMA representative), Pfizer,
Australia; Dr P. Aprea, Administración Nacional de Medicamentos, Alimentos
y Tecnología Médica, Argentina; Dr C. Blades, National Health Surveillance
Agency, Brazil; Mrs P. Chirachanakul, Ministry of Public Health, Thailand; Dr S.
Chong (Singapore Association of Pharmaceutical Industries representative), Roche
Singapore Technical Operations, Singapore; Ms J. Dahlan, National Agency of
Drug and Food Control, Indonesia; Dr H.J. Doh, Ministry of Food and Drug
Safety, Republic of Korea; Dr E. Griffiths, Consultant, Kingston-upon-Thames,
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Annex 3
the United Kingdom; Mr H. Hamel, Health Canada, Canada; Dr J. Jeong, Ministry

of Food and Drug Safety, Republic of Korea; Mrs T. Jivapaisarnpong, Ministry of
Public Health, Thailand; Dr J. Jung, Ministry of Food and Drug Safety, Republic
of Korea; Mrs Y. Jung, Lilly Korea, Republic of Korea; Dr H-N. Kang, World
Health Organization, Switzerland; Dr B. Kim, Ministry of Food and Drug Safety,
Republic of Korea; Dr D. Kim, Ministry of Food and Drug Safety, Republic of
Korea; Dr H. Meyer, Paul-Ehrlich-Institut, Germany; Dr Z. Munkombwe,
Zambia Medicines Regulatory Authority, Zambia; Dr I. Oh, Ministry of Food
and Drug Safety, Republic of Korea; Dr S. Ramanan (IFPMA representative),
Amgen, the USA; Ms J. Rodgers, Food and Drugs Authority, Ghana; Dr M.
Schiestl (IGBA representative and Medicines for Europe representative), Sandoz
Biopharmaceuticals, Austria; Dr T. Schreitmueller (IFPMA representative),
F. Hoffmann–La Roche Ltd, Switzerland; Dr T.J. Seng, Health Sciences Authority,
Singapore; Dr K-S. Seo, Dong-A Socio Holdings Co. Ltd, Republic of Korea;
Dr K.W. Seo, Ministry of Food and Drug Safety, Republic of Korea; Dr J. Shin,
WHO Regional Office for the Western Pacific, Philippines; Dr Y. Sohn, Ministry
of Food and Drug Safety, Republic of Korea; Mr S. Song, Celltrion, Republic of
Korea; Mr E. Spitzer (ALIFAR representative), Buenos Aires, Argentina; Dr S.K.
Suh, Ministry of Food and Drug Safety, Republic of Korea; Dr R. Volkova,
Ministry of Healthcare, Russian Federation; Dr M. Wadhwa, National Institute
for Biological Standards and Control, the United Kingdom; Dr W. Wei, China
Food and Drug Administration, China; Dr S. Xie, China Food and Drug
Administration, China; and Dr T. Yamaguchi, Pharmaceuticals and Medical
Devices Agency, Japan.
The document WHO/BS/2017.2311 was then posted on the WHO
Biologicals website for a second round of public consultation from 18 July to
15 September 2017 and comments were received from the following reviewers:
D. Baker (provided the consolidated comments of the PDA), the USA; Ms J.
Bernat (provided the consolidated comments of the IFPMA), Switzerland;
L. Feisee (provided the consolidated comments of the Biotechnology Innovation
Organization), the USA; Dr K. Gao, National Institutes for Food and Drug
Control, China; Dr Y. Jia, United States Food and Drug Administration, the
USA; Ms Z. Kusynová, (provided the consolidated comments of the International
Pharmaceutical Federation), The Hague, Netherlands; Dr D. Misztela (provided the
consolidated comments of the PPTA), Brussels, Belgium; J. Netterville (provided
the consolidated comments of AstraZeneca), the USA; Dr B. Nhaquila, Ministry of
Health, Mozambique; Dr I. Oh, Ministry of Food and Drug Safety, Republic of
Korea; Dr I. Schwarzenberger (provided the consolidated comments of the IGBA),
Brussels, Belgium; Dr M. Welin, Medical Products Agency, Sweden; and Mr T.
Zhen, Hovid Berhad, Malaysia.
Further changes were subsequently made to document WHO/BS/
2017.2311 by the WHO Expert Committee on Biological Standardization.
213
10. References
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on Biological Standardization: sixtieth report. Geneva: World Health Organization; 2013:
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WHO Expert Committee on Biological Standardization: sixty-seventh report. Geneva: World Health
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World Health Assembly, Geneva, 18–26 May 2014. Geneva: World Health Organization; 2014
(http://apps.who.int/gb/ebwha/pdf_files/WHA67/A67_R20-en.pdf, accessed 12 December 2017).
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de Janeiro, Brazil, 24–29 August 2014 (http://www.who.int/medicines/areas/quality_safety/
regulation_legislation/icdra/16_ICDRA_Recommendations2014.pdf?ua=1, accessed 12 December
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Expert Committee on Biological Standardization: sixty-fifth report. Geneva: World Health
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biologicals/vaccines/Annex4_Guidelines_changes_to_approved_vaccines_eng.pdf?ua=1,
Biological Standardization: sixty-sixth report. Geneva: World Health Organization; 2016: Annex 2
(WHO Technical Report Series, No. 999; http://www.who.int/biologicals/areas/vaccines/Annex_2_
WHO_Good_manufacturing_practices_for_biological_products.pdf?ua=1, accessed 12 December
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Expert Committee on Specifications for Pharmaceutical Preparations: forty-eighth report. Geneva:
World Health Organization; 2014: Annex 2 (WHO Technical Report Series, No. 986; http://www.
who.int/medicines/areas/quality_safety/quality_assurance/TRS986annex2.pdf?ua=1, accessed
12 December 2017).
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Committee on Specifications for Pharmaceutical Preparations: fiftieth report. Geneva: World
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11. Comparability of biotechnological/biological products subject to changes in their manufacturing

process. ICH Guideline Q5E. Geneva: International Conference on Harmonisation of Technical
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for Human Use. London: European Medicines Agency; 2007 (Document EMEA/CHMP/BMWP/
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(https://www.canada.ca/content/dam/hc-sc/migration/hc-sc/dhp-mps/alt_formats/pdf/
prodpharma/applic-demande/guide-ld/postnoc_change_apresac/noc_pn_quality_ac_sa_
qualite-final-eng.pdf, accessed 12 December 2017).
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Medicinal Products for Human Use. London: European Medicines Agency; 2012 (Document EMA/
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Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use;
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17. Guideline on real time release testing (formerly Guideline on parametric release). Committee
for Medicinal Products for Human Use. London: European Medicines Agency; 2012 (Document
EMA/CHMP/QWP/811210/2009-Rev1; http://www.ema.europa.eu/docs/en_GB/document_library/
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Appendix 1
Reporting categories and suggested review timelines
It is recommended that NRAs establish review timelines to allow marketing
authorization holders or applicants to plan the implementation of changes. The
review timelines are established taking into consideration the country or regional
situation, the capability of the NRA, the impact of the change and the amount of
data required to support the change. Consequently, the review time frames for
major changes should be longer than those for moderate changes. The suggested
review times in the table below are shown as examples; they are based on the
experience of several NRAs and apply to situations where the NRA performs a
full review or assessment of the supplement. The review time would start when
the supplement has been accepted for review and found to be complete, and
would end at the time when the initial assessment is shared with the marketing
authorization holder by the issuance of either a notification of approval or a
notification of noncompliance with a list of comments and deficiencies. In case
of the latter, the marketing authorization holder may seek approval for the change
by submitting an amendment to the supplement with responses to all the
comments in the notification of noncompliance. The NRA should also establish
timelines for the secondary review cycle following the receipt of responses from
the marketing authorization holder. If minor deficiencies are identified during
the initial review cycle, the NRA may communicate these to the marketing
authorization holder without stopping the review clock in order to try to finalize
the assessment within the established timeline (see section 8.1).
Expedited implementation procedures should be in place for dealing
with product labelling information changes which address urgent safety issues
(see section 8.3).
Reporting categories for post-approval changes and suggested review timelines

Quality changes
Reporting category Procedure Suggested review timeline
Major quality changes PAS 3–6 months
Moderate quality changes PAS 1–3 months
Minor quality changes Require notification to N/A
the NRA a, b
Quality changes with no Do not require N/A
impact notification to the NRA
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Table continued
Safety, efficacy and product labelling information changes
Reporting category Procedure Suggested review timeline
Safety and efficacy PAS 10 months
changes
Product labelling PAS 5 months
information changes
Urgent product labelling PAS for urgent safety Immediate implementation
information changes c restrictions on receipt of supplement
by the NRA
Administrative product PAS 30 days
labelling information
Do not require N/A
changes
approval prior to
implementation d
N/A: not applicable.
a
Each NRA is responsible for determining the timeline for reporting the notification (for example, annually).
However, NRAs should establish a mechanism to ensure that notifications are received no later than one year
post-implementation. In a case where a minor quality change results from the use of a comparability protocol, the
change should be notified to the NRA immediately after implementation.
b
Minor quality changes impacting the registered details may be bundled with moderate or major quality changes,
if needed.
c
Urgent product labelling information changes are applicable only to label changes which address urgent safety
updates or have the potential to have an impact on public health, with immediate implementation allowed after
prior agreement between NRAs and marketing authorization holders.
d
Administrative product labelling information changes that do not require approval prior to implementation and
that have been implemented since the last approved product labelling information change should be reported by
including all changes in subsequent PAS for safety and efficacy changes or product labelling information changes
when updated product labelling information is included.
NRAs that procure biotherapeutic products from countries other than their own
are encouraged to establish alternative accelerated timelines for changes that
have previously been approved by the other NRAs. Accordingly, those NRAs
should create a list of the NRA approvals they will recognize. On the basis of
the regulatory pathway options provided in section 8, the following examples
of accelerated timelines could be established:
■■ The NRA recognizes the decision of other regulatory authorities
and does not perform a review of supporting data but is informed
of the change. Using this approach, NRAs could allow changes to be
implemented immediately after receipt of the change notification.
217
■■ The NRA performs an assessment of the decision of the NRA of

the licensing country to determine if recognition of the latter NRA’s
decision is appropriate. Using this approach, NRAs could establish
abbreviated review timelines – such as 2 months for major quality
changes, 4 months for safety and efficacy changes, and immediate
implementation on receipt of the change notification for moderate
quality changes and product labelling information changes.
■■ The NRA performs a partial review and evaluation of a complete
supporting data package, as originally submitted to the licensing
country. Using this approach, timelines would be expected to be
shorter than the timelines described in the above table.
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Appendix 2
Changes to the drug substance
The examples presented in this appendix are intended to assist with the
classification of changes made to the quality information for the drug substance.
The information summarized in the table below provides guidance on:
■■ the conditions to be fulfilled for a given change to be classified as

major, moderate or minor (if any of the conditions outlined for a given
change are not fulfilled, the change is automatically considered to be
at the next higher reporting category – for example, if any conditions
recommended for a moderate quality change are not fulfilled, the
change is considered to be a major quality change);
■■ the supporting data for a given change, either to be submitted to the
NRA or maintained by the marketing authorization holder (if any
of the supporting data outlined for a given change are not provided,
are different or are not considered applicable, adequate scientific
justification should be provided); and
■■ the reporting category (major, moderate or minor quality change).
Marketing authorization holders should use scientific judgement,

leverage competent regulatory authority guidance or contact the NRA if a
change is not included in the table and has the potential to impact on product
quality. Marketing authorization holders should also contact the NRA when a
change is considered at the next higher reporting category because any of the
conditions outlined are not fulfilled and where the supporting data are not
described. NRAs should establish procedures, with appropriate timelines, on
the conducting and recording of communications between themselves and
Supporting data should be provided according to the submission format
accepted by the NRA – see for example (1, 2).
Additional information on data requirements to support quality changes
can be found in WHO good manufacturing practices for biological products
(3), WHO Guidelines on the quality, safety and efficacy of biotherapeutic
protein products prepared by recombinant DNA technology (4) and in relevant
International Conference on Harmonisation of Technical Requirements for
Registration of Pharmaceuticals for Human Use (ICH) guidelines (5, 6).
219
Quality changes to comply with updated

compendia and/or pharmacopoeias
NRAs should make a list of the recognized compendia and/or pharmacopoeias.
Manufacturers are expected to comply with the current versions of compendia/
pharmacopoeias, as referenced in the approved marketing authorization. Changes
linked to a change in the compendial/pharmacopoeial methods or specifications
for a drug substance do not need to be submitted for review if reference is made
to the current edition of the compendium or pharmacopoeia, but the changes
should be notified to the NRA with information on them available for inspection.
In some cases, changes introduced to comply with recognized compendia/
pharmacopoeias may require approval by the NRA prior to implementation
regardless of the timing of the change in relation to the date when the
compendium/pharmacopoeia was updated. For example, supplement submission
and approval by the NRA may be required for some changes to quality control
tests performed for product release (for example, to potency tests), for changes
that have an impact on any product labelling information items, and for changes
that may affect the quality, safety or efficacy of the product.
Quality changes affecting lot release

While WHO recognizes that independent lot release by NRAs or national
control laboratories is required for vaccines, in some countries this lot release
system also applies to other types of products such as plasma-fractionated
products. Where post-approval changes to the drug substance affect the lot
release protocol (for example, changes to test procedures, reference standards
or laboratory sites) or sample testing requirements for lot release, the marketing
authorization holder should inform the institution responsible for reviewing
the release of product lots. These procedures apply to changes that have been
authorized by the NRA in the case of major and moderate quality changes and
to changes that have been implemented in the case of minor quality changes.
For example, the qualification of a new lot of reference standard against the
approved reference standard may be considered a minor quality change if the
qualification of a new standard is performed in accordance with an approved
protocol and specification. Nevertheless, these changes must be reported to the
NRA or national control laboratory as appropriate.
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Manufacture
Description of change Conditions to Supporting Reporting
be fulfilled data category
1. Change to a drug substance manufacturing facility:
Note: For the purpose of this change, manufacturing refers to unit operations in the
manufacturing process of the drug substance and is not intended to refer to quality control
testing, storage or transportation.
a. Replacement or addition of a None 1–4, 6–8 Major
manufacturing facility for the
bulk drug substance or any 1–3 1–8 Moderate
intermediate
b. Conversion of a drug 4 9, 10 Moderate
substance manufacturing
facility from single-product
to multi-product
c. Deletion of a manufacturing 5, 6 None Minor
facility or manufacturer
of an intermediate drug
substance, or bulk
Conditions
1. The proposed facility is an approved drug substance facility for biotherapeutics (for
the same company/marketing authorization holder).
2. Any changes to the manufacturing process and/or controls are considered either
moderate or minor (for example, duplication of product line).
3. The new facility/suite is under the same quality assurance/quality control oversight.
4. The proposed change does not involve additional containment requirements.
5. There should remain at least one site/manufacturer, as previously authorized,
performing the same function as the one(s) to be deleted.
6. The deletion should not be due to critical deficiencies in manufacturing (for
example, recurrent out-of-specification events, environmental monitoring failures,
etc.).
Supporting data
1. Evidence of GMP compliance of the facility.
2. Name, address and responsibilities (for example, fermentation, purification) of the
proposed facility.
3. Summary of the process validation studies and results.
221
Table continued
4. Comparability of the pre-change and post-change drug substance with
respect to physicochemical properties, biological activity, purity, impurities and
contaminants, as appropriate. Nonclinical and/or clinical bridging studies may
be required if quality data alone are insufficient to establish comparability. The
extent and nature of nonclinical and/or clinical studies should be determined on
a case-by-case basis, taking into consideration the quality comparability findings,
the nature and level of the knowledge of the product, existing relevant nonclinical
and clinical data, and aspects of their use.
5. Justification for the classification of any manufacturing process and/or control
changes as moderate or minor.
6. Description of the batches and summary of in-process control and release testing
results as quantitative data, in a comparative tabular format, for at least three
consecutive commercial-scale batches of the pre-change and post-change drug
substance. Comparative pre-change test results do not need to be generated
concurrently; relevant historical testing results are acceptable. Matrixing,
bracketing, use of smaller-scale batches, use of fewer than three batches and/or
leveraging data from scientifically justified representative batches, or batches not
necessarily manufactured consecutively, may be acceptable where justified and
agreed by the NRA.
7. Comparative pre-change and post-change test results for the manufacturer’s
characterized key stability-indicating attributes for at least three commercial-scale
drug substance batches produced with the proposed changes and stored under
accelerated and/or stress conditions for a minimum of 3 months. Test results that
cover a minimum of 6 months in real-time/real-temperature conditions should
also be provided. A possibility of 3 months of real-time data could be acceptable if
properly justified (for example, it can be proven that the relevant effect, if present,
can already be observed within 3 months). Comparative pre-change test results
do not need to be generated concurrently; relevant historical results for batches
on the stability programme are acceptable. Additionally, the manufacturer should
commit to undertake real-time stability studies to confirm the full shelf-life/hold-
time of the drug substance under its normal storage conditions and to report
to the NRA any failures in these ongoing long-term stability studies. Matrixing,
bracketing, use of smaller-scale batches and/or use of fewer than three batches of
drug substance for stability testing may be acceptable where justified (6).
8. Updated post-approval stability protocol.
9. Information describing the change-over procedures for shared product-contact
equipment and the segregation procedures, as applicable. If no revisions, the
manufacturer should state that no changes were made to the change-over
procedures.
10. Cleaning procedures (including data in a summary validation report and
the cleaning protocol for the introduction of new products, as applicable)
demonstrating lack of carry-over or cross-contamination.
222
Annex 3

2. Change to the cell banks:
Note: New cell substrates that are unrelated to the licensed master cell bank (MCB) or
pre-MCB material may require a new application for marketing authorization or licence
application.
a. Adaptation of an MCB into a None 1, 2, 5–8, 10 Major
new culture medium
b. Generation of a new MCB 1 1, 2, 5–8 Moderate
c. Generation of a new working 2–4 1, 2 Minor
cell bank (WCB)
3. Change in the cell bank None 1, 2, 9 Moderate
manufacturing site
4. Change in the cell bank 5, 7 9 Minor
testing/storage site
5. Change in the cell bank None 3, 4 Moderate
qualification protocol
6 4 Minor
Conditions
1. The new MCB is generated from the original clone or from a pre-approved MCB
and is grown in the same culture medium.
2. The new cell bank is generated from a pre-approved MCB.
3. The new cell bank is at the pre-approved passage level.
4. The new cell bank is released according to a pre-approved protocol/process or as
described in the original licence.
5. No changes have been made to the tests/acceptance criteria used for the release
of the cell bank.
6. The protocol is considered more stringent (that is, addition of new tests or
narrowing of acceptance criteria).
7. No changes have been made to the storage conditions used for the cell bank, and
the transport conditions of the cell bank have been validated.
Supporting data
1. Qualification of the cell bank according to guidelines considered acceptable by
the NRA.
2. Information on the characterization and testing of the MCB/WCB, and cells from
the end-of-production passage or post-production passage.
3. Justification of the change to the cell bank qualification protocol.
4. Updated cell bank qualification protocol.
223
Table continued
5. Comparability of the pre-change and post-change drug substance with respect to
physicochemical properties, biological activity, purity, impurities and contaminants,
as appropriate. Nonclinical and/or clinical bridging studies may occasionally be
required when quality data are insufficient to establish comparability. The extent
and nature of nonclinical and/or clinical studies should be determined on a case-
by-case basis, taking into consideration the quality-comparability findings, the
nature and level of knowledge of the product, existing relevant nonclinical and
clinical data, and aspects of its use.
6. Description of the batches and summary of in-process and release testing results
as quantitative data, in a comparative tabular format, for at least three consecutive
commercial-scale batches of the drug substance derived from the new cell bank.
Matrixing, bracketing, use of smaller-scale batches, use of fewer than three
batches and/or leveraging data from scientifically justified representative batches,
or batches not necessarily manufactured consecutively, may be acceptable where
justified.
commit to undertake real-time stability studies to confirm the full shelf-life/hold-
time of the drug substance under its normal storage conditions and to report to the
NRA any failures in these ongoing long-term stability studies. Matrixing, bracketing,
the use of smaller-scale batches and/or the use of fewer than three batches of drug
substance for stability testing may be acceptable where justified (6).
9. Evidence that the new company/facility is GMP-compliant.

10. Supporting nonclinical and clinical data or a request for a waiver of in vivo studies
with justification.
224
Annex 3

6. Change to the fermentation or cell culture process:
a. A critical change (a change None 1–7, 9, 11 Major
with high potential to have
an impact on the quality
of the drug substance or
drug product; for example,
incorporation of disposable
bioreactor technology)
b. A change with moderate 1, 3 1–6, 8, 10 Moderate
potential to have an impact
on the quality of the drug
substance or drug product
(for example, extension of
the in vitro cell age beyond
validated parameters)
c. A noncritical change with 1–5, 7–10 1, 2, 4, 8 Minor
minimal potential to have
an impact on the quality of
the drug substance or drug
product, such as:
• a change in harvesting and/
or pooling procedures which
does not affect the method
of manufacture, recovery,
intermediate storage
conditions, sensitivity of
detection of adventitious
agents or production scale;
• duplication of a fermentation
train; or
• addition of similar/
comparable bioreactors
225
Table continued
7. Change to the purification process, involving the following:
a. A critical change (a change None 1, 2, 5–7, 9, Major
with high potential to have 11, 12
drug product, for example, a
change that could potentially
have an impact on the viral
clearance capacity of the
process or the impurity profile
of the drug substance)
b. A change with moderate 1, 3 1, 2, 5–7, Moderate
potential to have an impact 10–12
on the quality of the drug
substance or drug product
(for example, a change in the
chemical separation method,
such as ion-exchange HPLC 1
to reversed-phase HPLC)
c. A noncritical change with 1–4 1, 2 Minor
minimal potential to have
drug product (for example,
addition of an in-line filtration
step equivalent to the
approved filtration step)
8. Change in scale of the manufacturing process:

a. At the cell culture stage 3, 9–11 2, 3, 5–7, 9, 11 Moderate
b. At the purification stage 1, 2, 4, 6 2, 5–7, 9, 11 Moderate
9. Introduction of reprocessing 12, 13 8, 10, 11, 13 Minor
steps
10. Addition of a new holding None 5, 14 Moderate
step or change in the
parameters of an approved
holding step
1
HPLC = high-performance liquid chromatography.
226
Annex 3
Table continued
Conditions
1. The change does not have an impact on the viral clearance data or the chemical
nature of an inactivating agent.
2. There is no change in the drug substance specification outside the approved limits.
3. There is no change in the drug substance impurity profile outside the approved
limits.
4. The change is not necessitated by recurring events arising during manufacture or
because of stability concerns.
5. The change does not affect the purification process.
6. The change in scale is linear with respect to the proportionality of production
parameters and materials.
7. The new fermentation train is identical to the approved fermentation train(s).
8. There is no change in the approved in vitro cell age.
9. The change is not expected to have an impact on the quality, safety or efficacy of
the final product.
10. There is no change in the proportionality of the raw materials (that is, the change
in scale is linear).
11. The change in scale involves the use of the same bioreactor (that is, it does not
involve the use of a larger bioreactor).
12. The need for reprocessing is not due to recurrent deviations from the validated
process, and the root cause triggering reprocessing is identified.
13. The proposed reprocessing steps have been shown to have no impact on product
quality.
Supporting data
1. Justification for the classification of the change(s) as critical, moderate or
noncritical in terms of its impact on the quality of the drug substance.
2. Flow diagram (including process and in-process controls) of the proposed
manufacturing process(es) and a brief narrative description of the proposed
manufacturing process(es).
3. If the change results in an increase in the number of population doublings or
subcultivations, information on the characterization and testing of the post-
production cell bank for recombinant product or of the drug substance for non-
recombinant product.
4. For drug substance obtained from, or manufactured with, reagents obtained
from sources that are at risk of transmitting bovine spongiform encephalopathy/
transmissible spongiform encephalopathy (BSE/TSE) agents (for example,
ruminant origin), information and evidence that the material does not pose a
potential BSE/TSE risk (for example, name of manufacturer, species and tissues
from which the material is a derivative, country of origin of the source animals, use
and previous acceptance of the material) (7).
5. Process validation results.
227
Table continued
6. Comparability of the pre-change and post-change drug substance with
respect to physicochemical properties, biological activity, purity, impurities
and contaminants, as appropriate. Nonclinical and/or clinical bridging studies
may occasionally be required when quality data are insufficient to establish
comparability. The extent and nature of nonclinical and/or clinical studies should
be determined on a case-by-case basis, taking into consideration the quality–
comparability findings, the nature and level of knowledge of the product, existing
relevant nonclinical and clinical data, and aspects of its use.
as quantitative data, in a comparative tabular format, for at least three consecutive
commercial-scale batches of the pre-change and post-change drug substance.
Comparative pre-change test results do not need to be generated concurrently;
relevant historical testing results are acceptable. Matrixing, bracketing, the use
of smaller-scale batches, the use of fewer than three batches and/or leveraging
data from scientifically justified representative batches, or batches not necessarily
manufactured consecutively, may be acceptable where justified.
as quantitative data, in a comparative tabular format, for one commercial-scale
batch of the pre-change and post-change drug substance. Comparative pre-
change test results do not need to be generated concurrently; relevant historical
testing results are acceptable. Batch data on the next two full-production batches
should be made available on request and should be reported by the marketing
authorization holder if outside the specification (with proposed action). The use of
a smaller-scale batch may be acceptable where justified and.
also be provided. A possibility of 3 months and one batch of real-time data could
be acceptable if properly justified (for example, it can be proven that the relevant
effect, if present, can already be observed within 3 months). Comparative pre-

results for batches on the stability programme are acceptable. Additionally, the
manufacturer should commit to undertake real-time stability studies to confirm
the full shelf-life/hold-time of the drug substance under its normal storage
conditions and to report to the NRA any failures in these ongoing long-term
stability studies. Matrixing, bracketing, the use of smaller-scale batches and/or
the use of fewer than three batches of drug substance for stability testing may be
acceptable where justified (6).
228
Annex 3
Table continued
characterized key stability-indicating attributes with at least one commercial-scale
drug substance batch produced with the proposed changes under real-time/
real-temperature testing conditions. Comparative pre-change test results do
not need to be generated concurrently; relevant historical results for batches
on the stability programme are acceptable. Test results that cover a minimum of
6 months in real-time/real-temperature conditions should also be provided. A
possibility of 3 months of real-time data could be acceptable if properly justified
(for example, it can be proven that the relevant effect, if present, can already be
observed within 3 months). Additionally, the manufacturer should commit to
undertake real-time stability studies to confirm the full shelf-life/hold-time of the
drug substance under its normal storage conditions and to report to the NRA
any failures in these ongoing long-term stability studies. Matrixing, bracketing,
the use of smaller-scale batches and/or use of forced degradation or accelerated
temperature conditions for stability testing may be acceptable where justified.
11. Updated post-approval stability protocol and stability commitment to place the
first commercial-scale batch of the drug product manufactured using the post-
change drug substance into the stability programme.
12. Information assessing the risk with respect to potential contamination with
adventitious agents (for example, impact on viral clearance studies and BSE/TSE
risk) (7).
13. Data describing the root cause triggering the reprocessing, as well as validation
data (for example, extended hold-times, resistance to additional mechanical
stress) to help prevent the reprocessing from having an impact on the drug
substance.
14. Demonstration that the new or revised holding step has no negative impact on
the quality of the drug substance (data from one commercial-scale or scientifically
justified representative drug substance batch should be provided).

11. Change in equipment used in the drug substance manufacturing
process, involving the following:
Note: New bioreactor technology (for example, a change from stainless steel bioreactor
to disposable bioreactor) is excluded from this table and should be filed according to
change 6a.
a. Introduction of new None 1–5 Moderate
equipment with different
operating principles and 3, 4 1, 2, 5 Minor
different product contact
material
229
Table continued
b, Introduction of new None 1, 3–5 Moderate
equipment with the same
operating principles but 3, 4 1, 4, 5 Minor
different product contact
material
c. Introduction of new None 1–3, 5 Moderate
equipment with different
operating principles but 4 1, 2, 5 Minor
the same product contact
material
d. Replacement of product- None 3 Minor
contact equipment with
equivalent equipment
e. Change of product-contact 1, 2 1, 6 Minor
equipment from dedicated to
shared
f. Relocation of major 2, 4, 5 None Minor
equipment to another room
in the same facility/suite/
premises
Conditions
1. The site is approved as a multi-product facility.
2. The change has no impact on the risk of cross-contamination and is supported by
validated cleaning procedures.
3. The manufacturing process is not impacted by the change in product-contact
equipment.
4. The change has no impact on product quality.
5. Re-qualification of the equipment follows the original qualification protocol.
Supporting data
1. Information on the in-process control testing.
2. Process validation study reports.
3. Description of the batches and summary of results as quantitative data, in a
comparative tabular format, for one commercial-scale batch of the drug substance
produced with the approved and proposed product contact equipment/material.
Batch data on the next two full-production batches should be made available
on request and reported by the marketing authorization holder if outside
specification (with proposed action).
230
Annex 3
Table continued
4. Information on leachables and extractables.
5. Information on the new equipment and comparison of similarities and differences
regarding operating principles and specifications between the new and the
replaced equipment.
6. Information describing the change-over procedures for the shared product-
contact equipment.

12. Change in specification for the materials, involving the following:
a. Narrowing of the approved 1–4 1–3, 5 Minor
specification limits
for starting materials/
intermediates
b. Widening of the approved None 1–3, 5, 7 Moderate
specification limits
for starting materials/ 3–7 3–6 Minor
intermediates
13. Change in supplier of raw None 4, 6, 9, 10 Moderate
materials of biological
origin (for example, fetal 8 4, 6 Minor
calf serum, insulin, trypsin)
14. Change in source of raw None 4, 7, 9, 10 Moderate
materials of biological origin
(for example, bovine trypsin 8 4, 7 Minor
to porcine trypsin)
Conditions
1. The change in specification for the materials is within the approved limits.
2. The grade of the materials is the same or is of higher quality, where appropriate.
3. There is no change in the drug substance specification outside the approved
limits.
4. There is no change in the impurity profile of the drug substance outside the
approved limits.
5. The change has no significant effect on the overall quality of the drug substance
and/or drug product and there are no changes to the cell banks.
231
Table continued
7. The test does not concern a critical attribute (for example, content, impurity, any
critical physical characteristics or microbial purity).
8. The change is for compendial raw materials of biological origin (excluding human
plasma-derived materials).
Supporting data
1. Revised information on the quality and controls of the materials (for example,
raw materials, starting materials, solvents, reagents and catalysts) used in the
manufacture of the post-change drug substance.
2. Updated drug substance specification, if changed.
3. Copies or summaries of analytical procedures if new analytical procedures are used.
4. For drug substance obtained from, or manufactured with, reagents obtained
from sources that are at risk of transmitting bovine spongiform encephalopathy/
transmissible spongiform encephalopathy (BSE/TSE) agents (for example,
ruminant origin), information and evidence that the material does not pose a
potential BSE/TSE risk (for example, name of manufacturer, species and tissues
from which the material is a derivative, country of origin of the source animals, use
and previous acceptance of the material) (7).
5. Comparative table or description, where applicable, of pre-change and post-
change in-process tests/limits.
should be made available on request and reported by the marketing authorization
holder if outside specification (with proposed action). The use of a smaller-scale
batch may be acceptable where justified.
as quantitative data, in a comparative tabular format, for three consecutive

8. Justification/risk assessment showing that the attribute is non-significant.
adventitious agents (for example, impact on viral clearance studies and BSE/TSE
risk) (7).
10. Information demonstrating suitability of the auxiliary materials/reagents of both
sources through the comparability of the drug substance.
232
Annex 3

15. Change to in-process tests and/or acceptance criteria applied during
manufacture of the drug substance, involving the following:
a. Narrowing of approved in- 1, 3, 6, 7 1, 4 Minor
process limits
b. Addition of new in-process 2, 3, 6 1–5, 8 Minor
test and limits
c. Deletion of a non-significant 1–4, 6 1, 4, 7 Minor
in-process test
d. Widening of the approved None 1–4, 6, 8 Moderate
in-process limits
1–4 1, 4, 5, 8 Minor
e. Deletion of an in-process test None 1, 4, 6, 8 Moderate
which may have a significant
effect on the overall quality of
the drug substance
f. Addition or replacement of None 1–4, 6, 8 Moderate
an in-process test as a result
of a safety or quality issue
16. Change in the in-process 1–3, 5, 6 9 Minor
controls testing site
Note: Transfer of in-process control
testing to a different facility
within a GMP-compliant site is
not considered to be a reportable
change but is treated as a minor
GMP change and is reviewed
during inspections.
Conditions
1. No change in the drug substance specification outside the approved limits.
2. No change in the impurity profile of the drug substance outside the approved limits.
4. The test does not concern a critical attribute (for example, content, impurity, any
5. The replaced analytical procedure maintains or tightens precision, accuracy,
specificity and sensitivity, if applicable.
6. No change in the approved in-process controls outside the approved limits.
7. The test procedure remains the same, or changes in the test procedure are minor.
233
Table continued
Supporting data
1. Revised information on the controls performed at critical steps of the
manufacturing process and on intermediates of the proposed drug substance.
2. Updated drug substance specification, if changed.
4. Comparative table or description, where applicable, of pre-change and post-
change in-process tests/limits.
should be made available on request and reported by the marketing authorization
holder if outside specification (with proposed action). The use of a smaller-scale
batch may be acceptable where justified.
as quantitative data, in a comparative tabular format, for three consecutive
8. Justification for the new in-process test and limits.


17. Change in the approved design space, involving the following:
a. Establishment of a new None 1 Major
design space
b. Expansion of the approved None 1 Major
design space
c. Reduction in the approved 1 1 Minor
design space (any change
that reduces or limits the
range of parameters used to
define the design space)
234
Annex 3
Table continued
Conditions
1. The reduction in design space is not necessitated by recurring problems arising
during manufacture.
Supporting data
1. Manufacturing development data to support the establishment of, or changes to,
the design space.
Control of the drug substance

18. Change affecting the quality control (release and stability)
testing of the drug substance, involving the following:
Note: Transfer of testing to a different facility within a GMP-compliant site is not considered
to be a reportable change but is treated as a minor GMP change and is reviewed during
inspections.
a. Transfer of the quality control None 1, 2 Moderate
testing activities for a non-
pharmacopoeial assay to a 1–3 1, 2 Minor
new company not approved
in the current marketing
authorization or licence, or
to a different site within the
same company
b. Transfer of the quality None 1, 2 Moderate
control testing activities for
a pharmacopoeial assay to a 1 1, 2 Minor
authorization or licence
Conditions
1. The transferred quality control test is not a potency assay or bioassay.
2. No changes are made to the test method.
3. The transfer is within a facility approved in the current marketing authorization for
the performance of other tests.
Supporting data
1. Information demonstrating technology transfer qualification for the non-
pharmacopoeial assay or verification for the pharmacopoeial assay.
235

19. Change in the standard/monograph (that is, specifications)
claimed for the drug substance, involving the following:
a. A change from a None 1–5 Moderate
pharmacopoeial standard/
monograph to an in-house
standard
b. A change from an in-house 1–4 1–3 Minor
standard to a pharmacopoeial
standard/monograph or
from one pharmacopoeial
standard/ monograph to a
different pharmacopoeial
standard/monograph
20. Change in the specifications 1, 2 1, 2 Minor
for the drug substance in
order to comply with an
updated pharmacopoeial
standard/monograph
Conditions
1. The change is made exclusively in order to comply with a pharmacopoeial
monograph.
2. There is no change in drug substance specifications outside the approved ranges.
3. There is no deletion of tests or relaxation of acceptance criteria of the approved
specifications, except to comply with a pharmacopoeial standard/monograph.
4. There are no deletions or changes to any analytical procedures, except to comply
with a pharmacopoeial standard/monograph.
Supporting data
1. Revised drug product labelling information, as applicable.
2. Updated copy of the proposed drug substance specifications.
3. Where an in-house analytical procedure is used and a pharmacopoeial standard/
monograph is claimed, results of an equivalency study between the in-house and
pharmacopoeial methods.
4. Copies or summaries of validation reports if new analytical procedures are used.
5. Justification of specifications with data.
236
Annex 3

21. Changes in the control strategy of the drug substance,
involving the following:
a. Change from end-product None 1–3, 5 Major
testing to upstream
controls for some test(s) (for
example, real-time release
testing, process analytical
technology)
b. Addition of a new critical None 1–5 Moderate
quality attribute in the
control strategy
c. Deletion of a critical quality None 1, 5 Moderate
attribute from the control
strategy
Conditions
None
Supporting data
1. Information on the controls performed at critical steps of the manufacturing
process and on intermediates of the proposed drug substance.
2. Updated copy of the proposed drug substance specifications.
4. Copies or summaries of validation reports if new analytical procedures are used to
monitor the new CQA at release.
5. Justification and supporting data for each proposed change to the control strategy.

22. Change in the specification/analytical procedure used to
release the drug substance, involving the following:
a. Deletion of a test None 1, 5, 6 Moderate
b. Addition of a test 1–3 1–3, 5 Minor
c. Replacement of an analytical None 1–5 Moderate
procedure
5, 6, 8 1, 4, 5 Minor
d. Changes to an approved None 1–5 Moderate
analytical procedure
2, 4–6 1, 4, 5 Minor
237
Table continued
e. Change from an in-house None 1–5 Moderate
analytical procedure to a
recognized compendial/ 2, 6 1–3 Minor
pharmacopoeial analytical
procedure
f. Widening of an approved None 1, 5, 6 Moderate
acceptance criterion
g. Narrowing of an approved 1, 4, 7 1 Minor
Conditions
1. The change does not result from unexpected events arising during manufacture
(for example, new unqualified impurity, change in total impurity limits).
2. There is no change in the limits/acceptance criteria outside the approved limits for
the approved assays used at release/ stability.
3. The addition of the test is not intended to monitor new impurity species.
4. The method of analysis is the same and is based on the same analytical technique
or principle (for example, change in column length or temperature, but not a
different type of column or method) and no new impurities are detected.
5. The modified analytical procedure maintains or improves performance
parameters of the method.
6. The change does not concern potency-testing.
7. Acceptance criteria for residual solvent are within recognized or approved
acceptance limits (for example, within ICH limits for a Class 3 residual solvent, or
pharmacopoeial requirements).
8. The change is from one pharmacopoeial assay to another pharmacopoeial assay or
the marketing application holder has demonstrated an increased understanding

of the relationship between method parameters and method performance
defined by a systematic development approach including robustness studies.
Supporting data
1. Updated drug substance specifications.
3. Validation/qualification results if new analytical procedures are used.
4. Comparative results demonstrating that the approved and proposed analytical
procedures are equivalent.
5. Justification for the proposed drug substance specification (for example, tests,
acceptance criteria or analytical procedures).
6. Documented evidence that consistency of quality is maintained.
238
Annex 3
Reference standards or materials

23. Replacement of a primary None 1, 2 Moderate
reference standard
24. Change of the reference None 1, 2 Moderate
standard from
pharmacopoeial or
international standard to
in‑house (no relationship
with international standard)
25. Change of the reference 3 1, 2 Minor
standard from in-house
(no relationship with
international standard)
to pharmacopoeial or
international standard
26. Qualification of a new 1 1, 2 Minor
batch of reference standard
against the approved
reference standard (including
qualification of a new batch
of a secondary reference
standard against the
approved primary standard)
27. Change to reference None 3, 4 Moderate
standard qualification
protocol
28. Extension of the reference 2 5 Minor
standard shelf-life or re-test
period
Conditions
1. Qualification of the new reference standard is in accordance with an approved
protocol.
2. The extension of the shelf-life of the reference standard is in accordance with an
approved protocol.
3. The reference standard is used for a physicochemical test.
239
Table continued
Supporting data
1. Justification for the change in reference standard.
2. Information demonstrating qualification of the proposed reference standards
or materials (for example, source, characterization, certificate of analysis,
comparability data).
3. Justification of the change to the reference standard qualification protocol.
4. Updated reference standard qualification protocol.
5. Summary of stability testing and results to support the extension of reference
standard shelf-life.
Drug substance container closure system

29. Change in the primary None 1, 2, 4, 5 Moderate
container closure system(s)
for the storage and shipment 1 1, 3, 5 Minor
of the drug substance
Conditions
1. The proposed container closure system is at least equivalent to the approved
container closure system with respect to its relevant properties (including results
of transportation or compatibility studies, if appropriate).
Supporting data
1. Updated dossier sections describing information on the proposed container
closure system (for example, description, composition, materials of construction
of primary packaging components, specifications).
2. Data demonstrating the suitability of the container closure system (for example,
extractable/leachable testing) and compliance with pharmacopoeial standards, if

applicable.
3. Results demonstrating that the proposed container closure system is at least
equivalent to the approved container closure system with respect to its relevant
properties (for example, results of transportation or compatibility studies, and
extractable/leachable studies).
240
Annex 3
Table continued
characterized key stability-indicating parameters with commercial-scale drug
substance material using several container batches (for example, three different
batches) produced with the proposed changes and stored under accelerated and/
or stress conditions for a minimum of 3 months. Test results that cover a minimum
of 6 months in real-time/real-temperature conditions should also be provided. A
possibility of 3 months of real-time data could be acceptable if properly justified
(for example, it can be proven that the relevant effect, if present, can already be
observed within 3 months). Comparative pre-change test results do not need to
be generated concurrently; relevant historical results for batches on the stability
programme are acceptable. Additionally, the manufacturer should commit to
undertake real-time stability studies to confirm the full shelf-life/hold-time of the
drug substance under its normal storage conditions and to report to the NRA any
failures in these ongoing long-term stability studies. Matrixing, bracketing, the use
of smaller-scale batches and/or the use of fewer than three container batches for
stability testing may be acceptable where justified (6).
5. Comparative table of pre-change and post-change specifications of the container
closure system.

30. Change in the supplier for a primary container closure,
a. Replacement or addition of a None 1–3 Moderate
supplier
1, 2 None Minor
b. Deletion of a supplier None None Minor
Conditions
1. There is no change in the type of container closure, the materials of construction
or the sterilization process for a sterile container closure component.
2. There is no change in the specifications of the container closure component
outside the approved ranges.
Supporting data
1. Data demonstrating the suitability of the container closure system (for example,
extractable/leachable testing).
2. Information on the proposed container closure system (for example, description,
materials of construction of primary packaging components, specifications).
241
Table continued
3. Test results that cover a minimum of 6 months in real-time/real-temperature
conditions should also be provided. A possibility of 3 months of real-time data could
be acceptable if properly justified (for example, it can be proven that the relevant
effect, if present, can already be observed within 3 months). Comparative pre-
results for batches on the stability programme are acceptable. Additionally, the
manufacturer should commit to undertake real-time stability studies to confirm the
full shelf-life/hold-time of the drug substance under its normal storage conditions
and to report to the NRA any failures in these ongoing long-term stability studies.
Matrixing, bracketing, the use of smaller-scale batches and/or the use of fewer
than three batches of drug substance for stability testing may be acceptable where
justified (6).

31. Change in the specification/analytical procedure of the primary container
closure system for the drug substance, involving the following:
a. Deletion of a test 1, 2 1, 2 Minor
b. Addition of a test 3 1–3 Minor
c. Replacement of an analytical 6, 7 1–3 Minor
procedure
d. Minor changes to an 4–7 1–3 Minor
e. Widening of an acceptance None 1, 2 Moderate
criterion
f. Narrowing of an acceptance 8 1 Minor

criterion
Conditions
1. The deleted test has been demonstrated to be redundant compared to the
remaining tests or is no longer a pharmacopoeial requirement.
2. The change to the specification does not affect the functional properties of the
container closure component and does not result in a potential impact on the
performance of the drug substance.
3. The change is not necessitated by unexpected recurring events arising during
manufacture of the primary container closure system or because of stability
concerns.
4. There is no change in the acceptance criteria outside the approved limits.
5. The new analytical procedure is of the same type.
242
Annex 3
Table continued
6. Results of method validation demonstrate that the new or modified analytical
procedure is at least equivalent to the approved analytical procedure.
7. The new or modified analytical procedure maintains or tightens precision,
accuracy, specificity or sensitivity.
8. The change is within the range of approved acceptance criteria.
Supporting data
1. Updated copy of the proposed specification for the primary container closure
system.
2. Rationale for the change.
3. Description of the analytical procedure and, if applicable, validation data.
Stability
32. Change in the shelf-life of the drug substance or for a stored
intermediate of the drug substance, involving the following:
a. Extension None 1–5 Moderate
1–4 1, 2, 5 Minor
b. Reduction None 1–5 Moderate
5 2–4 Minor
Conditions
1. There are no changes to the container closure system in direct contact with the
drug substance with the potential of impact on the drug substance, or to the
recommended storage conditions of the drug substance.
2. Full long-term stability data are available covering the proposed shelf-life and are
based on stability data generated on at least three commercial-scale batches.
3. Stability data were generated in accordance with the approved stability protocol.
4. Significant changes were not observed in the stability data.
5. The reduction in the shelf-life is not necessitated by recurring events arising
during manufacture or because of stability concerns (Note: Problems arising during
manufacturing or stability concerns should be reported for evaluation).
Supporting data
1. Summary of stability testing and results (for example, studies conducted, protocols
used, results obtained).
2. Proposed storage conditions and shelf-life, as appropriate.
3. Updated post-approval stability protocol and stability commitment.
243
Table continued
4. Justification for the change to the post-approval stability protocol or stability
commitment.
5. Results of stability testing (that is, full real-time/real-temperature stability data
covering the proposed shelf-life generated on stability testing of at least three
commercial-scale batches unless otherwise justified). For intermediates, data to
show that the extension of shelf-life has no negative impact on the quality of the
drug substance. Under special circumstances, interim stability-testing results and
a commitment to notify the NRA of any failures in the ongoing long-term stability
studies may be provided. In such cases, the extrapolation of shelf-life should be
made in accordance with ICH Q1E guidelines (8).

33. Change in the post-approval stability protocol of the
drug substance, involving the following:
a. Substantial change to the None 1–5 Moderate
post-approval stability
protocol or stability 1 1, 2, 4, 5 Minor
commitment, such as deletion
of a test, replacement of
an analytical procedure
or change in storage
temperature
b. Addition of test(s) into the 2 1, 2, 4, 5 Minor
post-approval stability
protocol
c. Deletion of time point(s) from 3 4, 5 Minor
the post-approval stability

protocol within the approved
shelf-life
Conditions
1. In the case of replacement of an analytical procedure, the new analytical
procedure maintains or tightens precision, accuracy, specificity and sensitivity.
2. The addition of test(s) is not due to stability concerns or to the identification of
new impurities.
3. Deletion of time point(s) is made in accordance with relevant guidelines (for
example, (6)).
244
Annex 3
Table continued
Supporting data
2. Validation results if new analytical procedures are used.
3. Proposed storage conditions and/or shelf-life, as appropriate.
4. Updated post-approval stability protocol including justification for the changes,
and stability commitment.
5. If applicable, stability-testing results to support the change to the post-approval
stability protocol or stability commitment (for example, data to show greater
reliability of the alternative test).

34. Change in the storage conditions for the drug
substance, involving the following:
a. Addition or change to None 1–4 Moderate
storage conditions for the
drug substance (for example, 1, 2 1–3 Minor
widening or narrowing of a
temperature criterion)
b. Addition of a cautionary None 1, 3, 4 Moderate
statement
1 1, 3, 4 Minor
c. Deletion of a cautionary None 1, 3, 5 Minor
statement
Conditions
2. The change consists in the narrowing of a temperature criterion within the
approved ranges.
Supporting data
1. Proposed storage conditions and shelf-life.
3. Justification of the change in the storage conditions/cautionary statement.
4. Results of stability testing (that is, full real-time/real-temperature stability data
covering the proposed shelf-life generated on one commercial-scale batch).
5. Results of stability testing (that is, full real time/real temperature stability data
covering the proposed shelf-life generated on at least three commercial-scale
batches).
245
References
1. The common technical document for the registration of pharmaceuticals for human use:
quality – M4Q(R1) (Step 4 version). Geneva: International Conference on Harmonisation of
Technical Requirements for Registration of Pharmaceuticals for Human Use; 2002 (http://
www.ich.org/fileadmin/Public_Web_Site/ICH_Products/CTD/M4_R1_Quality/M4Q__R1_.pdf,
2. CTD Quality (M4Q) guideline. M4Q Implementation Working Group: Questions & Answers (R1) –
M4Q Q&As (R1). Geneva: International Conference on Harmonization of Technical Requirements
for Registration of Pharmaceuticals for Human Use; 2003 (http://www.ich.org/fileadmin/
Public_Web_Site/ICH_Products/CTD/M4_R1_Quality/M4_Quality_Questions_Answers_R1.pdf,
12 December 2017).
12 December 2017).
6. Stability testing of biotechnological/biological products. ICH Guideline Q5C. Geneva: International
Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for
Human Use; 1995 (https://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/
Quality/Q5C/Step4/Q5C_Guideline.pdf, accessed 12 December 2017).
7. WHO guidelines on transmissible spongiform encephalopathies in relation to biological and

8. Evaluation for stability data. ICH Guideline Q1E. Geneva: International Conference on
2003 (http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q1E/
Step4/Q1E_Guideline.pdf, accessed 12 December 2017).
246
Annex 3
Appendix 3
Changes to the drug product
The examples presented in this appendix are intended to assist with the
classification of changes made to the quality information of the drug product.
The information summarized in the drug product table provides guidance on:
■■ the conditions to be fulfilled in order for a given change to be

classified as major, moderate or minor (if any of the conditions
outlined for a given change are not fulfilled, the change is
automatically considered to be at the next higher reporting category
– for example, if any of the conditions recommended for a moderate
quality change are not fulfilled, the change is considered to be a
major quality change);
■■ the supporting data for a given change, either to be submitted to
the NRA and/or maintained by the marketing authorization holder
(if any of the supporting data outlined for a given change are not
provided, are different or are not considered applicable, adequate
scientific justification should be provided); and
■■ the reporting category (major, moderate or minor quality change).
Marketing authorization holders should use scientific judgement,

leverage competent regulatory authority guidance or contact the NRA if a
change is not included in the table and has the potential to impact on product
quality. Marketing authorization holders should also contact the NRA when a
change is considered at the next higher reporting category because any of the
conditions outlined are not fulfilled and where the supporting data are not
described. NRAs should establish procedures, with appropriate timelines, on
the conducting and recording of communications between themselves and
Supporting data should be provided according to the submission format
accepted by the NRA – see for example (1, 2).
Additional information on data requirements to support quality changes
can be found in WHO good manufacturing practices for biological products (3),
WHO Guidelines on the quality, safety and efficacy of biotherapeutic protein
products prepared by recombinant DNA technology (4) and in relevant ICH
guidelines (5, 6).
247
Quality changes to comply with updated

compendia and/or pharmacopoeias
NRAs should make a list of the recognized compendia and/or pharmacopoeias.
Manufacturers are expected to comply with the current version of compendia/
pharmacopoeias as referenced in the approved marketing authorization. Changes
in the compendial/pharmacopoeial methods or specifications for a drug product
do not need to be submitted for review if reference is made to the current edition
of the compendium or pharmacopoeia, but the changes should be notified to the
NRA, with information on them available for inspection.
In some cases, changes made to comply with recognized compendia/
pharmacopoeias may require approval by the NRA prior to implementation
regardless of the timing of the change in relation to the date when the
compendium/pharmacopoeia was updated. For example, supplement submission
and approval by the NRA may be required for some changes to quality control
tests performed for product release (for example, to potency tests), for changes
that have an impact on any product labelling information item, and for changes
that may affect the quality, safety or efficacy of the product.
Quality changes affecting lot release

While WHO recognizes that independent lot release by NRAs or national
control laboratories is required for vaccines, in some countries this lot release
system also applies to other types of products, such as plasma-fractionated
products. Where post-approval changes to the final product affect the lot
release protocol (for example, changes to test procedures, reference standards
or laboratory sites) or sample testing requirements for lot release, the marketing
authorization holder should inform the institution responsible for reviewing
the release of product lots. These procedures apply to changes that have been
authorized by the NRA in the case of major and moderate quality changes and
to changes that have been implemented in the case of minor quality changes.
For example, the qualification of a new lot of reference standard against the
approved reference standard may be considered a minor quality change if the
qualification of a new standard is performed in accordance with an approved
protocol and specification. Nevertheless, these changes must be reported to the
NRA or national control laboratory as appropriate.
248
Annex 3
Description and composition of the drug product

Note: Changes in dosage form and/or presentation may, in some cases, necessitate the filing
of a new application for marketing authorization or licensure. Marketing authorization
holders are encouraged to contact the NRA for further guidance.

35. Change in the description or composition of the
drug product, involving the following:
a. Addition of a dosage form or None 1–10 Major
change in the formulation (for
example, lyophilized powder
to liquid, change in the amount
of excipient, new diluent for
lyophilized product)
b. Change in fill volume (same None 1, 5, 7, 9, 10 Major
concentration, different
volume) 1, 2 1, 5, 7, 9 Moderate
1–3 5, 7, 9 Minor
c. Change in the concentration None 1, 5, 7, 9, 10 Major
of the active ingredient (for
example, 20 units/ml versus 2, 4, 5 1, 5, 7 Moderate
10 units/ml)
d. Addition of a new None 1, 5, 7–10 Major
presentation (for example,
addition of a new pre-filled
syringe where the approved
presentation is a vial for a
biotherapeutic in a liquid
dosage form)
Conditions
1. No changes are classified as major in the manufacturing process to accommodate
the new fill volume.
2. No change in the dose is recommended.
3. The change involves narrowing the fill volume while maintaining the lower limit of
extractable volume.
4. The new concentration is bracketed by existing approved concentrations.
5. More than two concentrations are already approved (that is, linear PK/PD profile of
the product from at least three different concentrations over the bracketed range
has been demonstrated and the two extreme concentrations of the bracketed
range have been shown to be bioequivalent or therapeutically equivalent).
249
Table continued
Supporting data
2. Characterization data demonstrating comparability of the new dosage form and/
or formulation.
3. Description and composition of the dosage form if there are changes to the
composition or dose.
4. Discussion of the components of the drug product, as appropriate (for example,
choice of excipients, compatibility of drug substance and excipients, leachates,
compatibility with new container closure system).
5. Information on the batch formula, manufacturing process and process controls,
controls of critical steps and intermediates, process validation results.
6. Control of excipients if new excipients are proposed (for example, specification).
7. Information on specification, analytical procedures (if new analytical methods are
used), validation of analytical procedures (if new analytical methods are used),
batch analyses (certificate of analysis for three consecutive commercial-scale
batches should be provided). Bracketing for multiple-strength products, container
sizes and/or fills may be acceptable if scientifically justified.
8. Information on the container closure system and leachables and extractables,
if any of the components have changed (for example, description, materials of
construction and summary of specification).
drug product batches produced with the proposed changes and stored under
commit to undertake real-time stability studies to confirm the full shelf-life/

hold-time of the drug product under its normal storage conditions and to report
bracketing, the use of smaller-scale batches and/or the use of fewer than three
batches of drug product for stability testing may be acceptable where justified (6).
10. Supporting clinical data or a justification for why such studies are not needed.
250
Annex 3
Description and composition of the drug

product: change to a diluent
36. Change to the diluent, involving the following:
a. Change in manufacturing None 1–5 Moderate
process
1, 3 1–4 Minor
b. Replacement of or addition to None 1–6 Moderate
the source of a diluent
1–3 1–3 Minor
c. Change in facility used to 1, 2 1, 3, 5 Minor
manufacture a diluent (same
company)
d. Addition of a diluent filling 1, 2, 4 1, 3, 5 Minor
line
e. Deletion of a diluent None None Minor
Conditions
1. The diluent is water for injection or a salt solution (including buffered salt
solutions) – that is, it does not include an ingredient with a functional activity such
as a preservative, and there is no change to its composition.
2. After reconstitution, there is no change in the drug product specification outside
the approved limits.
3. The proposed diluent is commercially available in the country/jurisdiction of
the NRA.
4. The addition of the diluent filling line is in an approved filling facility.
Supporting data
1. Flow diagram (including process and in-process controls) of the proposed
manufacturing process(es) and a brief narrative description of the proposed
manufacturing process(es).
2. Updated copy of the proposed specification for the diluent.
comparative tabular format, for at least three consecutive commercial-scale
batches of the approved and proposed diluent. Comparative test results for the
approved diluent do not need to be generated concurrently; relevant historical
testing results are acceptable.
4. Updated stability data on the product reconstituted with the new diluent.
5. Evidence that the facility is GMP-compliant.
251
Manufacture
37. Change in the approved design space, involving the following:
a. Establishment of a new None 1 Major
design space
b. Expansion of the approved None 1 Major
design space
c. Reduction in the approved 1 1 Minor
design space (any change
that reduces or limits the
range of parameters used to
define the design space)
Conditions
1. The reduction in design space is not necessitated by recurring problems that have
arisen during manufacture.
Supporting data
1. Pharmaceutical development data to support the establishment or changes to
the design space.

38. Change involving a drug product manufacturer/
manufacturing facility, involving the following:
a. Replacement or addition of None 1–7 Major
a manufacturing facility for

the drug product (including 1–5 1–3, 5–8 Moderate
formulation/filling and
primary packaging)
b. Conversion of a drug product None 9, 10 Moderate
manufacturing facility from
single-product to multi-
product facility
c. Replacement or addition 2, 3 1–3 Minor
of a secondary packaging
facility, including secondary
functional packaging (that is,
assembly) facility
252
Annex 3
Table continued
d. Deletion of a drug product 6, 7 None Minor
manufacturing facility or
packaging site
Conditions
1. The proposed facility is an approved formulation/filling facility (for the same
company/marketing authorization holder).
2. There is no change in the composition, manufacturing process and drug product
specification.
3. There is no change in the container/closure system and storage conditions.
4. The same validated manufacturing process at critical steps (that is, compounding
and filling) is used.
5. The newly introduced product is in the same family of product(s), or in the same
therapeutic classification, as the products already approved at the site, and also
uses the same filling process/equipment.
6. There should remain at least one site/manufacturer, as previously authorized,
performing the same function as the one(s) to be deleted.
7. The deletion should not be due to critical deficiencies in manufacturing (for example,
recurrent out-of-specification events, environmental monitoring failures, etc.).
Supporting data
1. Name, address and responsibilities (for example, formulation, filling, primary/
secondary packaging) of the proposed production facility involved in
manufacturing and testing.
2. Evidence that the facility is GMP-compliant.
3. Confirmation that the description of the manufacturing process of the drug
product has not changed (other than the change in facility), or submission of
supporting data on the revised description of the manufacturing process if the
process has changed.
4. Comparative description of the manufacturing process, if different from the
approved process, and information on the controls performed at critical steps of
the manufacturing process and on the intermediate of the proposed final product.
5. Summary of the process validation studies and results.
product. Comparative pre-change test results do not need to be generated
concurrently; relevant historical testing results are acceptable. Bracketing for
multiple-strength products, container sizes and/or fills may be acceptable if
scientifically justified.
253
Table continued
8. Rationale for considering the proposed formulation/filling facility as equivalent.
9. Information describing the change-over procedures for shared product-contact
equipment and the segregation procedures, as applicable. If there are no revisions,
the manufacturer should state that no changes were made to the change-over
procedures.
10. Cleaning procedures (including data in a summary validation report and the
cleaning protocol for the introduction of new products, as applicable)
demonstrating lack of carry-over or cross-contamination.

39. Change in the drug product manufacturing process,
a. Scale-up of the None 1–6 Major

manufacturing process at the
formulation/filling stage 1–4 1–6 Moderate
b. Addition or replacement of None 1–7 Moderate

equipment (for example,
formulation tank, filter 5 2, 7, 8 Minor
housing, filling line and head,
lyophilizer)
c. Addition of a new scale None 1, 3–5 Moderate
bracketed by the approved
scales or scale-down of the 1–4, 8 1, 4 Minor
manufacturing process
254
Annex 3
Table continued
d. Addition of a new step (for 3 1–6 Moderate
example, filtration)
e. Product-contact equipment 6, 7 2, 9 Minor
change from dedicated
to shared (for example,
formulation tank, filter
housing, filling line and head,
lyophilizer)
Conditions
1. The proposed scale uses similar/comparable equipment to the approved
equipment. Note: Change in equipment size is not considered as using similar/
comparable equipment.
2. Any changes to the manufacturing process and/or to the in-process controls
are only those necessitated by the change in batch size (for example, the same
formulation, controls and standard operating procedures are utilized).
3. The change should not be a result of recurring events that have arisen during
manufacture or because of stability concerns.
4. There is no change in the principle of the sterilization procedures of the drug
product.
5. Replacement of equipment with equivalent equipment; the change is considered
“like for like” (that is, in terms of product contact material, equipment size and
operating principles).
6. The site is approved as a multi-product facility.
7. The change has no impact on the risk of cross-contamination and is supported by
validated cleaning procedures.
8. The change does not affect the lyophilization step.
Supporting data
1. Description of the manufacturing process, if different from the approved process,
and information on the controls performed at critical steps of the manufacturing
process and on the intermediate of the proposed drug product.
2. Information on the in-process control testing, as applicable.
3. Process validation results (for example, media fills), as appropriate.
product. Comparative pre-change test results do not need to be generated
concurrently; relevant historical testing results are acceptable. Bracketing for
multiple-strength products, container sizes and/or fills may be acceptable if
scientifically justified.
255
Table continued
6. Information on leachables and extractables, as applicable.
7. Information on the new equipment and comparison of similarities and differences
regarding operating principles and specifications between the new and the
replaced equipment.
8. The rationale for regarding the equipment as similar/comparable, as applicable.
9. Information describing the change-over procedures for the shared product-
contact equipment.

40. Change in the controls (in-process tests and/or acceptance criteria) applied
during the manufacturing process or on intermediates, involving the following:
a. Narrowing of approved in- 2, 3, 7 1, 4 Minor
process limits
b. Addition of new in-process 2, 3, 6 1–5, 8 Minor
test and limits
c. Deletion of a non-significant 2–4 1, 4, 7 Minor
in-process test
d. Widening of the approved None 1–4, 6, 8 Moderate
in-process limits
1–3 1, 4, 5, 8 Minor
e. Deletion of an in-process test None 1, 4, 6,8 Moderate
which may have a significant
effect on the overall quality of
the drug product
256
Annex 3
Table continued
f. Addition or replacement of None 1–4, 6, 8 Moderate
an in-process test as a result
of a safety or quality issue
41. Change in in-process 1–3, 5, 6 9 Minor
controls testing site
Note: Transfer of in-process control
testing to a different facility
within a GMP-compliant site is
not considered to be a reportable
change but is treated as a minor
GMP change and reviewed during
inspections.
Conditions
1. There is no change in drug product specification outside the approved limits.
2. There is no change in the impurity profile of the drug product outside the
approved limits.
4. The test does not concern a critical attribute (for example, content, impurities, any
5. The replaced analytical procedure maintains or improves precision, accuracy,
specificity and sensitivity, if applicable.
6. There is no change in the in-process control limits outside the approved limits.
7. The test procedure remains the same, or changes in the test procedure are minor.
Supporting data
1. Revised information on the controls performed at critical steps of the
manufacturing process and on intermediates of the proposed drug substance.
2. Updated drug product specification if changed.
4. Comparative table or description, where applicable, of current and proposed
in‑process tests.
results as quantitative data, in a comparative tabular format, for one commercial-
scale batch of the pre-change and post-change drug product (certificates of
analysis should be provided). Comparative pre-change test results do not need to
be generated concurrently; relevant historical testing results are acceptable. Batch
data on the next two full-production batches should be made available on request
and reported by the marketing authorization holder if outside specification (with
proposed action). The use of a smaller-scale batch may be acceptable where justified.
257
Table continued
product (certificates of analysis should be provided). Comparative pre-change test
results do not need to be generated concurrently; relevant historical testing results
are acceptable.
8. Justification for the new in-process test and limits.

release the excipient, involving the following:
b. Addition of a test 4 1–3 Minor
c. Replacement of an analytical 1–3 1, 2 Minor
procedure
d. Minor changes to an None 1, 2 Minor
approved analytical
procedure
e. Change from an in-house None 1, 2 Minor
recognized compendial
f. Widening of an approved None 1, 3 Moderate

g. Narrowing of an approved 3, 4, 6, 7 1 Minor
Conditions
1. Results of method validation demonstrate that the proposed analytical procedure
is at least equivalent to the approved analytical procedure.
2. The replaced analytical procedure maintains or improves precision, accuracy,
specificity and sensitivity.
3. The change is within the range of approved acceptance criteria or has been made
to reflect the new pharmacopoeial monograph specification for the excipient.
258
Annex 3
Table continued
4. Acceptance criteria for residual solvents are within recognized or approved
acceptance limits (for example, within ICH limits for a Class 3 residual solvent or
6. The analytical procedure remains the same, or changes in the test procedure are
minor.
(for example, new unqualified impurity, change in total impurity limits).
8. An alternative test analytical procedure is already authorized for the specification
attribute/test and this procedure has not been added through a minor change
submission.
Supporting data
1. Updated excipient specification.
2. Where an in-house analytical procedure is used and a recognized compendial
standard is claimed, results of an equivalency study between the in-house and
compendial methods.
3. Justification of the proposed excipient specification (for example, demonstration
of the suitability of the monograph to control the excipient and potential impact
on the performance of the drug product).

43. Change in the standard/ None 1–4 Moderate
monograph (that is,
specifications) claimed for 1–5 1–4 Minor
the excipient
Conditions
1. The change is from a House standard to a pharmacopoeial standard/monograph.
2. The change is made exclusively to comply with a pharmacopoeial standard/
monograph.
3. There is no change to the specifications for the functional properties of the
excipient outside the approved ranges, and no change that results in a potential
impact on the performance of the drug product.
5. There is no deletion or change to any analytical procedures, except to comply with
a pharmacopoeial standard/monograph.
259
Table continued
Supporting data
1. Updated excipient specifications.
2. Where a House analytical procedure is used and a pharmacopoeial/compendial
standard/monograph is claimed, results of an equivalency study between the
House and compendial methods.
3. Justification of the proposed excipient specifications (for example, demonstration
of the suitability of the monograph to control the excipient and potential impact
on the performance of the drug product).
4. A declaration that consistency of quality and of the production process of the
excipient is maintained.

44. Change in the source of an None 2–7 Major
excipient from a vegetable or
synthetic source to a human
or animal source that may
pose a TSE or viral risk
45. Change in the source of an None 1, 3, 5, 6 Moderate
excipient from a TSE risk (for
example, animal) source
to a vegetable or synthetic
source
46. Replacement in the source 5, 6 2–7 Minor
of an excipient from a TSE
risk source to a different TSE
risk source (for example,
different animal source,

different country of origin)
47. Change in manufacture of a None 2–7 Major
biological excipient
2 2–7 Moderate
1, 2 2–7 Minor
48. Change in supplier for a None 3–8 Major
plasma-derived excipient
(for example, human serum 3, 4 5, 6, 9 Moderate
albumin)
260
Annex 3
Table continued
49. Change in supplier for an None 2, 3, 5–7 Moderate
excipient of non-biological
origin or of biological origin 1, 5, 6 3 Minor
(excluding plasma-derived
excipient)
50. Change in excipient 1 10 Minor
testing site
Note: Transfer of testing to a
different facility within a GMP-
compliant site is not considered
to be a reportable change but is
treated as a minor GMP change
and is reviewed during inspections.
Conditions
1. There is no change to the specification of the excipient or drug product outside
the approved limits.
2. The change does not concern a human plasma-derived excipient.
3. The human plasma-derived excipient from the new supplier is an approved
medicinal product and no manufacturing changes were made by the supplier of
the new excipient since its last approval in the country/jurisdiction of the NRA.
4. The excipient does not influence the structure/conformation of the active
ingredient.
5. The TSE risk source is covered by a TSE certificate of suitability and is of the same
or lower TSE risk as the previously approved material (7).
6. Any new excipient does not require the assessment of viral safety data.
Supporting data
1. Declaration from the manufacturer of the excipient that the excipient is entirely of
vegetable or synthetic origin.
2. Details of the source of the excipient (for example, animal species, country of
origin) and the steps undertaken during processing to minimize the risk of TSE
exposure (7).
3. Information demonstrating comparability in terms of physicochemical properties,
and the impurity profile of the proposed excipient compared to the approved
excipient.
4. Information on the manufacturing process and on the controls performed at
critical steps of the manufacturing process, and on the intermediate of the
proposed excipient.
comparative tabular format, for at least three commercial-scale batches of the
proposed excipient.
261
Table continued
adventitious agents (for example, impact on the viral clearance studies, or BSE/TSE
risk (7)), including viral safety documentation where necessary.
8. Complete manufacturing and clinical safety data to support the use of the
proposed human plasma-derived excipient.
9. A letter from the supplier certifying that no changes were made to the plasma-
derived excipient compared to the currently approved corresponding medicinal
product.
Control of the drug product

51. Change affecting the quality control testing of the drug

product (release and stability), involving the following:
Note: Transfer of testing to a different facility within a GMP-compliant site is not considered
to be a reportable change but is treated as a minor GMP change and is reviewed during
inspections.
a. Transfer of the quality None 1, 2 Moderate
a non-pharmacopoeial assay 1–3 1, 2 Minor
(in-house) to a new company
not approved in the current
marketing authorization or
licence or to a different site
within the same company
262
Annex 3
Table continued
b. Transfer of the quality None 1, 2 Moderate
a pharmacopoeial assay to a 1 1, 2 Minor
authorization or licence
Conditions
1. The transferred quality control test is not a potency assay or bioassay.
2. There are no changes to the test method.
3. The transfer is within a facility approved in the current marketing authorization for
the performance of other tests.
Supporting data
1. Information demonstrating technology transfer qualification for the non-
pharmacopoeial assays or verification for the pharmacopoeial assays.

52. Change in the standard/monograph (that is, specifications)
claimed for the drug product, involving the following:
a. A change from a None 1–5 Moderate
monograph to an in-house
standard
b. A change from an in-house 1–4 1–3 Minor
standard to a pharmacopoeial
standard/monograph or
from one pharmacopoeial
standard/ monograph to a
different pharmacopoeial
standard/monograph
53. Change in the specifications 1, 2 1–3 Minor
for the drug product to
comply with an updated
monograph
263
Table continued
Conditions
1. The change is made exclusively to comply with a pharmacopoeial monograph.
2. There is no change in drug product specifications outside the approved ranges.
4. There is no deletion or change to any analytical procedures, except to comply with
a pharmacopoeial standard/monograph.
Supporting data
2. An updated copy of the proposed drug product specifications.
3. Where an in-house analytical procedure is used and a pharmacopoeial standard/
monograph is claimed, results of an equivalency study between the in-house and
pharmacopoeial methods.
4. Copies or summaries of validation reports if new analytical procedures are used.
5. Justification of specifications with data.

54. Changes in the control strategy of the drug
product, involving the following:
a. Change from end-product None 1–3, 5 Major
testing to upstream
controls for some test(s) (for
example, real-time release
testing, process analytical
technology)
b. Addition of a new critical None 1–5 Moderate

quality attribute to the
control strategy
c. Deletion of a critical quality None 1, 5 Moderate
attribute from the control
strategy
Conditions
None
Supporting data
1. Information on the controls performed at critical steps of the manufacturing
process and on intermediates of the proposed product.
264
Annex 3
Table continued
2. An updated copy of the proposed drug product specifications.
4. Copies or summaries of validation reports if new analytical procedures are used to
monitor the new critical quality attribute at release.
5. Justification and supporting data for each proposed change to the control strategy.

release the drug product, involving the following:
a. Deletion of a test analytical None 1, 6, 7 Moderate
procedure and/or an
b. Addition of a test 1, 2, 7 1–3, 5 Minor
c. Replacement of an analytical None 1–5 Moderate
procedure
4, 5, 8 1, 4, 5 Minor
d. Changes to an approved None 1–5 Moderate
1, 3–5 2, 4, 5 Minor
e. Change from an in-house None 1–5 Moderate
recognized compendial 1, 5 1–3 Minor
f. Widening of an approved None 1, 5, 7 Moderate
g. Narrowing of an approved 1, 3, 6, 7 1 Minor
Conditions
1. There is no change to the limits/acceptance criteria outside the approved limits
for the approved assays used at release/ stability.
2. The additional test is not intended to monitor new impurity species.
3. The method of analysis is the same (for example, a change in column length or
temperature, but not a different type of column or method) and no new impurities
are detected.
4. The modified analytical procedure maintains or improves the performance
parameters of the method.
5. The change does not concern potency-testing.
265
Table continued
6. Acceptance criteria for residual solvents are within recognized or approved
acceptance limits (for example, within ICH limits for a Class 3 residual solvent, or
(for example, new unqualified impurity, or impurity content outside the approved
limits).
8. The change is from a pharmacopoeial assay to another pharmacopoeial assay or
the marketing application holder has demonstrated an increased understanding
of the relationship between method parameters and method performance
defined by a systematic development approach including robustness studies.
Supporting data
1. An updated copy of the proposed drug product specification.
3. Validation/qualification results if new analytical procedures are used.
5. Justification for the change to the analytical procedure (for example,
demonstration of the suitability of the analytical procedure in monitoring the drug
product, including the degradation products) or for the change to the specification
(for example, demonstration of the suitability of the revised acceptance criterion to
control the drug product).
6. Justification for the deletion of the test (for example, demonstration of the
suitability of the revised specification in controlling the final product).
7. Documented evidence that consistency of quality and of the production process is
maintained.
Reference standards

56. Replacement of a primary None 1, 2 Moderate
reference standard
57. Change of the reference None 1, 2 Moderate
standards from a
pharmacopoeial or
international standard to
in‑house (no relationship
with international standard)
266
Annex 3
Table continued
58. Change of the reference 3 1, 2 Minor
standard from in-house
(no relationship with
international standard)
to a pharmacopoeial or
international standard
59. Qualification of a new batch 1 2 Minor
of reference standard against
the approved reference
standard (including
qualification of a new batch
of a secondary reference
standard against the
approved primary standard)
60. Change to the reference None 3, 4 Moderate
standard qualification
protocol
61. Extension of the reference 2 5 Minor
standard shelf-life or re-test
period
Conditions
1. The qualification of a new standard is carried out in accordance with an approved
protocol.
2. The extension of the shelf-life of the reference standard is carried out in
accordance with an approved protocol.
3. The reference standard is used for a physicochemical test.
Supporting data
1. Revised product labelling to reflect the change in reference standard, as applicable.
2. Qualification data of the proposed reference standards or materials (for example,
source, characterization, certificate of analysis).
3. Justification of the change to the reference standard qualification protocol.
4. Updated reference standard qualification protocol.
5. Summary of stability testing and results or retest data to support the extension of
the reference standard shelf-life.
267
Drug product container closure system

62. Modification of a primary None 1–7 Moderate
container closure system
(for example, new coating, 4 3, 7 Minor
adhesive, stopper, type 1–3 3 Minor
of glass)
Note: The addition of a new
container closure system (for
example, addition of a pre-filled
syringe where the currently
approved presentation is only a
vial) is considered a change in
presentation (see change 35d).
63. Change from a reusable None 1, 3, 6 Moderate
container to a disposable
container with no changes
in product contact material
(for example, change
from reusable pen to
disposable pen)
64. Deletion of a container None 1 Minor
closure system
Note: The NRA should be notified
of the deletion of a container
closure system, and product
labelling information should be
updated, as appropriate.
Conditions
1. There is no change in the type of container closure or materials of construction.
2. There is no change in the shape or dimensions of the container closure.
3. The change is made only to improve the quality of the container and does not
modify the product contact material (for example, increased thickness of the glass
vial without changing interior dimensions).
4. The modified part is not in contact with the drug product.
268
Annex 3
Table continued
Supporting data
1. Revised product labelling information, as appropriate.
2. For sterilized products, process validation results, unless otherwise justified.
3. Update dossier containing information on the proposed container closure system,
as appropriate (for example, description, materials of construction of primary
packaging components).
4. Results demonstrating protection against leakage, no leaching of undesirable
substance, compatibility with the product, and results from the toxicity and
biological reactivity tests.
5. Summary of release testing results as quantitative data, in a comparative tabular
format, for at least three consecutive commercial-scale batches of the pre-change
and post-change drug product. Comparative pre-change test results do not need
to be generated concurrently; relevant historical testing results are acceptable.
Bracketing for multiple-strength products, container sizes and/or fills may be
acceptable if scientifically justified.
drug product batches produced (unless otherwise justified) with the proposed
changes and stored under accelerated and/or stress conditions for a minimum
of 3 months. Test results that cover a minimum of 6 months in real-time/real-
temperature conditions should also be provided. A possibility of 3 months of real-
time data could be acceptable if properly justified (for example, it can be proven
that the relevant effect, if present, can already be observed within 3 months).
relevant historical results for batches on the stability programme are acceptable.
Additionally, the manufacturer should commit to undertake real-time stability
studies to confirm the full shelf-life/hold-time of the drug product under its
normal storage conditions and to report to the NRA any failures in these ongoing
long-term stability studies. Matrixing, bracketing, the use of smaller-scale batches
and/or the use of fewer than three batches of drug product for stability testing
may be acceptable where justified (6).
7. Information demonstrating the suitability of the proposed container/closure
system with respect to its relevant properties (for example, results from last media
fills; results of interaction studies demonstrating preservation of protein integrity
and maintenance of sterility for sterile products; maintenance of sterility in
multidose containers; user testing).
269

65. Change in the supplier for a primary container closure
component, involving the following:
a. Replacement or addition of a 1, 2 1, 2 Minor
supplier
Note: A change in container closure
system involving new materials of
construction, shape or dimensions
would require supporting data,
such as is shown for change 62
on modification of a primary
container closure system.
b. Deletion of a supplier None None Minor
Conditions
1. There is no change in the type of container closure, materials of construction,
shape and dimensions, or in the sterilization process for a sterile container closure
component.
2. There is no change in the specification of the container closure component
outside the approved acceptance criteria.
Supporting data
1. Letter from the marketing authorization holder certifying that there are no
changes to the container closure system.
2. Certificate of analysis, or equivalent, for the container provided by the new
supplier and comparison with the certificate of analysis, or equivalent, for the
approved container.

66. Change in the specification used to release a primary container
closure component or functional secondary container closure
component, involving the following:
b. Addition of a test 3 1, 2 Minor
c. Replacement of an analytical 6, 7 1–3 Minor
procedure
d. Minor changes to an 4–7 1–3 Minor
270
Annex 3
Table continued
e. Widening of an acceptance None 1, 2 Moderate
criterion
f. Narrowing of an acceptance 8 1 Minor
criterion
Conditions
2. The change to the specification does not affect the functional properties of
the container closure component and does not have a potential impact on the
performance of the drug product.
4. There is no change to the acceptance criteria outside the approved limits.
5. The new analytical procedure is of the same type.
6. Results of method validation demonstrate that the new or modified analytical
procedure is at least equivalent to the approved analytical procedure.
7. The new or modified analytical procedure maintains or improves precision,
accuracy, specificity and sensitivity.
8. The change is within the range of approved acceptance criteria.
Supporting data
1. An updated copy of the proposed specification for the primary or functional
secondary container closure component.
2. Rationale for the change in specification for a primary container closure component.
3. Description of the analytical procedure and, if applicable, validation data.
Stability
67. Change in the shelf-life of the drug product,
a. Extension (includes extension None 1–5 Moderate
of shelf-life of the drug
product as packaged for
sale, and hold-time after
opening and after dilution or
reconstitution)
271
Table continued
b. Reduction (includes None 1–5 Moderate
reduction as packaged for
sale, after opening, and after
dilution or reconstitution)
Conditions
None
Supporting data
1. Updated product labelling information, as appropriate.
2. Proposed storage conditions and shelf-life, as appropriate.
4. Justification of the change to the post-approval stability protocol or stability
commitment.
5. Results of stability testing under real-time/real-temperature conditions covering
the proposed shelf-life generated on at least three commercial-scale batches
unless otherwise justified.

68. Change in the post-approval stability protocol of the
drug product, involving the following:
a. Substantial change to the post- None 1–5 Moderate
approval stability protocol or
stability commitment, such as
deletion of a test, replacement
of an analytical procedure, or
change in storage temperature
b. Addition of test(s) into the 1 1, 2, 4, 5 Minor
post-approval stability protocol
c. Deletion of time point(s) from 2 4, 5 Minor
the post-approval stability
protocol within the approved
shelf-life
d. Replacement of sterility None 1, 2, 4, 5 Moderate
testing by the container/
closure system integrity 3 4, 5 Minor
testing
272
Annex 3
Table continued
Conditions
1. The addition of the test(s) is not due to stability concerns or to the identification of
new impurities.
2. Deletion of time point(s) is done according to relevant guidelines (for example, (6)).
3. The method used to demonstrate the integrity of the container/closure system
has already been approved as part of a previous application related to the drug
product.
Supporting data
2. Validation results if new analytical procedures are used.
3. Proposed storage conditions and or shelf-life, as appropriate.
4. Updated post-approval stability protocol, including justification for the change,
and stability commitment.

69. Change in the labelled storage conditions for the drug product or the
diluted or reconstituted biotherapeutic products, involving the following:
a. Addition or change of None 1–4, 6 Moderate
storage condition(s) for the
drug product, diluted or
reconstituted drug product
(for example, widening or
narrowing of a temperature
criterion, addition of or change
to controlled temperature
chain conditions)
b. Addition of a cautionary None 1, 2, 4, 5 Moderate
statement (for example,
“Do not freeze”)
c. Deletion of a cautionary None 1, 2, 4, 6 Moderate
statement (for example,
“Do not freeze”)
Conditions
None
273
Table continued
Supporting data
1. Revised product labelling information, as applicable.
2. Proposed storage conditions and shelf-life.
4. Justification of the change in the labelled storage conditions/cautionary statement.
5. Results of stability testing under appropriate stability conditions covering the
proposed shelf-life, generated on one commercial-scale batch unless otherwise
justified.
6. Results of stability testing under appropriate conditions covering the proposed
shelf-life, generated on at least three commercial-scale batches unless otherwise
justified.
References
1. The common technical document for the registration of pharmaceuticals for human use:
quality – M4Q(R1) (Step 4 version). Geneva: International Conference on Harmonisation of
Technical Requirements for Registration of Pharmaceuticals for Human Use; 2002 (http://www.ich.
org/fileadmin/Public_Web_Site/ICH_Products/CTD/M4_R1_Quality/M4Q__R1_.pdf, accessed 12
December 2017).
2. CTD Quality (M4Q) guideline. M4Q Implementation Working Group: Questions & Answers (R1) –
M4Q Q&As (R1). Geneva: International Conference on Harmonization of Technical Requirements
for Registration of Pharmaceuticals for Human Use; 2003 (http://www.ich.org/fileadmin/
Public_Web_Site/ICH_Products/CTD/M4_R1_Quality/M4_Quality_Questions_Answers_R1.pdf,
12 December 2017).
12 December 2017).
6. Stability testing of biotechnological/biological products. ICH Guideline Q5C. Geneva: International
Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for
Human Use; 1995 (https://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/
Quality/Q5C/Step4/Q5C_Guideline.pdf, accessed 12 December 2017).
274
Annex 3
7. WHO guidelines on transmissible spongiform encephalopathies in relation to biological and

275
Appendix 4
Safety, efficacy and product labelling information changes
The examples of safety and efficacy changes, product labelling information
changes and administrative product labelling information changes in this
appendix are provided for clarification. However, such changes are not limited to
those included in this appendix. They may also result in changes to the product
labelling information for health-care providers and patients, and to inner and
outer labels.
Because the amount of safety and efficacy data needed to support
a change may vary according to the impact of the change, risk–benefit
considerations and product-specific characteristics there is no “one size fits all”
approach. This appendix therefore provides a list of examples of changes in the
various categories rather than a detailed table linking each change with the
required data needed to support that change (as is provided in Appendices 2
and 3 for quality changes). Marketing authorization holders or applicants are
encouraged to contact the NRA for guidance on the data needed to support
major changes if deemed necessary.
Safety and efficacy changes

Safety and efficacy change supplements require approval prior to implementation
of the change and are generally submitted for changes related to clinical practice,
safety and indication claims.
The following are examples of safety and efficacy changes requiring data
from clinical studies and/or nonclinical studies, post-marketing observational
studies or extensive post-marketing safety data:
■■ Change to the indication:

(a) addition of a new indication (for example, treatment of a
previously unspecified disease);
(b) modification of an approved indication (for example,
expansion of the age of use or restriction of an indication based
on clinical studies demonstrating lack of efficacy).
■■ Change in the recommended dose and/or dosing schedule.
■■ Change to the use in specific at-risk groups (for example, addition
of information on use in pregnant women or immunocompromised
patients).
276
Annex 3
■■ Change to add information on co-administration with other

medicines.
■■ Change to add a new route of administration.1
■■ Change to add a new dosage form 1 (for example, replacement of a
suspension for injection with a lyophilized cake).
■■ Change to add a new strength.1
■■ Change to add a new delivery device 1 (for example, adding a pre-
filled syringe or pen).
■■ Change in existing risk-management measures:
(a) (deletion of an existing route of administration, dosage form
and/or strength due to safety reasons;
(b) (deletion of a contraindication (for example, use in pregnant
women);
(c) changing a contraindication to a precaution.
Product labelling information changes

Supplements on product labelling information changes should be submitted
for changes which do not require clinical efficacy and/or safety data from
clinical studies but normally require extensive pharmacovigilance (safety
surveillance) data. Product labelling information changes require approval prior
to implementation.
The following are examples of product labelling information changes
that impact on the clinical use of a product:
■■ Addition of an adverse event that is identified as consistent with a

causal association with administration of the biotherapeutic product
concerned.
■■ Change in the frequency of occurrence of a given adverse reaction.
■■ Addition of a contraindication or warning (for example,
identification of a specific subpopulation as being at greater
risk, such as individuals with a concomitant condition or taking
concomitant medicines, or a specific age group). These changes may
include provision of recommended risk-management actions (for
example, ensuring patient awareness of certain risks).
1
Some NRAs consider that these changes may require a new application for a marketing authorization
or licence.
277
■■ Strengthening, clarification or amendment of the text of the product

labelling information relating to contraindications, warnings,
precautions and adverse reactions.
■■ Revisions to the instructions for use, including dosage,
administration and preparation for administration, to optimize the
safe use of the biotherapeutic product.
In some cases, the safety-related changes listed above may be urgent and
may require rapid implementation (for example, addition of a contraindication
or warning). To allow for the speedy processing of such requests, the supplements
for these changes should be labelled as “Urgent product labelling information
changes” and should be submitted after prior agreement between the NRA and
the marketing authorization holder (see section 8.3 and Appendix 1).
Administrative product labelling information changes

Administrative product labelling information changes are changes to any of
the labelling items which are not expected to have an impact upon the safe and
efficacious use of the biotherapeutic product. In some cases, these changes may
need to be reported to the NRA and approval received prior to implementation,
while in other cases reporting may not be required.
Examples of changes which do require reporting to the NRA and
receipt of approval prior to implementation by the marketing authorization
holder include:
■■ Change in the proper/nonproprietary name or trade name of the
biotherapeutic product.
Examples of changes which may not require approval by the NRA prior
to implementation include:
■■ Change in the name of the marketing authorization holder and/or

manufacturer (for example, change of name due to a merger).
■■ Updated contact information for the marketing authorization holder
(for example, customer service number or website address) or
distributor’s name.
■■ Minor changes to the layout of the product labelling information
items or revision of typographical errors without changing the
content of the label.
■■ Update of the existing information for referenced literature without
adding or removing references.
■■ Changes made to comply with an official compendium (for example,
change of the common name).
278
Annex 3
■■ Minor changes to the text to add clarity in relation to maintaining

consistency with common label phrase standards (for example,
change from “not recommended for children” to “not for use in
children”).
These administrative product labelling information changes (that is,
changes not subject to prior approval that have been implemented since the last
approved product labelling information) should be included when submitting
subsequent PAS for safety and efficacy changes or for product labelling
information changes (see section 8.4).
279
Annex 4
Technical Specifications Series (TSS) for WHO
Human immunodeficiency virus (HIV)

rapid diagnostic tests for professional use TSS–1
and/or self-testing

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281
Acknowledgements 283
List of contributors 283
Abbreviations 284
A Introduction 284
B How to apply these specifications 285
C Other guidance documents 286
D Performance principles for WHO prequalification 286
D.1 Intended use 286
D.2 Diversity of specimen types, users and testing environments and impact on
required studies 287
D.3 Applicability of supporting evidence to an IVD under review 287
E Table of Requirements 289
Part 1 Establishing analytical performance characteristics 291
Part 2 Establishing clinical performance characteristics (professional use and/or
self-testing) 305
Part 3 Qualification of usability (self-testing) 307
References 312
282
Annex 4
Acknowledgements
The document Technical Specifications Series 1 (TSS–1) for WHO Prequalification
– Diagnostic Assessment: Human immunodeficiency virus (HIV) rapid diagnostic
tests for professional use and/or self-testing was developed with support from the
Bill & Melinda Gates Foundation and UNITAID. The first draft was prepared
in collaboration with Dr M Lanigan, Geneva, Switzerland and Ms R Meurant,
WHO, Geneva, Switzerland, with input and expertise from Dr H Scheiblauer,
Paul-Ehrlich-Institut, Langen, Germany; Ms D Healy, Ms H Ardura, Dr R
Baggaley, Dr C Case, Dr C Figueroa, Dr M Nübling; and Ms A Sands, WHO,
Geneva, Switzerland. This document was produced under the coordination
and supervision of Ms R Meurant and Ms I Prat, Prequalification Team, WHO,
Geneva, Switzerland.
List of contributors
First-round comments were received from the following: Ms S Best, National
Serology Reference Laboratory, Victoria, Australia; Dr T Crucitti, Institute of
Tropical Medicine, Antwerp, Belgium; Dr J Duncan, London, the United Kingdom;
Dr F Gruszka, E-Meddia, Paris, France; Dr C Hill, CA, United States of America
(USA); Mr G James, Institute for Clinical Pathology and Medical Research, New
South Wales, Australia; Dr L Kestens, Institute of Tropical Medicine, Antwerp,
Belgium; Ms D Lepine, Medical Devices Bureau, Health Canada, Ottawa, Canada;
Dr A Reissinger, Paul-Ehrlich-Institut, Langen, Germany; and Dr M Nübling and
Ms M Perez Gonzalez, WHO, Geneva, Switzerland.
The draft technical specifications document was then posted on the WHO
Prequalification website for public consultation on 15 September 2016. Various
stakeholders, including manufacturers submitting to WHO prequalification
of in vitro diagnostic medical devices (IVDs), IVD manufacturing industry
associations, various national and international regulatory bodies, and IVD
standards organizations, were informed of the consultation in order to solicit
feedback. A two-month response period was provided.
Second-round public comments were then received from the following:
Dr P Akolkar, Center for Biologics Evaluation and Research, United States Food
and Drug Administration, MD, USA; Ms S Best, National Serology Reference
Laboratory, Victoria, Australia; Dr J Duncan, London, the United Kingdom;
Epicentre and Médecins sans Frontières International Office, Paris, France; Dr C
Kosack, Dr A Page and Ms E Tran, Geneva, Switzerland; Dr R Galli, bioLytical™
Laboratories Inc., Vancouver, Canada; Ms D Lepine, Medical Devices Bureau,
Health Canada, Ottawa, Canada; Dr M Nübling, WHO, Geneva, Switzerland;
OraSure Technologies Inc., PA, the USA; Dr H Scheiblauer, Paul-Ehrlich-Institut,
Langen, Germany; UNITAID/PSI HIV Self-Testing Africa (STAR) project
283
consortium members; and Dr E Cowan, Dr R Dacombe, Dr M Kumwenda, Dr M

Neuman, Professor R Peeling, Dr M Taegtmeyer and Dr V Watson, Liverpool
School of Tropical Medicine, Liverpool, the United Kingdom.
Following incorporation of the second-round public comments, a revised
draft was published on the WHO Biologicals website for a final round of public
consultation between 18 June and 18 September 2017. The comments received
were incorporated to produce the document WHO/BS/2017.2305. The document
was adopted by the WHO Expert Committee on Biological Standardization as a
WHO written standard on 20 October 2017.
Abbreviations
Ag antigen
CE Conformité Européenne (European Conformity)
CRF circulating recombinant form
IVD in vitro diagnostic medical device
A Introduction
The purpose of this document is to provide technical guidance to in vitro
diagnostic medical device (IVD) manufacturers that intend to seek WHO
prequalification of rapid diagnostic tests (RDTs) for the detection of human
immunodeficiency virus (HIV).
The minimum performance requirements for WHO prequalification
are summarized in this document, and apply equally to RDTs intended solely
for HIV detection and to those tests where HIV detection is one component
of a multi-detection assay (for example, an HIV/syphilis dual-detection RDT).
This document applies to RDTs intended to be used as an aid to diagnosis of
HIV infection. The current version of this document does not address IVDs
that discriminate between the detection of HIV-1 and HIV-2 infection, IVDs
intended as confirmatory tests, or the requirements for accompanying quality
control materials.
For the purpose of this document, the use of certain verbal forms is
as follows:
■■ “shall” indicates that the manufacturer is required to comply with
the technical specifications;
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Annex 4
■■ “should” indicates that the manufacturer is recommended to comply

with the technical specifications but it is not a requirement; and
■■ “may” indicates that the technical specifications are a suggestion but
not a requirement.
A documented justification and rationale shall be provided by the
manufacturer when the WHO prequalification submission does not comply with
the required technical specifications outlined in this document.
Minimum performance requirements for WHO prequalification
are summarized in this document– and where possible, WHO performance
requirements are aligned with published guidance, standards and/or regulatory
documents. Although references to source documents are provided, it should be
noted that WHO prequalification in some cases has additional requirements.
For WHO prequalification purposes, manufacturers shall provide
evidence in support of the clinical performance of an IVD to demonstrate that
reasonable steps have been taken to ensure that a properly manufactured IVD,
when correctly operated by the intended user, will detect the target analyte and
fulfil its indications for use.
The WHO prequalification requirements summarized in this document
do not extend to the demonstration of clinical utility – that is, the effectiveness
and/or benefits of an IVD, relative to and/or in combination with other measures,
as a tool to inform clinical intervention in a given population or health-care
setting. To demonstrate clinical utility, a separate set of studies is required.
Clinical utility studies usually inform programmatic strategy and are thus the
responsibility of programme managers, ministries of health and other related
bodies in individual WHO Member States. Such studies do not fall under the
scope of WHO prequalification.
B How to apply these specifications

For WHO prequalification purposes, an IVD intended for professional use only
(by a laboratory professional, health-care worker or trained lay provider) shall be
supported by studies outlined in Parts 1 and 2 of this document.
An IVD intended both for professional use and for self-testing shall be
supported by the studies outlined in Parts 1 and 2 of this document. In addition,
the claim for self-testing shall be supported by studies that qualify the usability
of the IVD among a broad range of self-testing users, as outlined in Part 3.
An IVD intended for self-testing only shall be supported by studies
outlined in Parts 1, 2 and 3.
For an IVD with an intended use that has been amended to include self-
testing, and for which performance in professional use is already established,
285
and Parts 1 and 2 of this document have already been satisfied, the additional
claim for self-testing shall be supported by studies outlined in Part 3.
These requirements are summarized below in Table 1.
Table 1
Summary of requirements for submission for WHO prequalification based on the
intended use of the IVD
Intended use Parts of the TSS to be fulfilled

Professional use Parts 1 and 2
Self-testing Parts 1, 2 and 3
Prequalified professional-use IVD with Part 3, with the provision that any
additional claim for self-testing adaptations made do not impact the
established safety and performance
C Other guidance documents

This document should be read in conjunction with other relevant WHO guidance
documentation, including:
■■ Technical Guidance Series for WHO Prequalification – Diagnostic
Assessment
■■ Sample Product Dossiers for WHO Prequalification – Diagnostic
Assessment
■■ Instructions for Compilation of a Product Dossier (WHO document
PQDx_018).
These documents are available at: http://www.who.int/diagnostics_laboratory/

evaluations/en/
D Performance principles for WHO prequalification

D.1 Intended use
An IVD intended for WHO prequalification shall be accompanied by a sufficiently
detailed intended use statement. This should allow for an understanding to be
gained of at least the following:
■■ the function of the IVD (for example, to detect antibodies to
HIV‑1, HIV-2 and/or HIV p24 antigen (Ag), etc.) and whether it is
qualitative, semi-quantitative or quantitative;
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Annex 4
■■ the testing population for which the functions are intended (for
example, detection of susceptible individuals) and the intended
operational setting (for example, for use in near-patient testing); and
■■ clinical indication (for example, aid to diagnosis of HIV infection).
D.2 Diversity of specimen types, users and testing

environments and impact on required studies
For WHO prequalification submission, clinical performance studies should be
conducted using the specimen types that are most likely to be used in resource-
limited WHO Member States (for example, capillary whole blood and oral fluid)
and claimed in the instructions for use. If this is not possible, substantial data
shall be presented to show the equivalence between specimen types used in
performance studies.
Prequalified RDTs in low- and middle-income countries are likely to be
used by laboratory professionals 1 and at point-of-care by health-care workers,
trained lay providers 2 or by individuals who self-test. Depending on the intended
use of an RDT, performance studies shall be designed to take into account not
only the diversity of knowledge and skills across the population of RDT users,
but also the likely operational settings in which testing will occur. For example,
studies that comprise the testing of left-over/repository specimens by research
and development staff at a manufacturer’s facility shall not, on their own, be
considered sufficient to meet many of the performance requirements summarized
in this document.
D.3 Applicability of supporting evidence to an IVD under review

Performance studies shall be undertaken using the specific locked-down version
of the IVD intended to be submitted for WHO prequalification. Where this is
not possible, a justification shall be provided and additional supporting evidence
may also be required. This may occur in the case of minor variations in design
where no negative impact on performance has been demonstrated.
Specific information is provided in Parts 1 and 2 of this document for
the numbers of lots required for particular studies. Each lot should comprise
different batches of critical components. It is a manufacturer’s responsibility to
ensure (via risk analysis of their IVD) that the minimum number of lots chosen
1
Medical technologists, medical laboratory technicians or similar, who have received a formal professional
or paraprofessional certificate or tertiary education degree.
2
Any person who performs functions related to health-care delivery and has been trained to deliver specific
services but has received no formal professional or paraprofessional certification or tertiary education
degree.
287
for estimating performance characteristics takes into account the variability

in performance likely to arise from the diversity of key components and their
formulation.
The true HIV status of a specimen shall be determined using a suitable
reference method, for which justification shall be provided. Estimation (and
reporting) of IVD performance shall include the rate of invalid test results.
For certain analytical studies it may be acceptable to use contrived specimens
(for example, normal human specimens that have been spiked with HIV
antibodies). Although all reasonable attempts should be made to use natural
specimens, justification should be provided where contrived specimens are used
in the submitted studies. Clinical studies should be based on testing in natural
specimens only.
For IVDs that include a claim for detection of multiple analytes, evidence
of performance shall be provided for each claimed analyte. It should be noted
that, depending on the design of an IVD, evidence generated in a similar, related
product will usually not be considered sufficient by WHO to support performance
claims in an IVD submitted for prequalification.
Example: an IVD designed to detect HIV antibodies only, and the same
IVD designed for the dual detection of HIV and syphilis. It is unlikely that
performance evidence presented for the HIV-only IVD would be acceptable
for supporting performance claims for the dual-detection IVD.
For an IVD with an intended use that has been expanded to include self-testing,
changes are usually required to improve the usability of the IVD for this new
testing population. Such changes may include the modification of:
■■ the instructions for use (for example, simplification of instructions
to reflect new intended users);
■■ buffer vials;
■■ collection procedures;
■■ reading times, etc.
It is a manufacturer’s responsibility to verify through testing (as
summarized in Parts 1 and 2 of this document) that any changes made do not
have an adverse impact on critical safety and performance characteristics of an
IVD. Usability studies are undertaken to optimize the presentation of an IVD and
the understanding of self-testing users. The minimum reporting requirements
summarized in Part 3 of this document are not intended to be an exhaustive list
or to indicate a particular order in which studies should be undertaken.
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E Table of Requirements
Part 1 Establishing analytical performance characteristics

1.1 Specimen type
1.1.1 Demonstration of equivalence between specimen types
1.1.2 Demonstration of equivalence of claimed anticoagulants
1.2 Specimen collection, storage and transport
1.2.1 Specimen stability
1.3 Precision of measurement
1.3.1 Repeatability, reproducibility
1.4 Performance panels
1.4.1 Subtype panels
1.4.2 Mixed titre panels
1.5 Validation of reading times
1.5.1 Validation of reading times
1.6 Analytical sensitivity
1.6.1 Seroconversion
1.6.2 Limit of detection for HIV-1 p24 Ag, where appropriate
1.7 Prozone/high-dose hook effect
1.7.1 Prozone/high-dose hook effect
1.8 Analytical specificity
1.8.1 Potentially interfering substances
1.8.1.1 Endogenous
1.8.1.2 Exogenous
1.8.2 Cross-reactivity
1.9 Metrological traceability of control material values
1.9.1 Metrological traceability of control material values
1.10 Stability
1.10.1 Shelf-life (including transport stability)
1.10.2 In-use stability
1.11 Flex studies
1.11.1 Flex studies
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Table continued
Part 2 Establishing clinical performance characteristics (professional use
and/or self-testing)
2.1 Diagnostic sensitivity and specificity
2.1.1 Diagnostic sensitivity
2.1.2 Diagnostic specificity
Part 3 Qualification of usability (self-testing)
3.1 Qualification of usability (self-testing)
3.1.1 Labelling comprehension study
3.1.2 Results interpretation study
3.1.3 Observed untrained user study
290
Part 1 Establishing analytical performance characteristics
Aspect Testing requirements Notes on testing requirements Source
documents
1.1 Specimen type
1.1.1 For each claimed specimen type, 1. The relationship between IVD performance in claimed Technical Guidance
Demonstration testing in at least: specimen types and reference materials used for Series for WHO
of equivalence • 25 HIV-positive specimens analytical studies shall be established. The design of Prequalification
between • 25 HIV-negative specimens. subsequent studies shall then take that relationship – Diagnostic
specimen types into account. Assessment (1)
2. If there is no equivalence between claimed specimen European
types then the impact that this will have on each Commission (2)
subsequent performance claim shall be fully
1.1.2 At least 25 HIV-positive and 25 understood and described. Where a significant
Demonstration HIV-negative specimens for each difference in performance exists between specimen
of equivalence claimed anticoagulant. types, equivalence may need to be investigated as
of claimed The equivalence of specimen part of a larger clinical study (see Part 2 below).
anticoagulants types shall be determined for all Example: an IVD intended for testing whole blood for
claimed analytes (for example, which seroconversion sensitivity is estimated using
HIV‑1 antibody, HIV-2 antibody, panels of serum/plasma specimens.
p24 Ag, as appropriate) (see • The relationship between seroconversion sensitivity
Note 3). in serum/plasma to that of the same characteristic
in whole blood shall be understood.
• This might be achieved by comparing titres
between end-point dilution series of matched
specimen types (whole blood versus serum/plasma)
from a set of positive patients.
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292
Table continued
documents
3. In some cases it may be acceptable to use diluted
or spiked specimens. This approach is acceptable in
early development work, but all reasonable attempts
should be made to use natural specimens. Justification
should be provided if diluted or spiked specimens are
used in the submitted studies.
4. Positive specimens (undiluted) shall be chosen so that
the majority of them are near the IVD cut-off.
5. Paired specimens should be used (for example, if
claiming equivalence of four anticoagulants then each
subject should provide four samples – one in each
anticoagulant).
1.2 Specimen collection, storage and transport
1.2.1 Real-time studies taking into 1. Evidence shall be provided which validates the Technical Guidance
Specimen account: maximum allowable time between specimen Series for WHO
stability • storage conditions (duration collection and its addition to the IVD in the setting Prequalification
at different temperatures, where testing takes place. – Diagnostic
temperature limits, freeze/ Assessment (3)
thaw cycles);
• transport conditions, where
applicable;
• intended use (see Note 1);
• specimen collection and/or
transfer devices intended to
be used with the IVD.
Table continued
documents
1.3 Precision of measurement
1.3.1 Both repeatability (within- 1. For example, within- or between-run, -lot, -day, -site, CLSI EP05-A3 (4)
Repeatability, condition – see Note 1) and etc. ISO 13612:2002 (5)
reproducibility reproducibility (between- 2. Precision shall be determined for each pathogen and/ CLSI EP12-A2 (6)
condition – see Note 1) shall or analyte for which detection is claimed (for example,
be estimated using panels of at HIV-1 antibody, HIV-2 antibody, HIV-1 p24 Ag, as
least: appropriate).
• 1 negative specimen; 3. The testing panel should be composed of natural
• 1 low-reactivity positive (that is, undiluted) specimens. Where this is not
specimen (near assay cut-off ); feasible, the stock specimens that are to be diluted
• 1 medium-reactivity positive should represent a range of stages of infection
specimen. (antibody maturation) in order to take into account
Each panel member shall be the limitations of mimicking low IVD reactivity with a
tested: high-avidity specimen.
• in 5 replicates; 4. IVDs which include whole blood as a specimen type
• using 3 different lots; shall include evidence of precision in (at a minimum)
• over 5 days (not necessarily spiked whole blood specimens (negative whole blood
consecutive) with one run spiked with highly reactive plasma/serum specimens
per day (alternating morning/ to produce an appropriate range of reactivities in the
afternoon); IVD).
• at each of 3 different testing 5. Where possible, the testing panel should be the same
sites. for all operators, lots and sites.
6. Lots shall comprise different batches of critical
components.
Annex 4
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294
Table continued
documents
The effect of operator-to- 7. Results shall be statistically analyzed to identify
operator variation on IVD and isolate the sources and extent of any variance.
performance should be In addition, the percentage of correctly identified,
included as part of the precision incorrectly identified and invalid results shall be
studies (see also Note 8). Testing tabulated for each specimen and be separately
should be done: stratified according to site, lot, etc. This type of analysis
• by personnel representative is especially important for rapid tests that may not have
of intended users; any numerical values.
• unassisted; 8. The effect of operator-to-operator variation on IVD
• using only those materials performance may also be considered as a human factor
provided with the IVD (for when designing robustness (flex) studies (see section
example, instructions for use, 1.11.1 below) and may be addressed as part of clinical
labels and other instructional studies in representative populations (see Part 2).
materials). 9. Users should be selected based on a pre-determined
and contextually appropriate level of education,
literacy and auxiliary skills that will challenge the

usability of the IVD and reflect the diversity of intended
users and operational settings.
1.4 Performance panels
1.4.1 Testing of WHO International 1. Testing should be performed using more than 1 lot of Health Products
Subtype panels Reference Preparations and/or the final design (locked-down). and Food Branch,
commercial HIV subtype panels 2. All confirmed subtype-positive specimens shall be Health Canada (7)
shall include: detected by the IVD.
Table continued
documents
• all HIV-1 subtypes (for 3. All reasonable attempts shall be made to test rare
example, A, B, C, D, G, subtypes.
etc.), HIV-2, HIV-1 group O, 4. For IVDs that include a claim for detection of HIV Ag,
and common circulating appropriate specimens for the same subtypes shall
recombinant forms (CRFs); also be included in the testing panel. The use of panels
• at least 10 each of the most of virus-like-particles (VLPs) or viral cultures may be
common subtypes (Subtype C, considered acceptable – however their use in place of
Subtype A, Subtype B, CRF02_ characterized specimens shall be justified.
AG, CRF01_AE, CRF07_BC and
Subtype G);
• at least 3 less‑common
subtypes (other CRFs and
unique recombinant forms.
1.4.2 Testing of a panel of specimens
Mixed titre with a range of analyte
panels concentrations (for example,
antibody “mixed-titre” panel).
1.5 Validation of reading times
1.5.1 For IVDs for which a reading 1. The ranges of humidity tested for shall be risk based, WHO
Validation of interval is specified (that is, time taking into consideration the likely operational Prequalification
reading times when result can first be read; settings. – Diagnostic
time beyond which result should 2. The intended operating temperature, upon which Assessment (8)
not be read), validation of critical reading time has been validated, shall be clearly stated
time points shall be provided.
Annex 4
in the instructions for use.
295
296
Table continued
documents
Performance studies shall 3. Some of these aspects could be evaluated within the
be conducted at each of 3 flex studies (see section 1.11.1 below).
temperatures (at the mid-point
and two extremes of the claimed
operating range); the effect of
humidity on reading times shall
also be investigated.
1.6 Analytical sensitivity
1.6.1 A minimum of 25 commercial 1. Specimens should have been collected at short European
Seroconversion or well-characterized intervals to cover the seroconversion period and Commission (2)
seroconversion panels shall be should also cover the whole window period. Health Products
tested: 2. Early seroconversion: and Food Branch,
• test at least 40 early – p24 Ag and/or HIV RNA-positive; Health Canada (7)
seroconversion specimens – not recognized by all Conformité Européenne (CE)- CLSI EP12-A2 (6)
(see Note 2); marked third-generation enzyme immunoassays;

• all seroconversion specimens – indeterminate or negative by confirmatory assays.
shall be reactive (see Note 3);
3. Seroconversion:
• start with a negative bleed(s)
and should have narrow – p24 Ag and/or HIV RNA-positive;
bleeding intervals. – recognized by all CE-marked third-generation
enzyme immunoassays;
– indeterminate or positive by confirmatory assays.
Table continued
documents
1.6.2 Analytical sensitivity estimated 4. Seroconversion sensitivity shall be reported to the
Limit of as the concentration of HIV-1 user in the instructions for use.
detection for p24 Ag at the assay cut-off. 5. Optimally, testing should be conducted using more
HIV-1 p24 The determination shall comprise than 1 lot of the final design (locked-down).
Ag, where a minimum of 15–20 replicate
appropriate tests of an 8-member dilution
panel of a suitable biological
reference material – for example,
the First WHO International
Reference Reagent for HIV-1 p24
antigen (NIBSC code 90/636).
1.7 Prozone/high-dose hook effect
1.7.1 For each claimed analyte, 1. Specimens shall be chosen that have a high analyte Health Products
Prozone/high- the potential for a prozone/ concentration, as determined using an IVD method and Food Branch,
dose hook high-dose hook effect shall be other than the IVD intended to be prequalified (for Health Canada (7)
effect determined: example, enzyme immunoassay). This second method Butch, AW (9)
• using multiple highly reactive shall be of a design not subject to prozoning.
specimens (minimum of 20); 2. An increase in signal upon dilution of a specimen
• using at least 2 different implies a hook effect.
concentrations (diluted by at
least a factor of 10);
• by the testing of several
replicates by the same
operator on the same day.
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298
Table continued
documents
1.8 Analytical specificity
1.8.1 Potentially The potential for false 1. The risk assessment conducted for an IVD shall identify Health Products
interfering results (false negatives and substances where the potential for interference and Food Branch,
substances false positives) arising from can reasonably be expected for the analyte being Health Canada (7)
interference from at least the detected (for example, HIV-1/2 antibodies and/or European
substances/conditions listed HIV‑1 p24 Ag). Commission (2)
below in sections 1.8.1.1 and 2. Where either the scientific literature and/or risk
1.8.1.2 (see Note 1) shall be CLSI EP07-A2 (10)
analysis identifies the potential for false results in
determined using: co-infected individuals (for example, decreased
• a minimum of 100 specimens sensitivity or specificity), further investigation shall be
(either naturally occurring or undertaken using both HIV-negative and HIV-positive
spiked to a low reactivity); specimens.
• each substance/condition 3. In addition to the substances listed here, IVDs that are
represented, where possible, used to test oral fluid shall take into account the effect
by at least 3–5 specimens from of oral infections, such as Candida, as well as tobacco,
different individuals. mouthwash, concomitant medications, dental fixtures,

Testing shall be undertaken toothpaste, food or drink (consumed immediately
using both HIV-negative prior to testing), consumption of alcohol and teeth
and HIV-positive specimens brushing.
(unspiked or spiked) with each 4. Any observed interference shall be investigated and
potentially interfering substance performance limitations of the IVD reported in the
at physiologically relevant instructions for use. Results shall be reported with
dosages. respect to each condition and not be reported as an
aggregate of the total number of specimens tested in
the study.
Table continued
documents
1.8.1.1 • human antibodies to the
Endogenous expression system (for
recombinants), for example,
anti-Escherichia coli or human
anti-mouse antibody (HAMA);
• recipients of multiple blood
transfusions, and pregnant
(including multiparous)
women;
• haemoglobin, lipids, bilirubin
and protein;
• elevated immunoglobulin G
and immunoglobulin M;
• rheumatoid factor;
• sickle-cell disease;
• other autoimmune conditions
including systemic lupus
erythematosus (SLE) and anti-
nuclear antibodies (ANA).
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300
Table continued
documents
1.8.1.2 • relevant medicines, including:
Exogenous antiparasitic, antimalarial,
antiretroviral and anti-
tuberculosis medications;
• common over-the-counter
anti-inflammatory medications
(aspirin, paracetamol and
ibuprofen);
• ethanol and caffeine.
1.8.2 The potential for false-positive 1. The types of interferences tested for shall be risk
Cross-reactivity results arising from cross- based, taking into consideration the operational
reactivity (see Note 1) shall be setting as well as the intended users for the analyte
determined for a minimum of being detected (for example, HIV-1/2 antibodies and/
100 specimens, including, where or HIV-1 p24 Ag).
possible, at least 3–5 specimens 2. Any observed interference shall be investigated and
representing each of the performance limitations of the IVD reported in the

following: instructions for use.
• non-HIV viral infections,
including: hepatitis B, C
infection and acute hepatitis
A infection, cytomegalovirus,
acute Epstein–Barr virus,
varicella zoster virus, yellow
fever virus post-immunization,
measles, influenza A and B and
tick-borne encephalitis;
Table continued
documents
• other retroviruses, including
human T-lymphotropic cell
virus-1 and -2;
• bacteria/parasites, including:
malaria, visceral leishmaniasis,
tuberculosis and human
African trypanosomiasis;
• influenza vaccine recipient;
• vaccine-induced HIV
seropositivity;
• other unrelated conditions
known to cause cross-
reactivity in HIV IVDs.
1.9 Metrological traceability of control material values
1.9.1 The traceability of an assay- 1. HIV RDT kits may not include external quality control WHO
Metrological specific quality control specimen specimens, but the IVD shall have a procedural control. Prequalification
traceability to a validated reference The extent to which a control band corresponds to – Diagnostic
of control material shall be demonstrated a valid test (identification of and traceability to a Assessment (8)
material values – for example, the First WHO suitable reference) should be demonstrated.
International Reference Panel for Comment 1: the nature of the procedural control
anti-human immunodeficiecy (specimen addition or only reagent addition) shall be
virus tests (NIBSC code 02/210) explained.
or the First WHO International
Comment 2: an external control specimen is one that
Reference Reagent for HIV-1 p24
is run in conjunction with the IVD, but is physically
antigen (NIBSC code 90/636).
Annex 4
separate from it – for example, an RDT cassette.
301
302
Table continued
documents
2. In some jurisdictions there is a requirement for the
use of a “National Testing Panel” for lot release and
IVD validation. Such a national requirement does
not remove the need for evidence of traceability to a
validated reference material as described here.
1.10 Stability
Replicate testing shall be 1. The testing panel shall include all claimed analytes ISO 23640:2011 (11)
undertaken using a panel (for and include whole blood specimens and/or oral CLSI EP25-A (12)
each claimed pathogen/analyte) fluid specimens, as appropriate, in accordance with
Technical Guidance
consisting of at least: intended use (for example to verify proper flow and
Series for WHO
• 1 non-reactive specimen; absence of background interference, and to account
Prequalification
• 2 low-reactivity specimens, for other variables).
– Diagnostic
near assay cut-off (see Note 2); 2. Where detection of multiple genotypes and/or Assessment (13)
• 1 medium-reactivity specimen. subtypes is claimed, equivalent performance (for
ASTM D4169 - 14
Wherever possible, specimens example, sensitivity and specificity) shall have been
(14)
chosen for the testing panel demonstrated; otherwise evidence of stability in these
shall reflect the main specimen genotypes/subtypes will need to be provided.
types intended for use with 3. Ideally, the stability testing panel shall be composed
the IVD (for example, capillary of natural (that is, undiluted) specimens. Where this
whole blood and/or oral fluid, is not feasible, stock specimens to be diluted should
as appropriate). represent a range of stages of infection (antibody
maturation) so as to take into account the limitations
of mimicking low IVD reactivity with a high-avidity
specimen.
Table continued
documents
1.10.1 Real-time, minimum of 3 lots of 4. Lots shall comprise different batches of critical
Shelf-life final design product and: components.
(including • transport stressed (simulated) 5. Determination of shipping stability shall be performed
transport before real-time studies are using simulated extreme stress conditions, ensuring
stability) undertaken; that application of those conditions is consistent and
• IVD in final packaging controlled.
subjected to drop-shock 6. Claims for stability shall be based on the second-last
testing. successful data point from the least-stable lot – with
1.10.2 • minimum of 1 lot, using (where lots are different) a statistical analysis showing
In-use stability panel(s) compiled as above; that the bulk of lots will be expected to meet the
• testing of all labile claimed life. For example, for testing conducted at 3, 6,
components (for example, 9, 12 and 15 months where stability was observed at
buffers vials, sealed cartridges, 15 months, then the maximum stability claim shall be
etc. – see Note 8). 12 months.
7. Accelerated studies do not replace the need for real-
time studies.
8. In-use stability of labile components shall be
determined using components in their final
configuration.
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304
Table continued
documents
1.11 Flex studies
1.11.1 The influence of the following 1. Refer to WHO document PQDx_018 “Instructions WHO
Flex studies factors on expected positive for compilation of a product dossier” for other flex Prequalification
and negative results shall be studies that may be relevant, taking into consideration – Diagnostic
considered: the broad range of operational and environmental Assessment (8)
• specimen and/or reagent conditions consistent with intended use.
volume; 2. The factors listed opposite should be investigated
• buffer pH (measure of in ways that not only reflect but also exceed likely
robustness – for example, as operating conditions in low- and middle-income
affected by evaporation of the countries, so that the limitations of the device can be
buffer); understood. For example, in addition to investigating
• reading time (that is, the deviations of temperature within those claimed in the
interval between when the instructions for use, temperature ranges should be
first and last readings can be investigated that exceed those of claimed operating
taken); conditions and which cause test failure (incorrect/
• IVD sturdiness, including invalid results).

robustness of packaging 3. The impact of lighting can be two-fold – that is, the
and labelling lighting and impact of lighting on packaging (for example, fading)
humidity (see Note 3); and on the sufficiency of lighting to read the test lines.
• operating temperature.
Part 2 Establishing clinical performance characteristics (professional use and/or self-testing)
documents
2.1 Diagnostic sensitivity and specificity
Diagnostic sensitivity and 1. Prequalified HIV RDTs are generally used by lay European
specificity shall be determined providers and health-care workers. For WHO Commission (2)
for each claimed specimen type. prequalification purposes, these should be Health Products
Testing should be conducted: considered as the intended user rather than a trained and Food Branch,
laboratory professional. Health Canada (7)
• at different geographical
settings (minimum of 2. Where an IVD is intended to detect multiple analytes
2 regions); without differentiating which analyte is detected,
• by a variety of intended users; the testing panel shall comprise specimens that are
• using more than 1 lot. reactive only for each individual analyte (that is, not
dual HIV-1/HIV-2-positive, etc).
2.1.1 Testing of: 3. A separate specimen shall be collected prior to
Diagnostic • at least 400 specimens testing to establish the reference result. The testing
sensitivity confirmed to be HIV-1 algorithm used to determine the reference results
antibody positive; shall include a state-of-the-art fourth-generation
• at least 100 specimens immunoassay, with all initially reactive specimens
confirmed to be HIV-2 reflexed for full characterization of HIV status.
antibody positive (where 4. Problematic specimens – that is, those with
HIV-2 detection is claimed; unexpected results but which otherwise meet
see Note 2); the selection criteria for a study – shall not be
• at least 50 specimens systematically excluded from analysis.
confirmed to be HIV p24 Ag
positive (where Ag detection is
claimed; see Note 2).
Annex 4
305
306
Table continued
documents
2.1.2 Testing of: 5. Consideration shall be given to the influence of
Diagnostic • at least 1000 HIV antibody/ antiretroviral medications present in a specimen on
specificity antigen negative specimens. the serostatus of such specimens, and to how this
might affect specimen selection.
6. Lots (locked-down design) shall comprise different
batches of critical components.
7. Where possible, all tests that produce a discrepant
result (between the assay under evaluation and the
reference results) shall be repeated using the same
lot, and then on all available lots and the variability
noted. Performance characteristics shall be reported
using initial results only. The results of further
testing of specimens with discrepant results shall be
reported separately as additional information on IVD
performance.
8. All indeterminate results shall be included in the

denominator data for analysis.
9. All invalid test results shall be recorded.
10. Estimates of diagnostic/clinical sensitivity and
specificity shall be reported with 95% confidence
intervals.
11. Results shall be expressed separately for each
specimen type and for each specimen type per
intended use (no aggregation of results).
Annex 4
Part 3 Qualification of usability (self-testing)

PURPOSE: Assessment of product design, instructions for use and usability of
RDTs for self-testing by analysis of the following:
■■ Results of a questionnaire to assess whether the key messages and
instructions from packaging and labelling would be understood and
easily followed by untrained intended users (that is, self-testers).
■■ Test results obtained by untrained users (that is, self-testers) of
simulated RDTs (for example, pre-made and with contrived results).
■■ Test results and interpretations when the assay is performed by
untrained intended users (that is, self-testers) (15–18).
ADDITIONAL POINTS:
■■ For each type of study summarized below, the study group shall
comprise untrained subjects whose age, gender, level of education,
literacy and additional supplementary skills may challenge the
usability of the IVD by its intended users, including in unfavourable
operational settings (for example, poor lighting).
■■ These assessment activities will determine the changes needed to
optimize the IVD for use by self-testers. Changes may range from
minor (simplification of instructions for use) to major. The impact
of any change on safety and performance shall be determined.
■■ Results from any one of the stages summarized below may indicate
that assay redesign is necessary. This may in turn result in a need to
revalidate the IVD or to perform additional specific performance
studies and to update the risk analysis.
307
308
documents
3.1 Qualification of usability (self-testing)
3.1.1 Questionnaire-based testing 1. Instructions for use and labelling shall be clear and European
Labelling of subjects representative of easy to understand; use of pictorial instructional Commission (2)
comprehension intended users, to assess the material is encouraged. ISO 18113-1:2009
study ability of such users to correctly (19)
comprehend key messages from
ISO 15197:2013 (20)
packaging and labelling with
regard to: IEC 62366-1:2015
(21)
• proper self-selection (whether
or not users understand if MHRA (22)
it is appropriate for them to Poffenberger, K (23)
undertake testing); FDA (24)
• understanding key warnings,
European
limitations and/or restrictions;
Parliament and
• proper test procedure;
European Council
• test result interpretation.
(25)
Questionnaire shall be
Center for Devices
administered to at least 200
and Radiological
subjects, representative of
Health, FDA (26)
intended users, in order to
demonstrate comprehension of WHO (27)
key messages. USAID and WHO
(28)
Center for Devices
and Radiological
Health, FDA (29)
Table continued
documents
3.1.2 A minimum of 400 subjects to 1. The study group may include subjects recruited as
Results interpret the results of contrived part of the labelling comprehension study.
interpretation IVDs (for example, static/pre-
study made tests) to assess their
ability to correctly interpret
pre-determined test results.
Contrived tests shall be made
to demonstrate the following
potential test results:
• non-reactive
• range of invalid results
• reactive
• weak reactive.
Study group to consist of at
least 200 self-testers from
2 high-prevalence (> 5%)
and geographically diverse
populations, and at least
200 self-testers from a low-
prevalence (< 5%) population
to demonstrate correct
interpretation of simulated
test results.
Annex 4
309
310
Table continued
documents
3.1.3 Testing by at least 900 self- 1. A separate venous whole blood specimen shall be
Observed testing subjects comprising collected prior to testing to establish the reference
untrained user at least 200 self-testers in results for HIV-1 status (and HIV-2 where detection is
study each of 2 high-prevalence claimed). The testing algorithm used to determine the
(> 5%) geographically diverse reference results shall include use of a state-of-the-
populations, and at least art fourth-generation immunoassay, with all initially
500 self-testers from a low- reactive specimens reflexed for full characterization of
prevalence (< 5%) population. HIV status.
• Each subject to self-collect 2. For WHO purposes, the term “professional use”
test specimen and perform encompasses a diversity of skills, training and
test according to only those experience, and does not necessarily imply “highest
materials provided with the standard of skills, training and experience”. It may be
IVD (for example, instructions a useful step in the evaluation of usability to compare
for use, labels and other the performance of self-testers with that of health-
instructional materials). care workers, lay providers and laboratory technicians.
However, concordance observed between the

different types of users may mask poor performance
within each user group. Consequently, such
comparisons do not replace the need for comparisons
to “clinical truth” based on the establishment of
reference results for each subject.
Table continued
documents
• Each such test to be observed 3. There may be a high likelihood of bias at the
by a trained laboratory or community level when simple study population
health-care professional. The sample methodologies are applied. Efforts shall be
observing professional does made to avoid convenience sampling of people
not tutor or interact with the (participants) who already know they are HIV positive.
subject conducting the test,
but notes errors and other
observations about the self-
tester. Observation may also
be conducted by viewing a
video recording of self-testing.
• The observing professional
also interprets the test result,
in a blinded fashion and
within the validated reading
time stated in the instructions
for use.
Annex 4
311
References
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of performance studies. TGS–3. Geneva: World Health Organization; 2017 (http://apps.who.int/
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Committee for Standardization; 2002.
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Second edition. Wayne (PA): Clinical and Laboratory Standards Institute; 2008.
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used in the laboratory. Ottawa: Health Canada; 2011 (http://hc-sc.gc.ca/dhp-mps/md-im/applic-
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HIV test kits intended to be used outside the laboratory, that is, at the point of care and/or for home
use. Separate guidance titled “Draft Guidance Document – HIV Simple/Rapid Test Kits” is available
for manufacturers of near-patient HIV test kits upon request from Health Canada’s Medical Devices
Bureau, Health Canada; email: [email protected]
8. Instructions for compilation of a product dossier. Prequalification of In Vitro Diagnostics
Programme. Geneva: World Health Organization; 2014 (PQDx_018 v3, 27 August 2014; http://
www.who.int/entity/diagnostics_laboratory/evaluations/141015_pqdx_018_dossier_instructions_
v4.pdf?ua=1, accessed 2 January 2018).

9. Butch AW. Dilution protocols for detection of hook effects/prozone phenomenon. Clin Chem.
2000; 46(10):1719–21 (http://clinchem.aaccjnls.org/content/46/10/1719, accessed 2 January
2018).
10. Interference testing in clinical chemistry; approved Guideline EP07-A2. Second edition. Wayne
(PA): Clinical and Laboratory Standards Institute; 2005.
11. ISO 23640:2011. In vitro diagnostic medical devices – evaluation of stability of in vitro diagnostic
reagents. Geneva: International Organization for Standardization; 2011.
12. Evaluation of stability of in vitro diagnostic reagents; approved Guideline EP25-A. Wayne (PA):
Clinical and Laboratory Standards Institute; 2009.
13. Technical Guidance Series (TGS) for WHO Prequalification – Diagnostic Assessment. Establishing
stability of an in vitro diagnostic for WHO Prequalification. TGS–2. Geneva: World Health
Organization; 2016 (http://www.who.int/entity/diagnostics_laboratory/guidance/160613_tgs2_
stability.pdf?ua=1, accessed 2 January 2018).
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14. Standard practice for performance testing of shipping containers and systems. ASTM D4169 - 14.
West Conshohocken (PA): ASTM International; 2014.
15. Blood Products Advisory Committee. 86th Meeting, 10 March 2006. Silver Spring (MA): United
States Food and Drug Administration; 2006 (https://www.fda.gov/ohrms/dockets/ac/06/
transcripts/2006-4206t2.pdf, accessed 2 January 2018).
16. Blood Products Advisory Committee. 85th Meeting, 3 November 2005. Silver Spring (MA):
United States Food and Drug Administration; 2005 (https://www.fda.gov/ohrms/dockets/ac/05/
transcripts/2005-4190t1.htm, accessed 2 January 2018).
17. Blood Products Advisory Committee. Open Session, 17 November 2009. Silver Spring (MA):
United States Food and Drug Administration; 2009 (https://wayback.archive-it.org/7993/2017011
3022625/http://www.fda.gov/downloads/AdvisoryCommittees/CommitteesMeetingMaterials/
BloodVaccinesandOtherBiologics/BloodProductsAdvisoryCommittee/UCM193386.pdf, accessed
2 January 2018).
18. Blood Products Advisory Committee. 102nd Meeting, 15 May 2012. Silver Spring (MA): United
States Food and Drug Administration; 2012 (https://wayback.archive-it.org/7993/2017011301
4805/http://www.fda.gov/downloads/AdvisoryCommittees/CommitteesMeetingMaterials/
BloodVaccinesandOtherBiologics/BloodProductsAdvisoryCommittee/UCM309516.pdf, accessed
2 January 2018).
19. ISO 18113-1:2009. In vitro diagnostic medical devices – information supplied by the
manufacturer (labelling) – Part 1: Terms, definitions and general requirements. Geneva:
International Organization for Standardization; 2009.
20. ISO 15197:2003. In vitro diagnostic test systems – requirements for blood-glucose monitoring
systems for self-testing in managing diabetes mellitus. Geneva: International Organization for
Standardization; 2003.
21. IEC 62366-1:2015. Medical devices – Part 1: Application of usability engineering to medical
devices. Geneva: International Electrotechnical Commission; 2015.
22. Guidance for notified bodies on the regulation of IVDs for self-testing. London: Medicines and
Healthcare products Regulatory Agency; 2012 (https://www.gov.uk/government/publications/
regulation-of-ivds-for-self-testing, accessed 2 January 2018).
23. Poffenberger K. Update: rapid HIV test approval requirements and standards [Slides]. Silver
Spring (MD): United States Food and Drug Administration; 2000 (http://www.fda.gov/ohrms/
dockets/ac/00/slides/3649s2.htm, accessed 2 January 2018).
24. OraQuick® In-Home HIV Test. Summary of safety and effectiveness. FDA BP120001. Silver Spring
(MA): United States Food and Drug Administration; 2012 (http://www.fda.gov/downloads/
BiologicsBloodVaccines/BloodBloodProducts/ApprovedProducts/PremarketApprovalsPMAs/
UCM312534.pdf, accessed 2 January 2018).
25. Directive 98/79/EC of the European Parliament and of the Council of 27 October 1998 on in
vitro diagnostic medical devices. OJ. 2012;L 331:1 of 7.12.1998 (http://data.europa.eu/eli/
dir/1998/79/2012-01-11, accessed 2 January 2018).
26. Recommendations: clinical laboratory improvement amendments of 1988 (CLIA) waiver
applications for manufacturers of in vitro diagnostic devices. Silver Spring (MD): United States
Food and Drug Administration; 2008 (http://www.fda.gov/MedicalDevices/DeviceRegulation
andGuidance/GuidanceDocuments/ucm079632.htm, accessed 2 January 2018).
27. Consolidated guidelines on HIV testing services. Geneva: World Health Organization; 2015
(http://who.int/hiv/pub/guidelines/hiv-testing-services/en/, accessed 2 January 2018).
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28. How to use a rapid diagnostic test (RDT). A guide for training at a village and clinic level.
Modified for training in the use of the Generic Pf-Pan Test for falciparum and non-falciparum
malaria). Bethesda (MD) and Geneva: The USAID Quality Assurance Project (QAP), University
Research Co., LLC, and the World Health Organization; 2009 (http://www.wpro.who.int/malaria/
NR/rdonlyres/23DD7DCB-48C4-4CFF-BD45-332F0BE3DCC7/0/generic_PfPan_training_manual_
web.pdf, accessed 2 January 2018).
29. Applying human factors and usability engineering to medical devices. Guidance for industry
and Food and Drug Administration staff. Silver Spring (MD): United States Food and Drug
Administration; 2011 (http://www.fda.gov/downloads/MedicalDevices/.../UCM259760.pdf,
accessed 2 January 2018).
314
Annex 5
Technical Guidance Series (TGS) for WHO Prequalification –
Diagnostic Assessment
Establishing stability of in vitro

diagnostic medical devices TGS–2

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315
Preface
WHO Prequalification – Diagnostic Assessment:
Technical Guidance Series
WHO WHO prequalification is coordinated through the

prequalification Department of Essential Medicines and Health
of IVDs Products. WHO prequalification of in vitro diagnostic
medical devices (IVDs) is intended to promote and
facilitate access to safe, appropriate and affordable IVDs
of good quality in an equitable manner. The focus is on
IVDs for priority diseases and on their suitability for
use in resource-limited settings. WHO prequalification
is based upon a comprehensive assessment of individual
IVDs using a standardized procedure that is aligned with
international best regulatory practice. It also involves
post-qualification activities for IVDs to ensure their
ongoing compliance with prequalification requirements.
Procurement Products that are prequalified by WHO are eligible for

of prequalified procurement by United Nations agencies. The products
IVDs are then commonly purchased for use in low- and
middle-income countries.
Prequalification IVDs prequalified by WHO are expected to be accurate,

requirements reliable and able to perform as intended for the lifetime
of the IVD under conditions likely to be experienced
by a typical user in resource-limited settings. Countries
in which WHO-prequalified IVDs are procured often
have minimal regulatory requirements, and the use of

IVDs in these countries presents specific challenges. For
example, IVDs are often used by health-care workers
who do not have extensive training in laboratory
techniques, in harsh environmental conditions, in the
absence of extensive pre- and post-test quality assurance
capacity, and for patients with a disease profile that
differs from the profiles encountered in high-income
countries. Therefore, the requirements of WHO
prequalification may differ from the requirements
of high-income countries, or those of the regulatory
authority in the country of manufacture.
316
Annex 5
About the The Technical Guidance Series (TGS) was developed

Technical following a WHO working group consultation held
Guidance Series on 10–13 March 2015 in Geneva, Switzerland. The
consultation was attended by experts from national
regulatory authorities, national reference laboratories,
and WHO prequalification dossier reviewers and
inspectors. The guidance series is a result of the efforts
of this and other international working groups.
Audience and This guidance is intended for manufacturers interested

scope in WHO prequalification of their IVD. It applies
in principle to all IVDs that are eligible for WHO
prequalification for use in WHO Member States. This
guidance should be read in conjunction with relevant
international and national standards and guidance.
The TGS documents are freely available on the WHO

web site.
317
Acknowledgements 320
List of contributors 320
1 Abbreviations 321
2 Definitions 322
3 Introduction 327
3.1 Key concepts 327
3.2 Rationale of stability studies 327
3.3 Purpose of this document 327
3.4 Standards 327
3.5 Limitations of this guidance 328
4 Considerations when applying for WHO prequalification 328
4.1 Manufacturer responsibility 329
4.2 Suitability for use in WHO Member States 329
4.3 Meeting customer requirements 329
5 Basic principles for stability testing 329
5.1 Critical characteristics or metrics of the IVD 329
5.2 Finalized product presentation 330
5.3 Environmental conditions 330
5.4 Minimum number of lots 331
5.5 Assessment of liquid components 331
5.6 Specimens for the stability testing panel 332
5.7 Validation of stability testing panel 332
5.8 Panel member selection and value assignment criteria 333
5.9 Time points 334
5.10 “Zero time” values and variance 335
6 Shelf-life studies 336
6.1 Requirements for determination of shelf-life 336
7 Component stability studies 337
7.1 General principles 337

7.2 Stability of control materials 338
7.3 Biocidal stability and efficacy 339
7.4 Desiccant functionality 339
8 Stability during transport 340
8.1 Rationale 340
8.2 Challenge conditions 341
8.3 Number of lots 341
8.4 Simulated versus actual challenge 341
8.5 Multiple stress test sequences (simulated transport challenges) 342
8.6 Physical conditions 342
9 In-use stability studies 342
9.1 Rationale 342
318
Annex 5
9.2 Conditions of use 343

9.3 Multiple in-use stability claims 343
10 Production lots used in stability studies 344
10.1 Considering variability 344
10.2 Testing the final configuration 344
10.3 Number of lots required for testing 345
10.4 Components of lots required for testing 346
11 Stability study plan 346
11.1 Responsibilities 347
11.2 Preparing the testing plan 347
11.3 Product storage 348
11.4 Documentation 348
11.5 Statistical methods 348
11.6 Stability testing protocol 349
11.7 Reading and recording results 351
11.8 Instability versus imprecision 352
11.9 Testing schedule 352
12 Stability study report 352
12.1 General 352
12.2 Link to claims 353
12.3 Consider variability 353
12.4 IVD stability versus component stability 353
13 Changes to a WHO prequalified IVD 353
13.1 Dealing with change 353
14 References 355
Appendix 1 Examples of stability protocols 358
Example 1: Evaluation of transport stability followed by real-time stability 359
Example 2: In-use stability protocol 364
Appendix 2 Suggested specimens for stability testing panels 367
1. Specimens to monitor NAT-based tests 367
2. Specimens to monitor tests that measure CD4 cells 368
3. Specimens to monitor tests for HIV antibodies 369
4. Specimens to monitor tests for HIV-1/2 and Treponema pallidum (TP) antibodies 370
5. Specimens to monitor tests for HCV antibodies 370
6. Specimens to monitor tests for HBsAg 371
Appendix 3 Summary table of standards relevant to stability studies 373
319
Acknowledgements
The document Technical Guidance Series 2 (TGS–2) for WHO Prequalification –
Diagnostic Assessment: Establishing stability of in vitro diagnostic medical devices
was developed with support from the Bill & Melinda Gates Foundation and
UNITAID. The document was prepared in collaboration with Dr RJS Duncan,
London, the United Kingdom; Ms S Best, Dr S Braniff and Dr M Lanigan,
National Serology Reference Laboratory, VIC, Australia; and Ms D Healy and
Ms R Meurant, WHO, Geneva, Switzerland, with input and expertise from Dr S
Hojvat, MD, United States of America (USA); Dr L Kestens, Institute of Tropical
Medicine, Antwerp, Belgium; and Ms D Lepine, Medical Devices Bureau, Health
Canada, Ottawa, Canada. This document was produced under the coordination
and supervision of Ms R Meurant and Ms I Prat, Prequalification Team, WHO,
Geneva, Switzerland.
List of contributors
First-round comments were received from the following: Dr JC Badciong,
Abbott Laboratories, Chicago, the USA; Mr K De Vore, Bio-Rad Laboratories,
France; Dr A Halim, Celldex Therapeutics, Hampton, NJ, the USA; Dr S Hojvat,
MD, the USA; Dr L Kestens, Institute of Tropical Medicine, Antwerp, Belgium;
Ms D Lepine, Medical Devices Bureau, Health Canada, Ottawa, Canada; Ms L
Ochs, Clinical and Laboratory Standards Institute (CLSI), Wayne, PA, the USA
and members of the CLSI Consensus Committee, ISO T212 WG3 committee;
Dr G Pennello, United States Food and Drug Administration, Silver Spring,
MD, the USA; Mr J Pierson-Perry, Siemens Healthcare Diagnostics, Erlangen,
Germany; Dr E Russek-Cohen, United States Food and Drug Administration,
Silver Spring, MD, the USA; Professor M Stevens Hardy, Medical Laboratory
& Technology Consultants, LLC, Washington, DC, the USA; Mr C Zang,
National Institutes for Food and Drug Control, Beijing, China; and the Japanese
Committee for Clinical Laboratory Standards, Tokyo, Japan.
The draft technical guidance document was posted on the WHO
Prequalification website for public consultation on 14 December 2015. Various
stakeholders, including manufacturers submitting to WHO prequalification
of IVDs, IVD manufacturing industry associations, various national and
international regulatory bodies, and IVD standards organizations, were informed
of the consultation in order to solicit feedback. A two-month response period
was provided.
Second-round public comments were received from the following: Ms A
Asahina, Alere Medical Co., Ltd, Chiba, Japan; Dr J Budd, Beckman Coulter Inc.,
Chaska, the USA; Dr C Candia Ibarra, Ministerio de Salud Pública y Bienestar
Social, Asunción, Paraguay; Dr NA Carrington, Roche Diagnostics, Indianapolis,
320
Annex 5
IN, the USA; Dr M Dreher, mdc medical device certification GmbH, Stuttgart,
Germany; Dr I Fijalkowska, United States Food and Drug Administration,
Silver Spring, MD, the USA; Ms J Goss, Sysmex Partec GmbH, Goerlitz,
Germany; Dr C Hill, Encinitas, CA, the USA; Dr L Kestens, Institute of Tropical
Medicine, Antwerp, Belgium; Dr M Kondratovich, United States Food and Drug
Administration, Silver Spring, MD, the USA; Dr M Leportier, Beckman Coulter,
Marseille, France; Ms K Máté‚ European Diagnostic Manufacturers Association,
Brussels, Belgium; Mr F Nyberg, Asia Pacific Medical Technology Association,
Singapore; Dr S Ortigoza, Ministerio de Salud Pública y Bienestar Social,
Asunciòn, Paraguay; Dr GP Payne, BD Diagnostics Point of Care, San Diego,
CA, the USA; Mr J Pierson-Perry, Siemens Healthcare Diagnostics, Erlangen,
Germany; Ms L Seixas, ALADDIV, Brasília, Brazil; Dr W-W Tsai and Ms P-W Tu,
Asian Harmonisation Working Party TC WG2, China, Hong Kong SAR; Dr NT
Wetherall, DAIDS/NIAID, Bethesda, MD, the USA; and Dr L Xu, Theranos, Inc.,
Palo Alto, CA, the USA.
Following incorporation of the second-round public comments, a
revised draft was published on the WHO Biologicals website for a final round
of public consultation between 18 June and 18 September 2017. The comments
received were incorporated to produce the document WHO/BS/2017.2304.
The document was adopted by the WHO Expert Committee on Biological
Standardization as a WHO written standard on 20 October 2017.
1 Abbreviations
ASTM ASTM International
CE Conformité Européenne (European Conformity)
CLSI Clinical and Laboratory Standards Institute
EIA enzyme immunoassay
HBsAg hepatitis B surface antigen
HBV, HCV hepatitis B virus, hepatitis C virus
IFU instructions for use
IgG, IgM immunoglobulin G, immunoglobulin M
ISO International Organization for Standardization
IVD in vitro diagnostic medical device
NAT nucleic acid test
NIBSC National Institute for Biological Standards and Control
321
NS3, NS4, HCV non-structural proteins

NS5
OD optical density
PEI Paul-Ehrlich-Institut
QA quality assurance
QC quality control
QMS quality management system
RPM revolutions per minute
R&D research and development
SOP standard operating procedure(s)
TGS WHO Technical Guidance Series
TP Treponema pallidum
2 Definitions
The definitions given below apply to the terms used in this document. They
may have different meaning(s) in other contexts. Common English dictionary
definitions apply to non-defined concepts, such as device, constituent, equipment,
evaluation, part, product, reaction, signal, substance, etc.
Accelerated stability evaluation: Study designed to increase the rate of chemical
and/or physical degradation, or change, of an IVD reagent by using stress
environmental conditions to predict shelf-life.
Note: The design of an accelerated stability evaluation can include extreme

conditions of temperature, humidity, light or vibration (1).
Acceptance criteria: A defined set of conditions that must be met to establish
the performance of a system (2, 3).
Numerical limits, ranges or other suitable measures for acceptance of the results
of analytical procedures (2, 3).
Accuracy of measurement: Closeness of the agreement between the result of a
measurement and a true value of the measurand.
Note 1: Accuracy of measurement is related to both trueness of measurement and
precision of measurement.
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Note 2: Accuracy cannot be given a numerical value in terms of the measurand,

only descriptions such as “sufficient” or “insufficient” for a stated purpose (4).
Arrhenius plot: Mathematical function that describes the approximate
relationship between the rate constant of a chemical reaction and the temperature
and energy of activation (2).
Batch/Lot: Defined amount of material that is uniform in its properties and has
been produced in one process or series of processes.
Note: The material can be either starting material, intermediate material or
finished product (5).
Biocidal products: Active substances and preparations containing one or more
active substance intended to destroy, deter, render harmless, prevent the action
of, or otherwise exert a controlling effect on any harmful organism by chemical
or biological means (6).
Characteristic: Distinguishing feature.
Note 1: A characteristic can be inherent or assigned.
Note 2: A characteristic can be qualitative or quantitative.
Note 3: Characterization: a description of the distinctive nature or features of
something (7).
Component: Part of a finished, packaged and labelled IVD (5).
Note: Typical kit components include antibody solutions, buffer solutions,
calibrators and/or control materials (5).
Constituent: Raw materials used to make a component.
Control material: Substance, material or article intended by its manufacturer to
be used to verify the performance characteristics of an IVD (5, 8).
Design input: The physical and performance requirements of an IVD that are
used as a basis for IVD design (9).
Drift: Characteristic slow change of a metrological value from a measuring
instrument (10).
Environmental factors: Variables that might affect the performance or efficacy
of IVD reagents – for example, temperature, airflow, humidity and light (2).
WHO note: For WHO purposes, this also includes altitude and microorganisms.
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Evidence: Information which can be proved true based on facts obtained

through observation, measurement, testing or other means (modified from (7).
Independent lots: lots with different production (or manufacturing, purification,

etc.) runs of critical reagents (for example, biological reagents prepared in
different syntheses, growths or purifications or other risk-defined critical reagents
from different manufactured lots or from different suppliers if applicable).
Instructions for use (IFU): Information supplied by the manufacturer to enable
the safe and proper use of an IVD.
Note: Includes the directions supplied by the manufacturer for the use,
maintenance, troubleshooting and disposal of an IVD, as well as warnings and
precautions (5).
WHO note: In order to avoid confusion, please note that, in the USA, the
acronym IFU also stands for “Indications for use”, and the acronym IU stands
for “Intended use” or “Indications for use” (the acronym PI is often used in the
USA to indicate the package insert, which may contain IFU). The International
Organization for Standardization (ISO) definition and requirements (5) for IFU
cover the intended use and the precise method of use and is the definition used
by WHO and throughout this and other TGS documents.
In-use stability: Duration of time over which the performance of an IVD
reagent within its expiration date remains within specified limits after opening
of the container system supplied by the manufacturer and use under standard
operation conditions (for example, storage on the instrument).
WHO note: For the purpose of this guidance document, WHO considers that it
includes the number of times the reagents can be removed, used and returned
to the storage condition without impact on test kit performance. It must reflect
the routine conditions of use (for example, on-board stability, reconstitution and
open-vial/bottle stability). A single product may have several different types of
in-use stability claim, each reflecting different aspects of its usage. For example,
an IVD reagent may have one in-use stability claim for unopened storage on
board its associated instrument system and another stability claim once it is
opened and put into active use. Another type of in-use life is the calibration
interval of an IVD reagent (2).
In vitro diagnostic medical device (IVD): A medical device, whether used alone
or in combination, intended by the manufacturer for the in vitro examination
of specimens derived from the human body solely or principally to provide
information for diagnostic, monitoring or compatibility purposes.
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Note 1: IVDs include reagents, calibrators, control materials, specimen

receptacles, software, and related instruments, apparatus or other articles, and
are used, for example, for the following test purposes: diagnosis, aid to diagnosis,
screening, monitoring, predisposition, prognosis, prediction, and determination
of physiological status.
Note 2: In some jurisdictions, certain IVDs may be covered by other

regulations (11).
IVD reagent: Chemical, biological or immunological components, solutions or

preparations intended by the manufacturer to be used as an IVD (5).
WHO note: This document uses the terms IVD and IVD reagent interchangeably.
Life-cycle: All phases in the life of a medical device, from the initial conception
to final decommissioning and disposal (12).
Metrological traceability: Property of the result of a measurement or the value

of a standard whereby it can be related to stated references (usually national or
international standards) through an unbroken chain of comparisons, all having
stated uncertainties.
Note: Each comparison is affected by a (reference) measurement procedure

defined in a calibration transfer protocol (4).
Performance claim: Specification of a performance characteristic of an IVD as

documented in the information supplied by the manufacturer.
Note: This can be based upon prospective performance studies, available

performance data or studies published in the scientific literature (5).
WHO note: “Information supplied by the manufacturer” includes but is not

limited to: statements in the IFU, in the dossier supplied to WHO and/or
regulatory authorities, in advertising and on the internet.
Referred to simply as “claim” or “claimed” in this document.
Precision: The closeness of agreement between independent test results obtained

under stipulated conditions (4).
Real-time stability evaluation: Study designed to establish or verify the shelf-life

of the IVD reagent when exposed to the conditions specified by the manufacturer.
Note: Conditions that can affect the stability of an IVD reagent include
temperature, transport conditions, vibration, light and humidity (1).
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Risk management: The systematic application of management policies,

procedures and practices to the tasks of analysing, evaluating, controlling and
monitoring risk (12).
Risk-management plan: For the particular IVD being considered, the
manufacturer shall establish and document a risk-management plan in
accordance with the risk-management process (12).
Shelf-life: Period of time until the expiry date, during which an IVD reagent,
in its original packaging, maintains its stability under the storage conditions
specified by the manufacturer.
Note: Stability and expiry date are related concepts (5).
WHO note: In this document “Labelled life” is considered to be the time up to
the expiry date printed on the label of an IVD or IVD component.
Stability: Ability of an IVD reagent to maintain its performance characteristics
within the limits specified by the manufacturer.
Note 1: Stability applies to:
■■ IVD reagents, calibrators and controls, when stored, transported
and used under the conditions specified by the manufacturer;
■■ reconstituted lyophilized materials, working solutions and materials
removed from sealed containers, when prepared, used and stored
according to the manufacturer’s IFU;
■■ measuring instrument or measuring system after calibration.
Note 2: Stability of an IVD reagent or measuring system is normally quantified
with respect to time:
■■ in terms of the duration of a time interval over which a metrological

property changes by a stated amount;
■■ in terms of the change of a property over a stated time interval.
WHO note: because definition restricts IVD reagent only. Refer to (1) definition
3.10.
Stability monitoring: Real-time stability testing at certain points in time during
shelf-life (or in-use life) to assure that an IVD reagent performs within specified
claims (2).
Note: A continuing stability monitoring programme (ongoing stability
monitoring) is required to verify that the stability claim is maintained over the
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life-cycle of the product. Data on stability must be obtained at end of shelf-life

(see (1); section 4.1) and ideally at the halfway point of assigned shelf-life so
that any problems that do occur can be dealt with in a timely fashion.
Trueness of measurement: Closeness of agreement between the average values
obtained from a large series of results of measurements and a true value (4).
Validation: Confirmation by examination and provision of objective evidence
that the requirements for a specific intended use or application have been
fulfilled (7).
Verification: Confirmation by examination and provision of objective evidence
that specified requirements have been fulfilled (7, 13).
3 Introduction
3.1 Key concepts
Stability is the ability of an IVD reagent to maintain its performance
characteristics over a defined time interval (12). The purpose of most stability
studies is to establish or verify the time interval, and the storage conditions that
can maintain stable IVD performance characteristics.
3.2 Rationale of stability studies

The stability of an IVD is fundamental to its reliable performance over a defined
period of time. It is a regulatory requirement for the manufacturer to provide
objective, scientifically sound evidence to support all claims made regarding
the stability of an IVD. In addition, a manufacturer can use stability studies to
demonstrate the probability that lots manufactured up to the end of the life-cycle
of the IVD will meet predetermined user needs (as identified in design inputs).
3.3 Purpose of this document

The purpose of this document is to provide IVD manufacturers with guidance
on possible approaches to determine stability. It also describes the expectations
of WHO prequalification in relation to stability studies.
3.4 Standards
WHO recommends the following standards for use in establishing stability
claims: International Organization for Standardization (ISO) 23640:2011 (1);
Clinical and Laboratory Standards Institute (CLSI) EP25-A (2) and ASTM
International D4169 - 14 (14). It is recommended that manufacturers be familiar
with these standards and consider them when designing and planning their
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stability studies. For other relevant standards see TGS–1: Standards applicable to
the WHO Prequalification of in vitro diagnostic medical devices 1.
3.5 Limitations of this guidance

This guidance document should not be taken as a prescriptive checklist of the
stability testing that must be performed, but as a guide on how to improve
processes and generate the evidence needed to ensure a comprehensive and
systematic procedure with an appropriate risk-management plan.
Depending on the particular categorization of the product and on the
particular jurisdiction, additional regulatory and/or legal requirements, beyond
the scope of this document, may apply.
The examples included throughout the document are not exhaustive and
apply to the principles outlined in this document only. Manufacturers must still
perform their own product-specific risk assessment for each of their IVDs, which
may identify other critical characteristics (for example, physical measurements).
4 Considerations when applying

for WHO prequalification
WHO requires that reports of studies used in establishing the stability claims
for the product be submitted as part of the prequalification application.2 As
part of the WHO prequalification assessment, manufacturers must describe the
rationale, the study methods, the stability monitoring programme followed and
the testing algorithms used, with references to the relevant standard operating
procedures (SOP). The information provided must demonstrate the link to the
predetermined user requirements and product development.
The expectations of WHO prequalification may be different from the
inputs of the users and from the requirements of the regulatory authority in the
country of manufacture. In addition, the expectations set out in this guidance

document may be additional to the requirements of ISO 23640 (1) and the
expectations of CLSI EP25-A (2). Wherever possible, this guidance document
explains the reasons for these additional expectations. Other approaches to
meeting these additional expectations, supported by rigorous risk assessment
or other evidence, may also be acceptable in dossiers submitted for WHO
prequalification.
1
Available at: http://www.who.int/diagnostics_laboratory/guidance/170808_tgs1_standards_2.0.pdf?ua=1
2
WHO documents PQDx_049 Product dossier checklist and PQDx_018 Instructions for compilation of a
product dossier are available on the WHO Prequalification – Diagnostic Assessment website: http://www.
who.int/diagnostics_laboratory/evaluations/en/
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4.1 Manufacturer responsibility

It is a manufacturer’s responsibility to ensure that the evidence supporting
performance claims regarding the end of the IVD shelf-life is objective and
scientifically rigorous.
4.2 Suitability for use in WHO Member States

The stability studies submitted to WHO prequalification shall accurately reflect
the expected environmental conditions and the normal usage conditions/
methods encountered by users in WHO Member States, such as:
■■ extremes of temperature under in-use conditions and during
transportation;
■■ extremes of humidity encountered under in-use conditions and
during transportation and storage;
■■ any affects that light may have on IVD functionality, especially on
the length of time for which a result is claimed to be stable; and
■■ prevalence of certain microorganisms.
4.3 Meeting customer requirements

By undertaking well-designed stability studies (including periodic verification
activities) the manufacturer can demonstrate that the product meets input
requirements (that is, customer requirements), as required by ISO 13485 (see
(15) section 7.2: Customer-related processes). Meeting predetermined user
expectations, not merely evaluating the capability of an IVD, is a fundamental
aspect of IVD development (see (9) definition (f); and (15) section 7.3.4). It is a
proactive means for the manufacturer to prevent quality problems at lot release
and in the post-production and marketing phase.
5 Basic principles for stability testing

5.1 Critical characteristics or metrics of the IVD
A well-designed stability study must generate evidence of the stability of each
of the critical constituents in the IVD (risk-evaluated critical constituents),
evidence of stability for each of the claimed analytes, and evidence for any
particular level of performance, including the precision, sensitivity and
specificity of the kit. A documented risk-based approach should be taken to
determine which claims and constituents must be evaluated over the stated
shelf-life.
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Examples:
1. A hepatitis C virus (HCV) assay containing the critical constituents
related to detection of NS3 or core proteins must have the stability of
all such constituents proven for the shelf-life of the IVD.
2. For an assay designed to detect both immunoglobulin G (IgG) and
immunoglobulin M (IgM) by use of protein A and protein L, the
stability of both protein A and protein L must be proven in the IVD.
3. For an IVD to quantitate CD4, all the constituent antibodies used
(for example, anti-CD3 and anti-CD4) must be shown to be stable in
the IVD.
4. For an IVD claimed to detect particular seroconversion specimens
or genotypes, or to have specified precision at particular analyte
concentrations, or a particular specificity, each of these claims at risk
or that change over time must be proven over the stated shelf-life (see
TGS–4: Guidance on test method validation for in vitro diagnostic
medical devices (16).
Other critical characteristics (also called critical metrics) identified in
the risk assessments may include physical measurement (for example, volume,
pH, flow rate, legibility and adhesion). These characteristics must be shown to
meet their specifications for the shelf-life of the IVD but are outside the scope of
this document.
5.2 Finalized product presentation

During stability testing, all IVD components (including the IVD, calibrator and/
or control material, etc.) must be made and tested to the finalized manufacturing
specifications and in the finalized packaging, including intended labels and
containers (see section 10.4). In most circumstances, all presentations (for
example, different buffer volumes used for different kit sizes) must be used during
stability testing. Where some presentations are not tested, the manufacturer
should document the rationale, justifying why all presentations have not
been tested.
5.3 Environmental conditions

The stability study must subject the IVD to a combination of conditions that
define, with predetermined confidence limits, the stability for lots marketed
during the life-cycle of the IVD. The combination of conditions and durations of
exposure and number of lots to be used will be driven by a manufacturer’s risk
assessment for the IVD and by research and development (R&D) data. The risk
assessment should, at a minimum, take into account the following:
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■■ the variability of the constituent materials (identifying the most

important sources of variation);
■■ an understanding of the nature of user environments; and
■■ the extremes of conditions (temperature, humidity, ambient
pressure and vibration) potentially occurring during transportation
to those users (see also section 4.2).
Boundary conditions for stability studies must reflect realistic extreme
conditions that are consistent with the design input requirements for the IVD. The
subsequent stability studies will prove the IVD capable of meeting performance
requirements up to the end of its stated shelf-life, after transportation to the users.
5.4 Minimum number of lots

The design of stability studies must take into consideration lot-to-lot variability,
with a risk assessment conducted to identify the most important sources of
variability. The degree of variation of individual lots affects the confidence that
a future production lot will remain within specification throughout its shelf-life.
Lot variability is most often caused by minor differences in the biological reagents
rather than by lack of reproducibility of the manufacturing process. Although
existing standards (1, 2) recommend the use of a single lot for certain stability
studies, the impact of lot-to-lot variability must be taken into consideration and
the use of additional lots may be necessary. Three lots, at a minimum, must be
used to establish or verify shelf-life; in-use claims require testing on a minimum
of one lot. To ensure that the potential for lot-to-lot variability is addressed,
independent lots must be used – that is, lots containing different batches of
critical constituents such as nitrocellulose membranes, recombinant antigens,
peptides, nucleic acids and the enzymes used in nucleic acid test-based (NAT-
based) testing technologies.
Example:
For NAT-based testing technologies, it is crucial to use independent lots of
enzyme for stability studies, as the manufacturing process can affect them.
Other components (including primer, probe and buffer) can also be affected
by the manufacturing process (for example, in terms of purity, pH, and
DNase and RNase contamination). Thus for these other components, the
use of independent lots that represent both material and process variability
are also recommended.
5.5 Assessment of liquid components

The orientation of the product during storage (that is, upright versus inverted or
horizontal) may need to be included in a protocol where contact of the product
with the different parts of the container (such as the closure system or the body
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of the container) may be expected to affect the stability of the products contained
(for example, liquid component). This is sometimes referred to as “inverted
container stability”. The product orientation may need to be moved occasionally
during the stability study to ensure that there is direct contact between the liquid
contents and all parts of the container. This aspect requires particular attention
during in-use stability studies of components that are diluted or reconstituted
from a freeze-dried state before use.
5.6 Specimens for the stability testing panel 3

The specimens used in the stability testing panel(s) must reflect the performance
claims related to the IVD. The specimen types most likely to be used in those
WHO Member States in which the IVD is intended to be used must be considered
and, as appropriate, included in the specimen panels used throughout the stability
studies (see Appendix 2). If a variety of specimen types (for example, serum,
plasma, whole blood and saliva) are claimed as being suitable for use in the IFU,
the stability study plan must be designed to provide evidence that the IVD will
meet its claims (for example, for sensitivity, specificity, proportion of valid runs
and precision) for each of the specimen types for the whole of the claimed shelf-
life, including during transport to the final users, unless an alternative approach
can be justified using a documented rationale. Evidence must be statistically
valid (see section 11.5). Regulatory requirements may also dictate the addition
of specified panel members.
5.7 Validation of stability testing panel

The stability testing panel(s) must be validated, and rejection and replacement
criteria must be established. The validation of the panel members used is crucial.
Panel members themselves must be stable and they must monitor parameters
that are useful in controlling the characteristic being tested.
Storage of a validated panel for testing stability is not always feasible.

For example, this is often the case for assays requiring fresh and/or whole blood
specimens (for example, assays for counting CD4 cells). When replacing panel
members, particularly for CD4 monitoring, the accuracy of results generated
using the replacement material must be confirmed using an appropriate reference
method (for example an instrument validated for use in an ISO 15189 (17)
accredited laboratory). Replacement criteria for unstable panel members must
include the duration for which a critical member will give valid results.
3
A panel is a collection of well-characterized specimens and other materials that are used to monitor
aspects of IVD and component function during stability studies, for in-process control, for some aspects
of design validation and at release to sale. The same materials might be used for each of these purposes
but be assigned different acceptance criteria for the different functions.
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5.8 Panel member selection and value assignment criteria

Panel members are chosen specifically to ensure that each member has an
attribute relevant to the intended use. The goal of stability testing is to ensure that
the test method appropriately monitors functionality at the end of the assigned
life (shelf-life or in-use life) of the antigens, epitopes and antibodies, along with
any physical specifications relevant to the intended use.
For example, an intended use claim may be that early seroconversion
specimens are detected. To show that this claim is true at the end of the product’s
shelf-life, a stability panel member representative of a very early seroconversion
specimen could be included. This might be a weakly reactive IgM specimen,
or some other specimen that has been shown to closely mimic the behaviour
of the IVD with the critical specimens. Rare and valuable specimens would
not be expected to be tested at all time points of stability studies. However,
evidence must be provided that key performance claims made in the IFU,
published material (including advertising) and dossiers submitted to WHO
prequalification are met at the end of the assigned shelf-life and in-use life.
Each panel member is assigned an expected value and this is used to
assign the acceptance criteria for that panel member. The expected value for
each panel member is assigned in a measurable manner that is relevant to the
outputs of the particular methodology. For example, the acceptance criteria for
each panel member may be assigned in terms of sample-to-cut-off ratio, cycle
time (CT) values or band intensity measured quantitatively/semi-quantitatively.
In the example of a weakly reactive IgM seroconversion specimen,
the specimen at the start of shelf-life may have an RDT reading of 1+ out of
4 assigned as its expected value using a semi-quantitative value based on band
intensity. The acceptance criteria assigned as a result may be that “all reactive
specimens remain reactive, and all non-reactive specimens do not react in
the assay”.
Panel members must be chosen so that they will not only be relevant in
demonstrating the intended use but will also have values that will appropriately
detect, and therefore monitor, any deleterious effects of storage. A strong positive
specimen that has a 4+ out of 4 semi-quantitative reading may continue to give
this reading despite decay in the assay, whereas a specimen with a reading of 1+
out of 4 (with an assigned acceptance criteria of “remaining positive”) is more
likely to give an indication of the ongoing stability of the assay.
Thus it is essential to know (and document) that whenever a panel
member meets the acceptance criteria, this is a true reflection of the stability of
the product and not due to the inability of the specimen result output to reflect
any change in the IVD.
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5.9 Time points

A simple study design requires a minimum of three testing intervals (2):
1. an initial baseline test;

2. a test at the time point beyond the claimed stability limit (see
section 5.9.1);
3. one point in between.
This simple study design is acceptable for submission to WHO

prequalification under some circumstances and for some IVDs based on:
■■ the manufacturer’s risk analysis;

■■ whether the manufacturer has prior-objective documented
experience of the stability of the product; and
■■ whether the statistical confidence in the result is sufficiently great
for all lots tested.
The benefits of a simple study design are that a small number of testing
intervals and fewer resources are required. However, such a simple design
represents a high-risk approach that has the potential to waste time and resources
if the IVD does not meet the acceptance criteria with an appropriate margin of
statistical confidence at the end of testing. If the acceptance criteria would have
been met at another intermediate time point then that might have been acceptable
as an assigned shelf-life.
A more effective and well-established approach routinely used is to
test at a number of additional predetermined intermediate time point intervals
(between 1 and 2 above). Typically, testing is carried out at relatively short
intervals (every 10 or 14 days) for the first 3 months, and then at monthly
intervals until at least one month beyond the design input-specified shelf-life.
This protocol provides information on whether the IVD ages more rapidly in the
period just after manufacture than later on in the shelf-life, and usually provides
sufficient data to enable the assignment of a confidence interval to the shelf-life.
The manufacturer could identify the most practical intermediate test
points from a risk evaluation of a specific IVD and include them in the stability
study plan/protocol. Such planning will also help manufacturers to estimate the
resources required to implement the testing.
Testing of all panel members is not expected at each of the test/time
points. However, testing with all stability testing panel members is expected at
the initial, the second to last and the last test/time point for all of the study types.
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The manufacturer should consider and document the rationale for the selection
of intermediate test points, and choose panel members to be tested at these
intermediate test points (for example, representative members, specimens that
are close to the medical decision points and those at the extremes of the assay
range tested).
5.9.1 Duration of testing

Testing conducted in stability studies should extend beyond the shelf-life
determined from user needs. At a minimum, testing should extend at least one
time point (one testing interval) beyond the predetermined user requirement
to provide a margin for uncertainty. The length of the time periods chosen
will depend on risk assessment, but should provide a safeguard in the event of
unexpected IVD failure during the testing period, where extrapolation from an
earlier time point would not be considered acceptable.
It is recommended that the standard relevant units of measurement
are used for the entire study (for example, unopened kit shelf-life is normally
measured in months; opened IVD/reagent stability in days or weeks; and allowed
reading times for enzyme immunoassay (EIA) and RDT in minutes or hours
after performing the assay).
5.10 “Zero time” values and variance

The value of each measured characteristic at the beginning of the stability
study and its variability over the course of the study are important pieces of
information. They should be measured independently for each lot of material in
the stability study. Analysis of the data will indicate if a statistically significant
change has occurred to any measured parameter from any lot during the
course of the study. A statistically significant change may not be of practical
significance. Relevant practical limits will have been predetermined in IVD
or process development. However, all statistically significant changes must be
thoroughly evaluated to decide whether they represent some important change
that would otherwise be undetected.
Zero time values could be obtained by evaluating each measured
characteristic for each lot on five or more occasions to establish the value and
its variance with freshly made materials. A definition of “occasion”, following
appropriate consideration, could be specified, for example, as involving a different
day, a different operator and a different set of equipment in order to investigate
potential sources of analytical variation. Later in the study, apparent differences
in the values of the characteristics can be detected reliably, relative to the “zero
time” value.
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6 Shelf-life studies
6.1 Requirements for determination of shelf-life
The stated shelf-life of an IVD must normally be based on real-time experimental
results. Accelerated stability studies are usually not sufficient to support a claimed
shelf-life, although they may be used in situations where experience already exists
with similar products (see (1) section 4.1) or when the stability of very similar
products is already known (see (2) section 7.3.1).
Note: If at the time of dossier submission for WHO prequalification the
real-time study outcome is not available, accelerated studies might be considered.
The manufacturer must justify why the accelerated study is acceptable as
supportive evidence until real-time experimental results become available.
In these cases, the results of real-time stability studies will be requested as a
condition of WHO prequalification. The shelf-life of the IVD could be extended
upon WHO review of real-time data.
6.1.1 Real-time stability studies

Real-time stability is determined using storage temperatures derived from user
requirements, over a period longer than the required life of the IVD.
Where a broad range of storage temperature is claimed (for example,
“Store at 4–40 °C”) WHO expects the studies will provide evidence for stability
over the whole of the temperature range for at least the length of the claimed
shelf-life. However, where claimed stability is restricted to a limited range (for
example, “Store at 2–8 °C”) it is acceptable for stability studies to be conducted
at a single temperature within this range.
It is recommended that a sequential approach be used (2) in which IVDs
are first submitted to stresses simulating transport before they are placed into
a shelf-life or in-use study. This approach best simulates the real-life situation,
where products will first be transported to the end user and then stored under
the recommended conditions before use, possibly until almost the end of their
labelled shelf-life.
It may be routine practice to store IVDs for an extended period after
manufacture before shipping. In this case, the IVDs would be kept first for a
defined period of time under recommended storage conditions, then taken
through the transport stress condition sequences, and finally put back into the
recommended storage conditions for the duration of the study (2).
6.1.2 Accelerated stability studies

Accelerated stability studies are designed to predict the shelf-life of an IVD
using increased rates of chemical and/or physical degradation caused by
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extreme environmental conditions (for example, elevated temperature at higher

humidity).
Accelerated stability studies provide results in a relatively short time.
However, the results of these studies are reached using assumptions about the
degradation of reagents and other IVD components that may not reflect their
observed performance under actual conditions of storage and use.
If the Arrhenius equation is used to calculate the expected life at
temperatures other than those actually used then the parameters of the equation
must be derived from the experimental data and not assumed (2). Manufacturers
must ensure that there are sufficient data (for example, for different temperatures
and test intervals) to allow for reliable extrapolation.
7 Component stability studies

7.1 General principles
7.1.1 Testing on final specifications
Component stability studies, including antimicrobial and desiccant studies,
must be performed using components made according to finalized and approved
manufacturing specifications (ideally to validated manufacturing scale) on
qualified manufacturing equipment and meeting finalized and approved
in‑process quality control (QC) specifications.
7.1.2 Considering component stability

IVD components are sometimes prepared in bulk and stored before being used
in several different lots of a completed IVD. The design-input documentation
should define how long components are likely to be stored before use. With that
information, component stability studies should be planned to provide evidence
that component shelf-lives will not restrict IVD shelf-life, since an IVD cannot
have a shelf-life beyond that of any of its dependent components.
The shelf-lives of components manufactured in bulk and used in several
different lots of an IVD can be verified using three lots of the component as a
minimum for shelf-life studies and, depending on documented risk assessment
related to variability, one or more lots subsequent to changes made to the
component. It is possible there will be two shelf-lives to evaluate: that of the bulk
material stored prior to transferring to the final packaging and that of the
component in its final packaging. The final contents of the evaluated lots of
the component must differ with regard to the batches of critical constituents
used (independent lots) but, subject to documented risk assessment, may all be
tested in their final presentation with a single set of the other components that
will be used together to constitute the IVD.
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Examples of stored components:

Wash solutions and substrates for EIA, amplification reagents for NAT and
calibrators for quantitative tests; all manufactured and stored in their final
labelled vials ready to be put into a kit.
Component stability can be assessed from the functionality of the lot
and also by factors related to the component that might change over time, such
as turbidity, colour, microbial contamination and the pH of liquid components.
Depending on the IVD and the conditions it is subjected to, it may be necessary
to distinguish between turbidity that arises from heat/cold denaturation and
turbidity that arises from microbial contamination.
7.1.3 Considering constituent stability

The stability study plan should consider whether components made from freshly
made constituents (for example, antigens, recombinant antigens, enzymes,
antibodies and membranes) will have the same shelf-lives as components made
from stored raw materials. Evidence should be provided to support the use of
stored constituents and detailing the lot-to-lot variability of critical constituents.
The stability study plan should also consider the choice of reagents or
methods to ensure that the most appropriate are used to measure the performance
of the component being studied (whether made from freshly made constituents
or from constituents with an already proven shelf-life).
Examples of stored constituents:
Purified recombinant antigens and monoclonal antibodies stored in aliquots
ready for use.
7.2 Stability of control materials

Assay-specific control materials provided by the manufacturer are used to show

that an IVD has performed as intended during use. These are often referred to as
“run controls” and are provided with some IVDs, along with an IFU statement
that if the control meets a certain criterion then the IVD will have functioned
as expected. “Control materials” does not refer to controls such as international
calibrators or those used in external quality assurance (QA) programmes.
The manufacturer must be able to demonstrate that the loss of signal from
control materials does not occur at a different rate from the loss of signal from a
validated panel member or from genuine, critical specimens; otherwise a failing
IVD might be regarded as still functional. Thus, the stability of control materials
must accurately reflect the stability of the IVD. The use of a control material that
is apparently more stable than the IVD and other components, or the use of
incorrectly assigned values for the control material, must be avoided (18).
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Example:
It is frequently seen in dossiers submitted for WHO prequalification that a
positive run control will produce a signal of > 2.0 optical density (OD) in
a freshly manufactured lot, and the IFU will state that an OD > 0.8 for the
same control qualifies a run. Thus the IVD may have lost more than half its
activity and still appear functional, even though some critical specimens are
shown in the dossier to have very weak signals on freshly made IVDs. This
is not considered appropriate unless data can be provided that demonstrate
that the critical specimens will still be detected at the end of shelf-life and
with a control material signal of 0.8 OD.
7.3 Biocidal stability and efficacy

7.3.1 Rationale
Bacterial and fungal organisms relevant to the environment of use must be
identified in the design input risk assessment, and antimicrobial preservatives
should be chosen, based on risk assessment, to prevent contamination of the
product in storage and in use. Antimicrobial preservative effectiveness must be
demonstrated throughout the shelf-life of the IVD.
If a new or modified preservative (for example, a different concentration)
is used as a result of further information on the conditions of intended use, the
manufacturer must obtain evidence that the new antimicrobial preservative or
concentration chosen does not negatively affect the stability of the IVD.
7.3.2 Study conditions

The studies should reflect expected in-use conditions for opened containers – the
stability of the IVD in the user environment, as intended by the manufacturer,
must be proven. On-board stability must be tested for an IVD used with an
instrument.
See (18) sections 51, 61 and 62; and (19) Appendix XI for suggested study
methods. Examples of bacterial groups to consider are spore-forming bacteria,
fungi, indigenous bacteria, bacteria found in the environment of the country of
manufacture and those found in the countries of intended use. Specific examples
outlined in references (18) and (19) include Aspergillus niger, Bacillus subtilis,
Candida albicans, Escherichia coli, Salmonella species, Pseudomonas aeruginosa,
Clostridium sporogenes and Staphylococcus aureus.
7.4 Desiccant functionality

Desiccants affect the stability of the entire IVD. Stability studies must show that
the desiccant will support the product over the whole claimed shelf-life within
the predetermined extremes of transport, storage and in-use conditions.
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Note: For WHO prequalification purposes:

1. It is recommended that a self-indicator (a humidity indicator that
changes colour upon saturation) be part of the desiccant design.
However, WHO strongly recommends against the use of cobalt
dichloride, the most commonly used humidity indicator, as it is a
carcinogenic substance.
2. Sachets are preferred to tablets, since the labelling instruction “Do
not eat” is more visible. There have been reports of desiccants in a
tablet formulation being mistaken for antimalarial medicine.
8 Stability during transport

8.1 Rationale
Transport stability studies evaluate the tolerance of an IVD to the types of
environmental conditions (for example, temperature and humidity) and physical
conditions (for example, inversion, vibration, physical handling and stacking) to
which it is likely to be subjected during and after shipping from the manufacturer
to the end user. These studies should provide evidence that there will be no
impact on IVD performance over the whole of its stated shelf-life as a result of
the transportation of the IVD by the recommended methods.
The manufacturer should assess the potential impact of multiple factors
and justify and document whether or not to include them in the evaluation. Final
transport conditions recommended by the manufacturer should reflect (and the
stability study plan document) the assessment of the conditions expected to be
encountered in the areas of use. The manufacturer should address any issues that
arise as a result of the transportation studies (for example, failing the stressed
conditions), and address these limitations in the manufacturer documentation
(for example, shipping documents and IFU if applicable).

WHO expects that a transportation challenge would precede the real-
time determination of shelf-life, and in-use studies. This will serve to determine
that transportation conditions do not reduce the shelf-life of the IVD (see
section 6.1.1).
In some cases it may be acceptable for the product to undergo
transportation-stability studies without a subsequent long-term real-time
stability study. In this case, shelf-life must be established under specified storage
conditions along with a stringent and evidence-based risk assessment of the
probabilities of extreme transport stress affecting IVD performance at the end
of the claimed life (see (2) section 4.2.3).
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8.2 Challenge conditions

Determination of the stability of an IVD during transportation should take
into consideration the local routes and means of transport used to supply the
IVD, which are usually defined in the design input risk assessment. It is not
necessary to test the IVD to the point where it is no longer usable, but merely to
validate the window of transport conditions within which the IVD will retain its
claimed performance to the end of its stated shelf-life. However, knowledge of
the possible limitations of an IVD and at what point the IVD becomes unusable
is useful to a manufacturer when trouble-shooting post-marketing problems.
WHO expects the manufacturer to take into consideration the possibility that
the product might continue to be subjected to suboptimal storage conditions
by the end user.
Example:
A static challenge of 45 °C for 3 days may represent conditions seen during
the actual transportation of an IVD – however, a more stringent challenge
of cyclical high and low temperatures (including freezing) for a longer
period of time, and followed or preceded by exposure to vibration might
better cover a “worst-case scenario” of shipment, storage and subsequent
transportation to the end user.
8.3 Number of lots

Where transport stability studies are incorporated into studies to establish shelf-
life, as recommended in this guidance document, a minimum of three lots of
the IVD must be used. For transport studies alone, a minimum of one lot of the
IVD may be used, however, as with shelf-life studies, more lots may be required
depending on lot-to-lot variability (see section 10.1).
8.4 Simulated versus actual challenge

An actual shipping challenge can be used to verify the conditions found in the
simulated transportation challenges. However, it may only replace a simulated
shipping challenge where there is an appropriate risk evaluation and where
experience and data have been actively collected for similar products and
documented in detail (for example, it is not sufficient to note “no complaints”).
In the R&D phase, actual data from shipping can be used to define the
conditions needed for an appropriate simulation of extremes. However, in the
post-production phase, actual shipping challenges often do not explore the full
range of shipping conditions that could be encountered, including extreme values.
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8.5 Multiple stress test sequences (simulated

transport challenges)
Proof of IVD performance after actual shipment is generally not sufficient
evidence of stability under all conditions and delay hazards. Multiple stress test
sequences are typically needed to address the range of transport conditions used
for global product delivery. Relevant guidance (14) recommends the evaluation
of several extreme conditions.
Appropriate stress test sequences may be developed on the basis of data
from actual product transport studies. Testing multiple stress sequences allows
a manufacturer to identify the most cost- and/or resource-effective transport
conditions from a set of alternatives, while ensuring adequate product stability
protection (see (2) section 4.2.3).
Note: For WHO prequalification, the environmental conditions
investigated as part of a stability study must reflect those likely to be encountered
in resource-limited WHO Member States. For example, temperatures at some
airport tarmacs in sub-Saharan Africa can exceed 40 °C, while temperatures
encountered during air transport fall below 0 °C. Significant delays can be
encountered at any time and especially during wet season transport to remote
health centres.
See Appendix 1 for an example of a protocol for simulated transport
challenges.
8.6 Physical conditions

Physical handling can be both manual and mechanical. The relevant user and
commercial factors should be identified as part of the design input risk assessment
and the packaging and shipping methods developed accordingly. Reference (14)
describes a number of factors to be considered, and their evaluation: drop, impact,
compression, vibration, repetitive shock, longitudinal shock, cyclic exposure,
vacuum, impact and inversion; along with the size, weight and composition of
the packaging. This should be regarded as part of stability testing.
9 In-use stability studies

9.1 Rationale
In-use stability of an IVD is the period of time over which components retain
adequate performance, after transport to the users, once they are opened,
reconstituted and/or diluted and exposed to the environmental conditions in
which they will be used.
As far as possible, the study should be designed to simulate the use of
the product in practice. If a range of conditions for use is stated in the IFU (for
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example, “use at 15–40 °C”) evidence must be provided to prove the stability
over that range with all the specimen types claimed (for example, serum, whole
blood and oral fluid), unless a documented rationale is provided. It is considered
best practice for the manufacturer to claim a stability range that includes an
appropriate safety margin (for example, test range 2–35 °C, claimed 4–30 °C) to
ensure that that the claimed stability range is acceptable. However, where claimed
in-use stability is restricted to a limited range (for example, “use at 35–37 °C”) it
is acceptable for in-use stability studies to be conducted at a single temperature
within this range, subject to evidence from documented robustness studies or
risk assessments.
It is good practice to perform the in-use stability testing at both the
start and end of the shelf-life of the IVD (or with components at the start and
end of their shelf-lives if any of the components have a longer shelf-life than
the complete IVD) and after simulated transport challenge (see section 8). This
will confirm that the IVD will have the claimed in-use life throughout its whole
shelf-life.
All studies should support precisely defined periods of in-use stability
claims.
Example:
An RDT test cassette may be labelled “Use immediately on opening”.
However, it is still necessary to determine the interval (one hour, one day,
etc.) over which IVD performance remains stable after the component
is opened.
9.2 Conditions of use

Determination of the in-use stability of an IVD and/or its components must
reflect the routine conditions of use of the IVD. Freeze-thaw stability should be
considered to address situations in which reagents may be exposed to multiple
freeze-thaw cycles during use.
Note: For WHO prequalification, in-use stability studies should take
into account the environmental conditions and usage conditions encountered
in WHO Member States and by users, such as exposure to extreme temperature,
humidity and light and to microorganisms.
9.3 Multiple in-use stability claims

Depending on the way in which the IVD is used it may be necessary to have
several in-use stability claims. In situations where multiple stability claims are
made, a manufacturer must provide evidence (from testing that investigates
routine use) supporting each of the claims.
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Examples:
1. A reagent may have a stated period of stability once it has been placed
on board an instrument and another period of stability once it is in
active use (that is, during actual use/testing).
2. Multiple-use reagents (for example, buffers) may repeatedly be exposed
to high temperatures during the day while in use and exposed to lower
temperatures when not in use and stored in the refrigerator. The actual
use of the multiple-use reagent – squeezing of bottles, exposure of the
lid and tip to working surfaces and hands, and exposure to dust and
light – may also affect stability. Stability studies and associated risk
assessments should take all of these factors into account.
10 Production lots used in stability studies

10.1 Considering variability
As noted in section 12.3 below, planning for stability studies must take into
consideration all possible sources of variation within and between manufactured
lots. For most IVDs it is likely that differences between batches of the biological
reagents will cause the most variation. Factors to consider include apparently
minor and technically uncontrollable differences in the culture and purification
of recombinant antigens and antibodies; synthesis and purification of primers,
probes and peptides; undocumented production changes of an outsourced
buffer component; and lot variability of nitrocellulose membrane used in
lateral-flow IVDs.
At a minimum, lots chosen for stability studies must be independent
lots – that is, they must differ in the source lot of their critical constituents,
for example, different purification and/or culture batches for all recombinant
antigens and monoclonal antibodies. If pilot or small-scale lots are chosen,
special attention must be paid to the potential for variability (see also section
12.3). However, the sources of variation will depend on the particular process,
product and component, and should be identified during product development
risk analyses.
Use of different batches of critical components ensures that the stability
evidence obtained is more likely to be representative of long-term manufacture.
Any variability found can be taken into consideration when assessing the
outcome of the studies against the design input requirements and when making
claims. This minimizes user problems and hence complaints.
10.2 Testing the final configuration

Shelf-life, in-use and transport stability must be determined for the finalized
approved product in terms of:
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■■ manufacturing specifications
■■ release-to-market QA criteria
■■ packaging and labelling (see section 10.4)
■■ validated manufacturing scale on qualified manufacturing
equipment.
Note 1: For WHO prequalification, it is important that the stability
studies have been conducted using the IVD intended to be prequalified, and
not surrogates and/or closely related products. Changes perceived as small (for
example, change in production scale, bulk container materials, supplier of a
critical biological or vial stopper) can have unexpected effects on stability and
other performance characteristics. After such changes, a new documented risk
assessment and, if necessary, a stability plan and study, is needed. Manufacturers
should have change-control procedures in place compliant with ISO 13485 (15).
Note 2: Stability studies undertaken in the R&D phase of the product
life-cycle provide an important understanding of how to design the product
so that it will meet the final stability requirements identified in the input
documentation. However, these studies are usually not sufficient for submission
to WHO prequalification assessment since they may not reflect the final design
and manufacture of the IVD.
10.2.1 Exceptions
If any of the above criteria are not met (for example if “pilot lots” or small-scale
lots are used, or if the method of use described in the IFU is not finalized), strong
evidence must be provided that the materials that were evaluated will perform
exactly the same as the final commercial product.
Note: In some exceptional circumstances, where it is not possible to sample
from actual production lots, samples from pre-production or development lots
might be used. If this is the case, manufacturers should justify why production
lots were not used, and provide robust evidence that the lots chosen are expected
to behave identically to the production lots. Data concerning lot-to-lot variability
must still be submitted. Although WHO will consider the available evidence
on its merits, this preliminary information must be followed by stability claims
conducted on fully qualified production lots.
10.3 Number of lots required for testing

Current guidance (1, 2) recommends that three product lots at a minimum
must be used to establish or verify shelf-life; in-use claims require testing on
a minimum of one lot. The actual minimum number of lots to be used must
be determined by a stringent risk assessment based on evidence of variability
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obtained during R&D (see section 10.1). However, the minimum will never be
less than three lots for shelf-life verification.
WHO note: It is not acceptable to sample IVDs from a single production
lot but to label them so that they appear to have been taken from three separately
manufactured production lots. This is true for all performance evaluation and
regulatory submission purposes. WHO prequalification investigates batch
records during on-site inspections. Non-compliance with this requirement may
result in a critical non-conformity grading.
10.4 Components of lots required for testing

Current guidance (1, 2) recommends that stability work be performed using
materials in their final packaging. Labelling is a significant factor of packaging
and is known to present stability issues in some cases. For example, some label
adhesives diffuse through some plastics, enter vials and affect the function of
the reagents over time. Other label types lose adhesion over time; while some
printing inks fade. The physical stability of packaging requires the same degree of
risk evaluation and subsequent experimental verification as its chemical stability,
with attention given to the countries of intended use. This is most important
for primary packaging but must also be considered for secondary packaging,
particularly for transport stability studies.
If there is more than one configuration or version of the IVD (for
example, pack size differences, or Conformité Européenne (CE) marked and
non-CE marked) then any potential effects on performance, including stability,
must be assessed. In particular, if different reagent-container sizes are used in
packs with different volumes of reagent (for example, different volumes for single
use and multiple use), stability evidence should be obtained on all variants, even
if the contents of the containers are identical, unless stringent risk evaluation
supported by physical or chemical evidence indicates otherwise.
Once component shelf-lives are assigned, it is expected that both
relatively fresh components and components which have progressed into their
assigned shelf-life will be used when selecting the different production lots for
use in studies to establish the product shelf-life (1, 2).
11 Stability study plan

Stability studies should be well designed, scientifically sound, well implemented,
well recorded and able to deliver meaningful conclusions concerning IVD
performance. This will minimize the time and resources required by the
manufacturer to generate appropriate evidence and by the regulatory authority
to assess it.
It is good practice to prepare, within the mechanisms of a quality
management system (QMS), a plan for the investigation of each characteristic
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of IVD stability. A well-developed study plan, with clearly defined objectives,

responsibilities and pass/fail criteria, should be developed, reviewed and
internally approved in advance of testing. The plan should be based on the
design input requirements.
It is essential that the stability study plan takes into account the intended
use of the product to ensure that the relevant critical characteristics are all
captured by the plan. The results of the stability studies should support the
claims made in the IFU.
Careful forward planning will help to ensure that sufficient resources
are made available, effective experiments are performed, and both experimental
results and associated documentation are recorded in an appropriate manner.
11.1 Responsibilities
The study plan should outline the responsibilities and applicable training for all
staff involved in the study. The responsibilities for implementing the study plan
must be assigned to appropriately qualified and trained staff. Responsibilities to be
allocated include study set up, testing, monitoring, validation of equipment and/or
processes, sample selection, risk assessment and corresponding documentation.
In addition, the manufacturer must nominate a person responsible for
investigating failures and a person responsible for conducting risk assessments
if the IVD fails to meet the requirements of the design inputs.
11.2 Preparing the testing plan

A complete, detailed description should be prepared that documents all of the
required testing and procedures to be undertaken and the expected outcomes.
Authorization of the plan should be obtained internally in advance of
commencing work. The plan should include the following details:
■■ the qualification and training of technical staff performing the work;
■■ any biohazard issues identified with reagents;
■■ aspects of instrumentation, including storage facilities or rooms,
validation, calibration, monitoring and servicing;
■■ the lot/batch numbers of kits to be used, with justification for any
manufacturing anomalies or deviations from documented
procedures;
■■ the expected life of the kit from the input documentation;
■■ any proposal, with justification, to launch a kit with a shelf-life
based on accelerated data, or to launch with a shorter shelf-life than
in the input documentation while awaiting the conclusion of real-
time testing;
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■■ documentation of the nature and extent of in-use testing;

■■ the justification for the selection of lots and components, taking into
account lot-to-lot variability and the critical characteristics;
■■ the number of units (test cassettes, bottles, tablets, etc.) of each
component to be collected and stored under each condition;
■■ the nature of the panel to be used, justifying each panel member’s
inclusion and defining the volume and characterization of the bulk
specimen to be used, and the aliquot size and number to be stored
for the testing;
■■ the expected criteria for each panel member at the beginning and
end of the product’s proposed shelf-life;
■■ the statistical methods to be used for data analysis, including those
used to identify outlying values and to establish criteria (see section
11.5); and
■■ the methods for approval and justification of any deviations from
the plan.
11.3 Product storage

A sufficient number of product components from the identified lots should be
reserved and stored separately to ensure that the study will be completed with
identified products. Sufficient numbers of the testing IVDs should be retained to
allow for additional testing, calculated from estimated invalid result rates.
11.4 Documentation
The plan should make reference to the preparation of a study report that will
be used to summarize the interim, and ultimately final, study findings and
conclusions. The study plan, the testing protocol, the study report and all
associated documentation (worksheets, etc.) should be controlled within the

manufacturer’s QMS. At the end of the study, the manufacturer should be able
to confirm whether or not the design input requirements have been met.
Any changes from the methods identified in the plan must be recorded
and undergo risk assessment. The plan should refer to the development of a
detailed and valid testing protocol that includes all information and material
relevant to testing.
11.5 Statistical methods

Statistical methods are used to support stability claims by providing estimates of
the probability of results being as stated. For example, prior to the stability studies
on an EIA, it has been documented that if a panel member has at least a particular
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OD then the IVD will meet a particular claim. Given the results of the stability
study using that panel member and showing the variability within and between
lots of the IVD, the probability of future similar production of the IVD meeting
claims at the assigned life can be estimated. The derivation of valid criteria and
the probability of maintenance of all claims can be estimated by appropriate
statistical methods.
There is a wealth of information available on the statistical methods used
in the R&D of IVDs, from both ISO (20–22) and CLSI (2, 23–26). Although
most of these methods apply to quantitative assays, information on statistical
methods for qualitative assays is also available (27).
The fundamental considerations for stability testing are the number of
replicates required at each time point and the number of different production
lots required which together will produce an “acceptable overall probability
estimate” of the likelihood of future production lots meeting claims (and hence
user input requirements) at the end of the shelf-life.
However, consideration must also be given to what represents “an
acceptable overall probability limit”. “Acceptability” is a decision critical to
quality and must be decided upon in advance based on the input requirements
(for example, 80% confidence that 95% of lots will meet the claims). This is a
tolerance interval as described in ISO 16269-6:2014 (22). The consideration can
then be phrased as: “How many replicates and how many different production
lots can then be derived from the tolerance interval required?”
It is strongly recommended that manufacturers seek advice from a
professional statistician once the quality-critical requirements have been defined
and before beginning any experimental work.
The statistical methods to be used must be documented in the plans
and protocols of any stability study and consideration given to the treatment of
unexpected and atypical results. In general, all results must be used unless there
is a documented physical reason that the result can be ignored – for example,
known operator error, too little volume, incorrect timing or use of an unqualified
instrument (one lacking maintenance or calibration). Any ignored results must
nevertheless be recorded and included in the report of the stability study.
11.6 Stability testing protocol

As part of an approved study plan for the determination of IVD stability, a
detailed testing protocol should be prepared as appropriate (examples of stability
protocols are provided in Appendix 1: Examples of stability protocols). The
protocol should include the following as a minimum:
■■ QMS identifiers (for example, experiment name, document references,
etc.) that allow traceability to both the overarching study plan and to
the records/documents generated, such as result worksheets.
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■■ The training requirements for operator(s).

■■ The expected dates and times when the data will be collected.
■■ The objectives of the study (that is, determination of shelf-life,
determination of in-use stability of a component, etc.).
■■ The name and lot number of the IVD and/or components to be
investigated.
■■ Specification of how the components will be sampled from the
production department.
■■ The panel members to be used and their characterization, including
valid test methods which reflect the IFU claims.
■■ The experimental method that will be used for testing. This must
follow the finalized testing method from the IFU where appropriate.
It must describe clearly how the experiment is to be performed in
terms of:
–– required storage and/or challenge conditions
–– duration of storage/challenge
–– schedule of testing intervals (see (2) section 4.3)
–– stability testing panel
–– numbers of replicate tests performed for each panel member.
■■ How and where results are to be recorded.
■■ The acceptance criteria.
■■ How aberrant, discordant or invalid results will be dealt with.
■■ How storage/challenge conditions are to be applied:
Example: For determination of stability during transportation it should
be made clear that each IVD will be subjected to a sequence of stated

temperatures.
■■ How actual storage/challenge conditions are recorded:
Example: Recording of temperature not as “room temperature” but as an
actual numerical value obtained from calibrated instrumentation.
Note: Statements of a general nature can be unclear to a regulatory
or WHO reviewer. For example: “Sample buffer was stored at the required
temperature and tested each month”. This statement raises questions such as:
(a) were the bottles of sample buffer stored open at the required temperature for
the entire testing period; or (b) were the bottles stored capped and refrigerated,
and only reopened briefly at the required temperature at each schedule test point?
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To avoid confusion, the details of actual storage and use procedures are required
in the testing report.
11.7 Reading and recording results

11.7.1 Avoiding reader bias
It is good practice to use approaches that make the reading of results as objective
as possible, such as using a documented scoring system. For IVDs for which a
subjective element forms part of the result (for example, reading the intensity
of an RDT band within a specified time frame) the results should always be
reviewed by both a first and a second reader to avoid operator bias. Both readers
must be blinded to the expected results and the second reader must also be
blinded to the first reader’s results. If a validated band intensity scoring tool is to
be included in the final RDT kit, this should be used to record results.
11.7.2 Recording actual individual results

The results of a test (not only the test interpretation) should be recorded. An
interpretation on its own provides insufficient detail to detect the degradation
of a signal over time. Photographic records of qualitative tests are recommended,
as appropriate.
Some IVDs (for example, line-blots) may require the presence of
particular band patterns to allow an interpretation to be reached, and several
different patterns may yield the same final result. Recording only the final
interpretation of a test specimen may cause the failure of particular bands to go
unnoticed, while allowing the IVD to pass stability assessment.
Quantitative assays, for example EIAs or NATs, should be tested with
sample panels containing concentrations of analyte across the quantitative range
of the assay. Numerical results should be reported and statistical methods should
be applied to ensure that the assay is measuring the analyte appropriately across
the quantitative range.
Qualitative assays, for example EIAs and NATs, should also be tested
with samples at several different analyte concentrations, including samples at low
concentration near the cut-off level of the assay. Results should be recorded as
positive or negative according to the predetermined cut-off level of the assay.
Example:
Some RDTs may stipulate that the strength of test band is not correlated
with the strength of antibody titre. Nevertheless, the following should be
recorded: (a) the intensity of observed patterns according to a predetermined
and validated intensity scoring system with as fine a gradation as possible;
and (b) the final result interpretation.
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11.7.3 Retention of records

WHO recommends the retention of photographic records, machine printouts and
electronic data, or physical retention of membranes from opened test cassettes, as
appropriate. Records should be retained for the period of time equivalent to the
commercial lifetime of the IVD but not less than two years (modified from (15)
section 4.2.4).
11.8 Instability versus imprecision

Testing at more than two time points can be important for avoiding confusion
between imprecision and instability. For example, if a 10% decrease (compared
to the zero time value) is recorded from testing at the end of the shelf-life, it may
not be possible to judge if the difference was due to imprecision or instability.
The inclusion of additional test points (for example one or more between
the zero time and the end of the shelf-life) allows for fluctuation caused by
imprecision to be distinguished from drift due to instability.
Increased clarity between instability and imprecision can be gained by
increasing the number of replicates and runs, primarily with reference to the
zero time values (see sections 5.9 and 5.10).
11.9 Testing schedule

Testing intervals should be selected to detect any trending of results over
the testing period. Different testing intervals may be required for different
components. For example, it may be appropriate to test an IVD test cassette
against a panel on a monthly or quarterly basis, but to test for open vial stability
on a weekly basis.
11.9.1 Acceptance criteria for results

The acceptance criteria to establish what is acceptable or not acceptable should
be defined according to the panel criteria for both qualitative and quantitative
test methods. Results from failed (invalid) test runs must not be used in the
determination of the stability claim. However, the invalid results should be
recorded and included in the report of the stability testing.
12 Stability study report

12.1 General
After testing has been completed, the findings should be summarized in a stability
study report. The report should clearly identify the IVD that was tested, the
objectives of the study, the conditions under which the IVD was tested and the
conclusions that were drawn from the findings. The report should be traceable
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to the study plan, testing protocol and input requirements. It should make clear
references to other supporting documentation (for example, result worksheets).
12.2 Link to claims

The results and conclusions of stability studies presented in the report must
support the claims of IVD stability reported in the IFU and elsewhere in the
WHO prequalification dossier.
12.3 Consider variability

An overall stability claim (whether for shelf-life, in-use stability or stability
during transportation) must be based on the expected stability when taking into
account inter-lot variability.
Example:
The manufacturer should evaluate the variability between the different lots
studied (see section 10.1) and assume that any differences in shelf-life are
inherent to the manufacturing process. The claimed life should be calculated
so that a known and stated proportion of all lots (usually > 95%) will meet
the claimed shelf-life. Frequently, more than three lots are needed to obtain
a realistic idea of the variability of the results.
12.4 IVD stability versus component stability

A claim of stability for an IVD as a whole must not exceed any individual
component stability.
Example:
For an IVD claimed to detect HIV-1 and HIV-2 antibodies – if detection of
HIV-1 antibodies is stable to 24 months but that of HIV-2 to only 18 months
then the shelf-life must be based on the shorter time of 18 months.
13 Changes to a WHO prequalified IVD

13.1 Dealing with change
Any critical or major modification to a WHO prequalified IVD or to its process
of manufacturing will require the provision of new direct evidence of stability.
An appropriate risk assessment and an accelerated stability study
comparing the original product and the modified product for usability,
performance and lot-to-lot variability may serve to assess the impact of the
changes to product formulation or manufacture.
It would be necessary to validate the stability of the modified IVD
on a minimum of one lot of the IVD (subject to risk assessment) in order to
353
demonstrate equivalence between the original and modified IVDs. Testing of

further lots may be appropriate depending on the product nature, variability
of components and failure risk (see (2) section 7.1.2). WHO expects the results
of accelerated testing to be confirmed by real-time studies.
If there are different presentations, evidence of the stability of each one
must be provided (see also section 10.4).
The following examples illustrate the scope for considering the
performance evidence from one IVD as support for the performance of another.
It should be noted that the observations discussed here refer specifically to
IVD stability. Other aspects of IVD performance should still be validated
as appropriate.
Examples:
1. An HIV RDT uses an identical test cassette and physical components
as a manufacturer’s existing, fully validated, HCV RDT, but the
reagent formulations are different (antigen/antibodies, buffers,
conjugates, etc.) – evidence of stability of the HCV RDT would not
suffice for the HIV RDT. Even if the manufacturer claims that both
IVDs have been sold in a number of countries for several years and
no adverse feedback has been reported, this would not constitute
evidence in support of the stability of either IVD.
2. From an HIV RDT that has been fully validated for detection of
HIV-1 antibodies, a new product is developed that includes detection
of HIV-2 antibodies. The stability of any sample buffers that are
identical between the two IVDs would, most likely, not need to be
validated. However, other components (conjugates, antigens or
antibodies) that are different between the two IVDs would need to be
tested; it would not be sufficient to assume that HIV-1 reagents will
have the same stability in the new IVD. An IVD modification of this
nature is likely to require substantial new validation of stability.

3. An HIV RDT previously intended for testing serum/plasma has a claim
added for detection of HIV-1 in whole blood. The only substantive
design change associated with the new claim is the addition of a
small filter pad near the sample port which acts as a filter for whole
blood specimens. Depending on the nature of the material, it may be
reasonable to argue that the pad material would not be expected to age;
that it is not, in any practical sense, chemically labile. Consequently,
shelf-life and in-use stability may not necessarily need to be retested
in full. However, stability during transportation may need to be
determined to provide confidence that the modification is able to
withstand likely shipping conditions (for example, that the extra square
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of filter pad material does not dislodge when packages are jostled and
bumped in transit).
4. Based on an HIV RDT that has been fully validated for the detection
of HIV-1 antibodies, a new IVD is developed which includes detection
of antibodies to Treponema pallidum (TP). Detection of TP-specific
antibodies occurs on a completely separate membrane (and associated
architecture) to that of HIV-antibody detection. Additional handling
steps may have an impact on the stability of the HIV-1 antibodies
and retesting may be required. It may be necessary to review evidence
of stability during transportation to ensure that new components are
not affected by transit (for example, where a new packaging concept
is used).
–– If a new machine is used for striping of the HIV-1/TP IVD,
validation of the new machine (installation qualification,
operational qualification and performance qualification) would
be required to show that the stability studies are still valid.
–– If the IVD is designed in a way that HIV and TP detection occurs
either on the same membrane and/or using most of the same
architecture (and assuming that sample buffers are identical
between IVDs) it is likely that this new IVD would need to be
fully validated.
14 References
1. ISO 23640:2011. In vitro diagnostic medical devices – evaluation of stability of in vitro diagnostic
reagents. Geneva: International Organization for Standardization; 2011.
2. Evaluation of stability of in vitro diagnostic reagents; approved Guideline EP25-A. Wayne (PA):
Clinical and Laboratory Standards Institute; 2009.
3. Specifications: test procedures and acceptance criteria for biotechnological/biological products.
Q6B (Step 4). ICH Harmonised Tripartite Guideline. Geneva: International Conference on
1999.
4. ISO 17511:2003. In vitro diagnostic medical devices – measurement of quantities in biological
samples – metrological traceability of values assigned to calibrators and control materials.
Geneva: International Organization for Standardization; 2003.
5. ISO 18113-1:2009. In vitro diagnostic medical devices – information supplied by the
manufacturer (labelling) – Part 1: Terms, definitions and general requirements. Geneva:
6. Directive 98/8/EC of the European Parliament and of the Council of 16 February 1998
concerning the placing of biocidal products on the market). OJ. 1998;L 123:1–63 of 24.4.98
(http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:1998:123:0001:0063:en:PDF,
355
7. ISO 9000:2005. Quality management systems – fundamentals and vocabulary. Geneva:

8. ISO 15198:2004. Clinical laboratory medicine – in vitro diagnostic medical devices – validation
of user quality control procedures by the manufacturer. Geneva: International Organization for
Standardization; 2004.
9. Code of Federal Regulations Title 21. Section 820.3 Definitions. Washington (DC): United States
Food and Drug Administration; 2010.
10. ISO/IEC Guide 99:2007. International vocabulary of metrology – basic and general concepts and
associated terms (VIM). Geneva: International Organization for Standardization; 2007.
11. Glossary and definitions of terms used in GHTF documents. GHTF/SC/N4:2012 (Edition 2). Global
Harmonization Task Force (GHTF) Steering Committee; 2012 (http://www.imdrf.org/docs/ghtf/
final/steering-committee/procedural-docs/ghtf-sc-n4-2012-definitions-of-terms-121109.pdf,
12. ISO 14971:2007. Medical devices – application of risk management to medical devices. Geneva:
13. Quality management systems – process validation guidance. GHTF/SG3/N99-10:2004 (Edition 2).
Global Harmonization Task Force (GHTF) Steering Committee; 2004 (http://www.imdrf.org/docs/
ghtf/final/sg3/technical-docs/ghtf-sg3-n99-10-2004-qms-process-guidance-04010.pdf, accessed
21 December 2017).
14. Standard practice for performance testing of shipping containers and systems. ASTM D4169 - 14.
West Conshohocken (PA): ASTM International; 2014.
15. ISO 13485:2003. Medical devices – quality management systems – requirements for regulatory
purposes. Geneva: International Organization for Standardization; 2003.
16. Technical Guidance Series (TGS) for WHO Prequalification – Diagnostic Assessment. Guidance
on test method validation for in vitro diagnostic medical devices. TGS–4. Geneva: World Health
Organization; 2017 (http://apps.who.int/iris/bitstream/10665/258971/1/WHO-EMP-RHT-PQT-
TGS4-2017.04-eng.pdf?ua=1, accessed 21 December 2017).
17. ISO 15189:2012. Medical laboratories – requirements for quality and competence. Geneva:
18. United States Pharmacopeia 31 - National Formulary 26 (USP 31-NF 26). Rockville (MD): The
United States Pharmacopeial Convention; 2008.
19. Pharmacopoeia of the People’s Republic of China English edition. Beijing: State Pharmacopoeia
Commission of the People’s Republic of China; 2000.
20. ISO 5725-1,2,3,4,6:1994 and ISO 5725-5:1998 Accuracy (trueness and precision) of measurement
methods and results – Parts 1–6. Geneva: International Organization for Standardization; 1994
and 1998.
21. ISO 3534-1,2:2006 and ISO 3534-3:2013. Statistics – vocabulary and symbols – Parts 1–3. Geneva:
International Organization for Standardization; 2006 and 2013.
22. ISO 16269-4:2010, ISO 16269-6:2014, ISO 16269-7:2001 and ISO 16269-8:2004. Statistical
interpretation of data – Parts 4 and 6–8. Geneva: International Organization for Standardization;
2010, 2014, 2001 and 2004.
23. Evaluation of the linearity of quantitative measurement procedures: a statistical approach;
approved Guideline EP06-A. Wayne (PA): Clinical and Laboratory Standards Institute; 2003.
24. Interference testing in clinical chemistry; approved Guideline EP07-A2. Second edition. Wayne
(PA): Clinical and Laboratory Standards Institute; 2005.
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25. Evaluation of detection capability for clinical laboratory measurement procedures; approved
Guideline EP17-A2. Second edition. Wayne (PA): Clinical and Laboratory Standards Institute; 2012.
26. Evaluation of precision of quantitative measurement procedures; approved Guideline EP05-A3.
Third edition. Wayne (PA): Clinical and Laboratory Standards Institute; 2014.
27. Valcárcel M, Cárdenas S, Barceló D, Buydens L, Heydorn K, Karlberg B et al. Metrology of qualitative
chemical analysis. Brussels: Directorate-General for Research and Innovation (European
Commission); 2002 (http://bookshop.europa.eu/en/metrology-of-qualitative-chemical-analysis-
pbKINA20605/, accessed 22 December 2017).
357
Appendix 1
Examples of stability protocols
This appendix uses the example of a wholly fictitious IVD to illustrate the kinds
of experimental design that would be required to adequately determine:
1. the stability of whole kits during transport followed by the stability of whole
kits during shelf-life; and
2. the in-use stability of whole kits including reagents.
The information provided in these examples should be used as a guide
to possible approaches for generating evidence of a standard sufficient to satisfy
the expectations of WHO prequalification. Further examples can be found in
the WHO Prequalification: Sample Product Dossiers available on the WHO
Prequalification website 1.
WHO expects that a transportation challenge would precede the real-
time determination of shelf-life and in-use studies.
Description of the fictitious IVD
The fictitious IVD used in the examples below is an RDT for the detection of
antibodies to HIV-1, HIV-2 and Treponema pallidum (TP) in serum, plasma
and whole blood, and is referred to as the HIV/TP RDT.
The IVD kit components are: a test cassette sealed in a foil pouch (with
desiccant) and a bottle of specimen buffer/diluent for use.
It is recommended that the kit be stored at 8–40 °C and brought to 15–30 °C

before use.
It is recommended that once the sealed foil pouch of the test cassette is
opened that the test cassette be used immediately.
The specimen buffer is expected to have similar stability to the sealed and
pouched test cassette. The stability of the opened bottle of specimen buffer is
determined below (see Example 2: In-use stability protocol).
1
http://www.who.int/diagnostics_laboratory/guidance/sample_product_dossier/en/
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Stability study plan:

The manufacturer has developed a stability study plan to determine the
stability of the HIV/TP RDT. As part of this plan, a preliminary determination
of accelerated stability has been made at several extremes of temperature,
which suggests that the IVD would be stable to an equivalent of 12 months
following manufacture. The plan calls for the development of real-time
stability protocols that will form the basis of subsequent testing of the IVD.
Preliminary work has shown that the variability between lots is minimal.
As a result, three independent lots (with no critical constituents in common)
will suffice to enable a reasonable estimation of shelf-life, taking lot-to-lot
variability into account.
Example 1: Evaluation of transport stability

followed by real-time stability
Objective
To determine the stability after transportation of the HIV/TP RDT in real-time
using simulated shipping conditions, and to generate components that have
already undergone stress testing to be used in real-time shelf-life studies as
proposed in Stability Study Plan XZY00001.
Preparation
Acquire sufficient numbers of the IVD kits from three independent production
lots using a predetermined sampling protocol (for example, random, first X
number of kits in first box, every 100th kit, etc.). Allow at least 10% overage for
unexpected requirements and re-testing.
Note 1: To provide security against unforeseen events, duplicate tests
should be performed as a minimum. However, testing in triplicate will
provide more statistical confidence in the observed test result.
The IVD kits chosen for testing must be in their final packaging including all
labelling (see section 10.4).
The IVD kits are stored so that the reagents are in contact with all
elements of the packaging (for example, the bottles in the IVD kits are stored
horizontally, lying flat on their sides, allowing liquids to remain in contact with
the bottle closures).
Acquire sufficient volume of each panel member for the duration of the
testing schedule (see testing schedule below).
359
The protocol for these studies specifies the number of IVD kits to be
picked, the statistical sampling plan to be used and the required panel members
and their volumes.
Documentation
In Worksheet XYZ00001 record the following:
■■ the lot numbers from which the IVD kits were sampled;
■■ the number of IVD kits sampled from each lot; and
■■ details (including manufacturing/lot information) for each of the
IVD kit components that will be tested as part of this protocol (test
cassette and specimen buffer).
Testing schedule: for transport simulation

Testing will be conducted at 0, 3, 6, 9, 12 and 13 months.
Note 1: Testing beyond 13 months will allow for an understanding of

when, in real-time, the IVD is likely to “fail” and may allow for an
extension of the proposed shelf-life.
Note 2: For determination of shelf-life, a fresh bottle of specimen
buffer must be opened at each testing point – though there may be
circumstances in which multiple sampling could be taken from the same
bottle after it has been opened.
The IVD kits will be divided into two groups. One group will be stored at
40 ± 5 °C, the other at 8 ± 2 °C. IVD kits from each group will then be subjected
to the following conditions:
Condition 1: Temperature and humidity sequence; all IVD kits will be taken
through a temperature and humidity sequence consisting of:
i) Ambient humidity (X% RH)
–– Put at IFU storage temperature for 24 ± 4 hours followed by

–– 30 ± 5 °C for 24 ± 4 hours, followed by
■■ IFU storage temperature for 24 ± 4 hours
Followed by
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ii) Desert humidity (30% RH)

–– IFU storage temperature for 24 ± 4 hours
Followed by
iii) Tropical humidity (85% RH)
–– IFU storage temperature for 24 ± 4 hours
Followed by
iv) Ambient humidity (X% RH)
–– IFU storage temperature for 24 ± 4 hours.
Note 1: It is important to make clear that the above complete sequence
of temperatures will be used, as opposed to separate IVD kits being
held at individual temperatures. The actual temperatures, durations and
the nature of the sequence will depend on the IVD and on the kinds of
conditions expected to be encountered during shipping.
Note 2: Freezing temperatures are not considered in this example
but should be included if the IVD kits could be exposed to freezing
temperatures during transport.
Note 3: If transport by air is anticipated, the effect of reduced pressure
should be included in the protocol for a period of time at least 10%
longer than the longest anticipated flight, and at a pressure expected in
aircraft holds.
361
Note 4: The protocol should call for testing of at least five individual IVD
kits after each stress condition, using the stability panel members giving
the most informative results. This approach will enable verification that
the IVD kits are sufficiently stable to progress to the next condition –
though this should already be known from preliminary experiments and
R&D work.
Condition 2: Transport stress conditions – shaking; each IVD kit will be placed
on a shaking table at X revolutions per minute (rpm) for X hours/days at
42 ± 5 °C as defined by ASTM D4169 section 12.2
After the simulated shipping challenge, each IVD kit will be returned to
its corresponding storage temperature (40 ± 5 °C or 8 ± 2 °C).
Testing schedule for real-time stability studies

Testing will be conducted at 0, 3, 6, 9, 12 and 13 months. At each scheduled time
point, the allotted number of IVD kits will be brought to 15–30 °C and used to
test each member of the panel in triplicate.
Note 1: The test at 0 months will provide evidence that the IVD kit is
stable under extreme conditions of shipping (but similar to those likely
to be experienced); the testing at later time points will provide evidence
to support the claimed shelf-life after transport; and testing beyond the
claimed shelf-life will provide evidence that the IVD kit is stable and not
close to a failure point.
Documentation for transport stress conditions

■■ the lot numbers of the IVD kits used to conduct the test;
■■ the operator(s) name(s);

■■ the dates of testing;
■■ identifying details for each member of the panel being tested;
■■ the temperature at which the IVD kits are stored;
■■ the values of temperature and humidity for each of the challenge
conditions;
■■ instrument settings for the shaking apparatus and duration of
operation for challenge conditions;
2
See: Standard practice for performance testing of shipping containers and systems. ASTM D4169 - 14. West
Conshohocken (PA): ASTM International; 2014.
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■■ the ambient temperature and humidity during testing;

■■ each test result as an interpretation according to the IFU;
■■ each test result as a band intensity – band intensity should be scored
using the calibrated scale described in Protocol ZXY00001 (for
example, 0; faint/trace; +1; +2; +3;…+10) even though the IFU do
not give scores to results);
■■ any aberrations or deviations from the protocol, the reason for the
deviation and any remedial action undertaken. Results from invalid
assays must be recorded but not included in the calculations of
shelf-life. Apparently aberrant results, unless the underlying cause
can be positively identified as not related to a problem with the IVD,
must be included in the calculations of shelf-life.
Panel for monitoring stability

See the suggestions in Appendix 2: Suggested specimens for stability testing
panels.
Acceptance criteria
Each panel member should show a band intensity result that matches its
expected result at each tested time point. The expected result must be validated
so that if the IVD fails to meet the claims (for example, fails to detect critical
specimens, has unacceptable performance at medical decision concentrations
or has unacceptable specificity) the panel member would also fail to meet its
specified result.
The stability after transportation of the IVD kit will be taken as the time
point before the last time point to have met the acceptance criteria – for example,
if the IVD is stable to 13 months, the stability after transportation will be deemed
to be 12 months.
The stability after transportation should be identical to the claimed shelf-
life of the IVD kit – that is, the extremes of possible conditions to which the
IVD kit is likely to be subjected during transport must not affect the shelf-life of
the IVD.
Calculation of results
Detailed statistical instruction must be obtained from a professional statistician
with an understanding of the expectations of the stability study plan and outcome.
Professional statistical input is particularly recommended when calculating
confidence limits for discrete data such as readings from a graduated scale.
Each of the following applies at each time point:
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The variance of the results for all replicates within and between all the lots
must be calculated for each panel member. From the overall variance between
lots, the confidence with which future lots of the IVD kit will detect the panel
member at that time point after manufacture and transport can be calculated. If
the confidence that the panel member will meet its specification is less than some
pre-defined value (normally 95%), it must be deemed to have failed at that time
point and the shelf-life of the IVD kit should be restricted accordingly.
If regression analysis is used to define the time point at which a panel
member would not meet its criterion, then lot-to-lot variability must be included
when setting the confidence limits around the regression line. However, real-
time data must extend beyond the claimed shelf-life so that the intercept of the
regression confidence limit and the expected value must be at a time period
longer than the claim. It is usually more appropriate to calculate as discussed
in the previous paragraph, particularly if the regression cannot be proven to
be linear.
Example 2: In-use stability protocol

Objective
To determine the stability of opened bottles of the specimen buffer used in the
IVD kit in real-time when stored at 15–30 °C as proposed in Stability Study Plan
XYZ00001.
In this example, the manufacturer recommends that the test cassette be used
immediately upon opening; this claim should also be validated in a separate
experiment, so that it can be confirmed that the IVD will still perform
satisfactorily after the test cassette has been removed from its pouch and left
at room temperature for 1, 2, 6 and 24 hours, etc., as appropriate.
Acquire sufficient numbers of IVD kits from one production lot using a
predetermined sampling protocol (for example, random, first X number of kits
in the first box, every 100th kit, etc.).
Acquire sufficient volume of each panel member for the duration of the
testing schedule. Establish a method for randomizing the panel for testing.
■■ the lot numbers from which the IVD kits were sampled;
■■ the number of IVD kits sampled from each lot; and
■■ details (including manufacturing/lot information) for each of the
IVD kit components that will be tested as part of this protocol (test
cassette and specimen buffer).
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Preparation
Two lots of specimen buffer are to be tested. One lot of the component must be
freshly made, while the other should be towards the end of the assigned shelf-life
of the IVD kit.
The component is to be tested in its final packaging.
The IVD kits are stored so that the reagents are in contact with all
elements of the packaging (for example, the bottles in the IVD kits are stored
horizontally, lying flat on their sides, allowing liquids to remain in contact with
the bottle closures).
Half of each lot will be stored at 30 ± 5 °C, the other half at 15 ± 5 °C. At
the start of testing, each bottle will be brought to room temperature (20 ± 2 °C),
opened, used for testing and then recapped and returned to the stated storage
temperature.
Note: It is important that the components under test are opened and
used under circumstances likely to occur in users’ laboratories (that is,
not in rooms with HEPA-filtered air) thus mimicking, as far as possible,
genuine use.
Testing schedule
At each subsequent scheduled time point the allotted number of bottles will be
brought to room temperature and used to test each panel member in triplicate.
Testing will be conducted at 0, 1, 2, 3, 4 weeks, etc., up to the end of the claimed
in-use life.
Documentation
■■ the lot number of the IVD kit used to conduct the test;
■■ the operator(s) name(s);
■■ the dates of testing;
■■ the temperature at which the IVD kits are stored;
■■ the ambient temperature during testing;
■■ identifying details for each member of the panel being tested;
■■ each test result as a band intensity – band intensity should be scored
using the calibrated scale described in Protocol ZXY00001 (for
example, 0; faint/trace; +1; +2; +3;…+10);
■■ each test result as an interpretation according to the IFU;
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■■ any aberrations or deviations from the protocol, the reason for the
deviation and any remedial action undertaken.
Panel for testing stability

See the suggestions in Appendix 2: Suggested specimens for stability testing
panels.
Acceptance criteria
Each panel member should show a band intensity result that matches its expected
result at each tested time point. The in-use stability of the sample buffer will be
taken as the time point before the last time point to have met the acceptance
criteria – for example, if the IVD kit is observed to be stable to 5 weeks, the in-use
stability will be deemed to be 4 weeks.
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Appendix 2
Suggested specimens for stability testing panels
Examples in this section
Not all of the specimens in the examples that follow will be necessary for all IVDs,
and nor is the list exhaustive. Panels must be composed according to strict risk-
management principles and all decisions must be documented and traceable.
The minimum set of specimens recommended for inclusion in a testing
panel for different types of products are outlined below.
1. Specimens to monitor NAT-based tests

If a proprietary nucleic acid preparation/extraction system is provided, the
recovery must be shown to meet claims for each genotype from each of
the specimen types claimed (for example, dried blood spots, whole blood
and plasma). Successful removal of inhibitory substances, if intended, must
be demonstrated for appropriate specimen types. Unless potentially variable
biological reagents are involved, this system would be expected to be verified in
manufacture and not necessarily tested at release.
Specimens Remarks
Specimens to demonstrate Traceability is required to one of the WHO
maintenance of sensitivity international standards 1 if available – for example,
and/or limit of detection, and/ the Third WHO International Standard for HIV1-RNA
or accuracy, and precision for NAT-based assays (National Institute for Biological
Standards and Control (NIBSC) code 10/152); or the
Fourth WHO International Standard for hepatitis C
virus RNA for NAT-based assays (NIBSC code 06/102).
More than one genotype may be required to
validate these claims: see the First WHO International
Reference Panel for hepatitis B virus genotypes for
NAT-based assays (Paul-Ehrlich-Institut (PEI) code
5086/08).
This may be required on each of the claimed
specimen types.
1
The catalogue of WHO International Reference Preparations is available at: http://www.who.int/
bloodproducts/catalogue/en/
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Table continued
Specimens Remarks
Specimens to demonstrate Sufficient negative specimens should be included
specificity and validity of to ensure that the claims will be met at end of
runs shelf‑life.
Specimens (or reagents) to If more than one part of the genome is to be
demonstrate stability of each detected, both systems must be shown to
of the critical components of be stable.
the IVD If both DNA and RNA are measured the complete
system must be shown to be stable.
2. Specimens to monitor tests that measure CD4 cells

Rationale
CD4 measurements are quantitative, and accuracy at the clinical decision point
is crucial. The design input should have information on the accuracy and other
parameters required, and the panel must be designed to provide evidence
that these parameters are maintained over the assigned life of the reagent and
measuring IVD.
Parameters
The panel used in stability work must be able to demonstrate the following:
■■ stability of all the antibodies used in the IVD (frequently anti-CD4
and anti-CD3 antibodies; any other critical components must also
be covered);
■■ accuracy and trueness of measurement maintained at the critical
level (at least five specimens required);
■■ claimed linearity over the required range of CD4 count (at least five
specimens required); and
■■ measure drift.
Specimens
Artificial specimens (such as stabilized blood specimens) can be used if a risk
assessment based on R&D work indicates that they are effective. Fresh specimens
are usually required. Measurements should be compared to an approved
reference system.
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Examples of approaches
Aged or in-use lots may be compared with a reference – for example, a new lot.
Precision studies can be performed as described elsewhere.2
3. Specimens to monitor tests for HIV antibodies
Specimens Remarks
IgM first seroconversion Possible approaches to obtain samples:
specimens and IgG first • Study the early data from commercial
seroconversion specimens seroconversion panels where the
seroconversion was frequently monitored
by IgM and IgG blots.
• Study the responses to second and third
generation assays or protein A and protein L
assays (this approach is less useful).
All other parts of the HIV
proteome included – for example,
reverse transcriptase (RT)
Late stage specimens – usually This might serve to monitor any kit run
a high-dilution set near the control.
sample-to-cut-off ratio Note: HIV serology is not particularly genotype
dependent. It is usually not necessary to
include controls for genotype detection
unless risk assessment or experiment shows
that it is required for a particular IVD.
HIV-2, diluted to near the sample- Seroconversion specimens are very rare.
to-cut-off ratio
HIV-1 (O), if claimed
Difficult specimens to monitor 100 negatives at release subject to risk analysis
specificity and invalidity rate and statistical analysis of the allowable
(relative to the claimed) false-reactive rate and
invalidity rate.
2
Evaluation of precision of quantitative measurement procedures; approved Guideline EP05-A3. Third
edition. Wayne (PA): Clinical and Laboratory Standards Institute; 2014.
369
4. Specimens to monitor tests for HIV-1/2 and

Treponema pallidum (TP) antibodies
Specimens Remarks
Specimens to detect HIV See the above section 3. Specimens to
monitor tests for HIV antibodies.
Specimens to detect all the critical Note: Each of these epitopes plays a role in
epitopes in the IVD – for example, detecting syphilis in different stages of the
TpN47, TpN17 and TpN15 infection. It is necessary to have a panel
member to monitor each epitope system
present (and possibly each stage of infection)
even if poly-fusion proteins are used. This
can be avoided if the manufacturer can
demonstrate that each epitope system is
equally stable.
Specimens able to show that the Note: It would not be sufficient for WHO
invalidity and specificity rates prequalification to extrapolate to the stability
do not fall outside the claims, of HIV-2/TP detection by testing only HIV-1-
particularly if whole blood is a positive specimens.
claimed specimen type
5. Specimens to monitor tests for HCV antibodies
Specimens Remarks
NS3 first seroconversion specimens
and core first seroconversion
specimens
Specimens to monitor any other Results can be obtained from line

antibodies claimed (frequently immunoassays that differentiate antibody
against NS5 and NS4) responses to the different proteins.
A late-stage dilution near the Note: HCV serology is not particularly
sample-to-cut-off ratio genotype dependent in terms of anti-core and
anti-NS3, but it is possible to make serotyping
assays based on NS4 that mimic genotyping
reasonably well. It is usually not necessary
to include controls for genotype detection
unless risk assessment or experiment for a
particular IVD show otherwise.
370
Annex 5
Table continued
Specimens Remarks
Difficult specimens to monitor 100 negative specimens subject to risk
specificity and invalidity rate analysis and statistical analysis of the
allowable (relative to the claimed) false-
reactive rate and invalidity rate.
6. Specimens to monitor tests for HBsAg
Specimens Remarks
Specimens to define sensitivity Traceability is required to one of the WHO
relative to the claim international standards 1 – for example,
the Third WHO International Standard for
hepatitis B virus surface antigen (genotype B4;
HBsAg subtypes ayw1/adw2); NIBSC code
12/226) for one or more specimens and
probably also to the ad and ay standards
available from a commercial supplier.
Commercially available seroconversion
specimens are almost all of the adw2 subtype
– different from the Third WHO International
Standard – so claims of critical threshold
specimen detection must be proven by
specimens in the panel.
Specimens to monitor the These will almost certainly be traceable to the
maintenance of the claims for a First WHO International Reference Panel for
variety of serotypes/genotypes hepatitis B virus genotype for HBsAg assays
and mutant forms (PEI code 6100/09).
Specimens to control against
prozone/high dose hook effect if
found or if theoretically an issue
If detection of HBsAg in the
presence of anti-HBsAg is claimed
(current best practice) proof
of maintenance of the claim
is required
3
The catalogue of WHO International Reference Preparations is available at: http://www.who.int/
bloodproducts/catalogue/en/
371
Table continued
Specimens Remarks
Specimens to monitor the critical If the monoclonal antibodies used have a
components of the IVD particular function or bias, such as against
the ayr or adr subtypes (not controlled by the
standards) or are used to detect mutant forms
of the antigen, then each must be monitored
to ensure viability at end of shelf-life. These
may be the same specimens as mentioned in
the previous paragraphs.
If there are critical dissociation chemicals or
red-cell capture or rupture agents used then
these must also be monitored.
Difficult specimens to monitor 100 negative specimens subject to risk
specificity and invalidity rate analysis and statistical analysis of the
allowable (relative to the claimed) false-
reactive rate and invalidity rate.
372
Annex 5
Appendix 3
Summary table of standards relevant to stability studies
Recommendation Comment Standard
Studies must be compliant The minimum expected standards. CLSI EP25-A
with CLSI EP25-A and ISO ISO 23640:2011
23640:2011
Studies must be fully Risk assessment must be specific to CLSI EP25-A
documented with the analyte, type of physical device (many sections)
risk evaluations, plans and assay format, and previous ISO 23640:2011
and protocols prior to manufacturing experiences, not (section 2)
initiation generic nor by rote. ISO 14971:2007
Studies and risk This is particularly important for
management must transport stress where extreme
take into consideration conditions must be evaluated.
the conditions likely to
be encountered in the
geographical and health-
care settings in which the
IVD is intended to be used
IVDs must be subjected This is particularly important to CLSI EP25-A
to simulation of transport WHOPQ as transport will always (section 4.2.3)
stress before being used be involved before use of an IVD, ISO 23640:2011
to establish any form of and transport conditions cannot be (section 5.2)
stability guaranteed nor predicted.
Transport simulation must It is most unlikely that actual transport CLSI EP25-A
cover the extremes of will involve all extreme conditions (section 4.2.3)
environmental conditions that might occur during the
ascertained during risk marketing life of the IVD, or that the
evaluations conditions during actual transport
can be adequately documented.
IVDs used in any stability If IVDs are not made to final validated Good
studies must be made to and documented manufacturing manufacturing
finalized manufacturing scales, stringent proof must be practice (GMP)
specifications, to final presented that the scale change will CLSI EP25-A
scale and in the packaging not affect any parameters of the IVD,
(including labelling) in nor any of the manufacturer’s claims.
which the IVDs will be Pre-production lots can only be used
made available for stability work if these conditions
are met.
373
Table continued
If several presentations If, for example, two pack sizes are CLSI EP25-A
of the IVD are to be to be provided then each pack size
presented, all aspects of must be evaluated completely, even
stability must be shown though the contents are identical
for each except for vial size.
Sufficient numbers of “Independent lots” means lots CLSI EP25-A
independent lots of the with different production (or (section 4.4)
IVD must be evaluated manufacturing, purification, etc.)
to enable each form of runs of critical reagents (for example,
stability to be evaluated biological reagents prepared in
in terms of inter-lot different syntheses, growths or
variability purifications or other risk-defined
critical reagents from different
manufactured lots or from different
suppliers if applicable).
CLSI EP25-A and ISO 23640:2011
specify minimum numbers of lots
to be used but give no guidance to
recommended numbers beyond
documented risk evaluation.
If critical components of It must be documented that stored CLSI EP25-A
the IVD are assigned lives materials (for example, freeze-thawed (section 4.4)
independently of the life biological reagents) operate as
of the IVD, the various expected during the whole of the
forms of stability of the assigned shelf-life.
IVD must be proven
with those reagents
at different stages of
their lives
Each form of stability If any lot-to-lot variability is found, the
must be defined manufacturer must provide evidence
statistically with respect that subsequent lots will not have
to any inter-independent worse stability than that claimed.
lot variability, not just
assigned to the minimum
stability found among the
lots that happened to be
evaluated experimentally
374
Annex 5
Table continued
If any control material If the analytic function of the IVD is
with a claim to prove the out of specification from any cause,
functionality of the IVD including stability failure, the control
is provided to users that material must be demonstrated to
claim must be justified be able to alert the user to that fact.
in stability studies in
addition to any other
studies
Use of accelerated Accelerated stability is acceptable CLSI EP25-A
stability, even to provide in providing interim life if the (section 7.3 &
interim life assignments, parameters of the Arrhenius Appendix B)
must be justified equation, or any other method ISO 23640:2011
scientifically used, are adequately proven and (section 5.3.1;
documented. notes 1 & 2)
WHO/EMP/RHT/PQT/TGS2/2017.02
The Technical Guidance Series (TGS) for WHO Prequalification – Diagnostic
Assessment is intended to assist manufacturers in meeting WHO prequalification
requirements for their IVD. For further information on this guidance and other
TGS documents email: [email protected]
375
Annex 6
Biological substances: WHO International Standards,
Reference Reagents and Reference Panels
The provision of global measurement standards is a core normative WHO
activity. WHO reference materials are widely used by manufacturers, regulatory
authorities and academic researchers in the development and evaluation of
biological products. The timely development of new reference materials is crucial
in harnessing the benefits of scientific advances in new biologicals and in vitro
diagnosis. At the same time, management of the existing inventory of reference
preparations requires an active and carefully planned programme of work to
replace established materials before existing stocks are exhausted.
The considerations and guiding principles used to assign priorities
and develop the programme of work in this area have previously been set out
as WHO Recommendations.1 In order to facilitate and improve transparency
in the priority-setting process, a simple tool was developed as Appendix 1 of
these WHO Recommendations. This tool describes the key considerations taken
into account when assigning priorities, and allows stakeholders to review and
comment on any new proposals being considered for endorsement by the WHO
Expert Committee on Biological Standardization.
A list of current WHO International Standards, Reference Reagents and
Reference Panels for biological substances is available at: http://www.who.int/
biologicals.
At its meeting in October 2017, the WHO Expert Committee on
Biological Standardization made the changes shown below to the previous list.
1
Recommendations for the preparation, characterization and establishment of international and other
biological reference standards (revised 2004). In: WHO Expert Committee on Biological Standardization:
fifty-fifth report. Geneva: World Health Organization; 2006: Annex 2 (WHO Technical Report Series, No. 932;
http://www.who.int/immunization_standards/vaccine_reference_preparations/TRS932Annex%202_
Inter%20_biol%20ef%20standards%20rev2004.pdf?ua=1, accessed 27 March 2018).
377
Additions 2
Preparation Activity Status
Biotherapeutics other than blood products
Parathyroid hormone 0.914 mg/ampoule Second WHO
1-34 (recombinant, 9140 IU/ampoule International Standard
human)
Rituximab In vitro biological activities: First WHO International
1000 IU/ampoule (CDC activity) Standard
1000 IU/ampoule (ADCC activity)
1000 IU/ampoule (cell-binding
activity)
1000 IU/ampoule (apoptotic
activity)
Infliximab 500 IU/ampoule (TNF- First WHO International
neutralizing activity) Standard
500 IU/ampoule (binding activity)
50 µg/ampoule for use in
therapeutic drug monitoring
Blood products and related substances
Activated blood 10.5 IU/ampoule Second WHO
coagulation factor IX International Standard
(human)
Blood coagulation FXII:C = 0.86 IU/ampoule First WHO International
factor XII (plasma, FXII:Ag = 0.80 IU/ampoule Standard
human)
(via assignment of
additional analytes to
the current Second

WHO International
Standard for blood
coagulation factor XI)
Activated blood 6.7 U/ampoule First WHO Reference
coagulation factor X Reagent
(human)
2
Unless otherwise indicated, all materials are held and distributed by the National Institute for Biological
Standards and Control, Potters Bar, Herts, EN6 3QG, the United Kingdom. Materials identified by an * in the
above list are held and distributed by the Paul-Ehrlich-Institut, 63225 Langen, Germany.
378
Annex 6

In vitro diagnostics
anti-cytomegalovirus 46.4 IU/vial First WHO International
immunoglobulin G Standard*
Chikungunya virus 2.5 x 10 6 IU/ml First WHO International
RNA for NAT-based Standard*
assays
Lupus anti-dsDNA 100 U/ampoule First WHO Reference
serum Reagent
Genomic KRAS codons Consensus mutation %; First WHO Reference
12 and 13 mutations consensus mutant KRAS copy Panel
number; and consensus total
KRAS copy number provided for
KRAS mutations p.G12A; p.G12C;
p.G12D; p.G12R; p.G12S; p.G12V;
and p.G13D
Human herpes virus 7.75 log10 IU/ml First WHO International
6B DNA for NAT-based Standard
assays
Hepatitis A virus RNA 4.42 log10 IU/ml Third WHO
for NAT-based assays International Standard
HIV-1 RNA for NAT- 5.10 log10 IU/ml Fourth WHO
based assays International Standard
Ebola virus antibodies 1.5 IU/ml First WHO International
(plasma, human) Standard
Ebola virus antibodies [no assigned units] First WHO Reference
(plasma, human) Panel
Plasmodium HRP2 = 1000 IU/ampoule First WHO International
falciparum antigens pLDH = 1000 IU/ampoule Standard
Vaccines and related substances
mOPV type 1 7.32 log10 TCID 50 /ml First WHO International
Standard
Standard
Standard
379

bOPV type 1+3 7.19, 6.36 and 7.32 log10 TCID50/ First WHO International
ml for serotypes 1 and 3 and total Standard
virus content, respectively
Pertussis toxin 1881 IU/ampoule (histamine Second WHO
sensitization test) International Standard
680 IU/ampoule (CHO cell
clustering assay)
Vi polysaccharide of 1.94 ± 0.12 mg/ampoule First WHO International
Citrobacter freundii Standard
Vi polysaccharide of 2.03 ± 0.10 mg/ampoule First WHO International
Salmonella Typhi Standard
Anti-typhoid capsular 100 IU/ampoule First WHO International
Vi polysaccharide Standard
immunoglobulin G
(human)
Antiserum to 1000 IU/vial First WHO International
respiratory syncytial Standard
virus
380
SELECTED WHO PUBLICATIONS OF RELATED INTEREST

The World Health Organization was established in 1948 as a specialized agency of the
Sixty-seventh report.
United Nations serving as the directing and coordinating authority for international
WHO Technical Report Series, No. 1004, 2017 (xviii + 591 pages)
health matters and public health. One of WHO’s constitutional functions is to
provide objective and reliable information and advice in the field of human health, WHO Expert Committee on Biological Standardization
a responsibility that it fulfils in part through its extensive programme of publications Sixty-sixth report.
WHO Technical Report Series, No. 999, 2016 (xix + 267 pages)
The Organization seeks through its publications to support national health strategies
and address the most pressing public health concerns of populations around the world. WHO Expert Committee on Biological Standardization
To respond to the needs of Member States at all levels of development, WHO publishes Sixty-fifth report.
practical manuals, handbooks and training material for specific categories of health WHO Technical Report Series, No. 993, 2015 (xvi + 262 pages)
workers; internationally applicable guidelines and standards; reviews and analyses of
health policies, programmes and research; and state-of-the-art consensus reports that WHO Expert Committee on Biological Standardization
offer technical advice and recommendations for decision-makers. These books are Sixty-fourth report.
closely tied to the Organization’s priority activities, encompassing disease prevention WHO Technical Report Series, No. 987, 2014 (xviii + 266 pages)
and control, the development of equitable health systems based on primary health
care, and health promotion for individuals and communities. Progress towards better WHO Expert Committee on Biological Standardization
health for all also demands the global dissemination and exchange of information Sixty-third report.
that draws on the knowledge and experience of all WHO’s Member countries and the WHO Technical Report Series, No. 980, 2014 (xv + 489 pages)
collaboration of world leaders in public health and the biomedical sciences. WHO Expert Committee on Biological Standardization
To ensure the widest possible availability of authoritative information and guidance on Sixty-second report.
health matters, WHO secures the broad international distribution of its publications WHO Technical Report Series, No. 979, 2013 (xiii + 366 pages)
and encourages their translation and adaptation. By helping to promote and protect WHO Expert Committee on Biological Standardization
health and prevent and control disease throughout the world, WHO’s books contribute Sixty-first report.
to achieving the Organization’s principal objective – the attainment by all people of the WHO Technical Report Series, No. 978, 2013 (xi + 384 pages)
highest possible level of health.
The WHO Technical Report Series makes available the findings of various international Sixtieth report.
groups of experts that provide WHO with the latest scientific and technical advice on WHO Technical Report Series, No. 977, 2013 (viii + 231 pages)
a broad range of medical and public health subjects. Members of such expert groups
serve without remuneration in their personal capacities rather than as representatives WHO Expert Committee on Biological Standardization
of governments or other bodies; their views do not necessarily reflect the decisions or Fifty-ninth report.
the stated policy of WHO. WHO Technical Report Series, No. 964, 2012 (viii + 228 pages)
For further information, please contact: WHO Press, World Health Organization, 20 Website: http://www.who.int/biologicals
avenue Appia, 1211 Geneva 27, Switzerland (tel. +41 22 791 3264; fax: +41 22 791 4857;
email: [email protected]; order on line: www.who.int/bookorders).
Further information on these and other WHO publications can be obtained from
WHO Press, World Health Organization, 1211 Geneva 27, Switzerland
(tel.: +41 22 791 3264; fax: + 41 22 791 4857; email: [email protected];
order online: www.who.int/bookorders)
This report presents the recommendations of a WHO Expert
Committee commissioned to coordinate activities leading to the
1011
adoption of international recommendations for the production W H O Te c h n i c a l R e p o r t S e r i e s
and control of vaccines and other biological substances, and the
establishment of international biological reference materials. 1011
Following a brief introduction, the report summarizes a number

of general issues brought to the attention of the Committee. The
next part of the report, of particular relevance to manufacturers
and national regulatory authorities, outlines the discussions
held on the development and adoption of new and revised
WHO Recommendations, Guidelines and guidance documents.
Following these discussions, WHO Guidelines on the quality,
safety and efficacy of Ebola vaccines, and WHO Guidelines
on procedures and data requirements for changes to approved
biotherapeutic products were adopted on the recommendation
of the Committee. In addition, the following two WHO

guidance documents on the WHO prequalification of in vitro
diagnostic medical devices were also adopted: (a) Technical
Specifications Series (TSS) for WHO Prequalification –
Diagnostic Assessment: Human immunodeficiency virus
(HIV) rapid diagnostic tests for professional use and/or self-
testing; and (b) Technical Guidance Series (TGS) for WHO
on Biological
Prequalification – Diagnostic Assessment: Establishing stability
of in vitro diagnostic medical devices. Standardization
Subsequent sections of the report provide information on the
current status, proposed development and establishment of
international reference materials in the areas of: antibiotics,
biotherapeutics other than blood products; blood products Sixty-eighth report
and related substances; in vitro diagnostics; and vaccines and
related substances.
A series of annexes are then presented which include an
updated list of all WHO Recommendations, Guidelines and
WHO Technical Report Series

other documents on biological substances used in medicine
(Annex 1). The above four WHO documents adopted on
the advice of the Committee are then published as part
of this report (Annexes 2–5). Finally, all additions and
discontinuations made during the 2017 meeting to the list of
International Standards, Reference Reagents and Reference
Panels for biological substances maintained by WHO are
summarized in Annex 6. The updated full catalogue of
WHO International Reference Preparations is available at:
http://www.who.int/bloodproducts/catalogue/en/.
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This report presents the recommendations of a WHO Expert

Committee commissioned to coordinate activities leading to the

WHO Expert Committee on Biological Standardization

WHO Expert Committee

WHO Technical Report Series

ISBN 978 92 4 121020 1

WHO Expert Committee on Biological Standardization

WHO Expert Committee

© World Health Organization 2018

Dr L. Elmgren, Biologics and Genetic Therapies Directorate, Health Canada, Ottawa,

Representation from non-state actors

Developing Countries Vaccine Manufacturers Network 8

EUAL WHO emergency use assessment and listing (procedure)

HPA human platelet antigens

IFU instructions for use

PTx pertussis toxin

TSS WHO Technical Specifications Series

A wide range of stakeholders had participated in the Expert Consultation

2.1.2 Vaccines and biotherapeutics: recent and planned

specific (or relevant to all vaccines), a number specifically cover biotherapeutic

establishment at the present meeting, its suitability as a standard in the mAb

An update was then provided on the revision of the WHO Guidelines

speaking countries, organized by the WHO Regional Office for Europe in

2.1.3 Blood products and in vitro diagnostics: recent and

in vitro diagnostics (see sections 7.1.1–7.1.10).

approaches) and to propose solutions to specific blood-related issues. After

2.2.2 Report from the WHO network of collaborating centres on

Proposal to simplify the structure of type-specific vaccine written standards

2.2.3 Report from the WHO network of collaborating centres

2.3 Feedback from custodian laboratories

National Institute for Biological Standards and Control

distributed to 75 different countries. Of the standards distributed, approximately

European Directorate for the Quality of Medicines &

Paul-Ehrlich-Institut (PEI), Langen, Germany

further extension of the First WHO Repository of platelet transfusion relevant

a CBER-WHO Cooperative Agreement was being used to support the revision

2.4 Cross-cutting activities of other WHO committees and groups

2.4.2 WHO Global Benchmarking Tool

2.4.3 Development of WHO guidelines on good regulatory practices

2.4.4 Snake-bite envenoming

2.4.5 Update from the WHO Product Development

2.4.6 Pilot WHO prequalification of biosimilar monoclonal antibodies

2.4.7 Model NRA Lot Release Certificate for prequalified vaccines

2.4.8 Planned proficiency testing study of a standardized

identified standardized HPAEC-PAD test protocol that is applicable to all eight

2.4.9 Vaccine prequalification – establishment of the WHO-NNB

system faced a number of challenges, including: (a) the increasing number of

2.5 Strategic issues

A number of gaps in regulatory preparedness had also been identified

or further guidance on prime-boost vaccines should also be kept under review

2.5.2 International standards and reference preparations

strongly encouraged to establish requirements that are commensurate with their

3.2 Cellular and gene therapies

modified viable cells should be considered CTPs. However, products containing

3.3 In vitro diagnostics

based assays for the management of a number of bloodborne diseases (including

and scrutiny of assessment.

During discussions the Committee raised the issue of the degree of

3.3.2 Human immunodeficiency virus rapid diagnostic