Clearance Services

A guide to planning your

Cleaning Validation Study

O-0040508

Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Why undertake a cleaning validation study? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Process validation for removal and inactivation of bacteria, fungi, and viruses . . . . . . . . . . . . . . . . . . . 2 Selection criteria for microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Study process. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Pre-study protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Study protocol including microbial spiking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Cleaning technique. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Coupons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Swabbing validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Cleaning agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 TSE agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8

Calculation methods: logarithmic reduction factor. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Cleaning validation study results. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Data handling and record maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Delivering the study report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

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A guide to planning your Cleaning Validation Study

Introduction
Why undertake a cleaning validation study?
Cleaning validation studies are performed to establish documented evidence which demonstrateswith a high degree of assurancethat an equipment-specific cleaning process will consistently yield results meeting specifications and quality attributes. GMP regulations explicitly state that manufacturers of finished pharmaceuticals must properly clean their facilities and equipment to ensure product safety: 21 CFR 211.67(a) Equipment and utensils shall be cleaned, maintained, and sanitized at appropriate intervals to prevent malfunctions or contamination that would alter the safety, identity, strength, quality, or purity of the drug product beyond the official or other established requirements. The FDA has further clarified its definition of a properly validated cleaning method.1 The expectations are very rigorous and include having written procedures for how cleaning processes are validated, who approves the validation studies, the acceptance criteria applied to these studies, and preparation of a final validation report indicating that residues have been reduced to an acceptable level. Analytical methods and sampling procedures need to be written into the validation protocols. Cleaning validation studies are typically performed as a product goes through phase III clinical trials. Common situations that lead manufacturers to conduct cleaning validation studies include: When the same facility is used to manufacture multiple biologic products, especially when change-over validation is essential When both animal-derived and Animal Origin Free (AOF) supplies are used in the same facility When plant-derived materials are used, as they can be a source of mycoplasmas and a variety of adventitious agents When operating inside viral vaccine manufacturing environments (different viral constructs)

BioReliance has performed hundreds of cleaning validation studies in accordance to criteria set by various global regulatory bodies and we have processes in place to address differing requirements. Furthermore, we validate the cleaning procedures in our own manufacturing facilities using the same methodologies that we utilize for you. The information provided in this brochure outlines these procedures for everything from cleaning techniques to calculation methods used to validate cleaning study results, all of which are designed with your process in mind.

1. Guide to Inspections Validation of Cleaning Process, http://www.fda.gov/ora/inspect_ref/igs/valid.html.

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A guide to planning your Cleaning Validation Study

Process validation for removal and inactivation of bacteria, fungi, and viruses
BioReliances cleaning validation studies are designed to quantify the elimination of bacteria, fungi, and viruses during the cleaning procedures used at your manufacturing facility. Our reports feature specific reduction factors for each cleaning measure studied, a method of reporting that is preferred by regulators, compared to a simpler plus or minus (+/) result for the presence or absence of bacteria after a cleaning step. BioReliances studies incorporate methodology and design analogous to the rigorous requirements for viral clearance studies, and are designed to withstand scrutiny by worldwide regulatory agencies. All work is performed under U.S. Food and Drug Administration Good Laboratory Practices per 21 CFR, part 58, and the U.K. GLP Regulations, the Japanese GLP Standard, and the OECD Principles of Good Laboratory Practice.

Selection criteria for potential contaminants

The selection of model microorganisms for validation studies is a critical part of developing a removal/inactivation protocol. The selection should take into account the nature and origin of equipment and raw materials used in production processes, and the model microorganisms should be known contaminants or appropriate related models. For example, bacterial and fungal species selected should be representative of environmental, human, and material source-derived microbial flora, and should include species of known antimicrobial resistance. An additional factor to consider for a model microorganism selection is its ability to grow as a high-titer stock in both standard microbiological and cell culture media, and its ease of detection in a sensitive and reliable assay. A combination of United States Pharmacopoeia (USP) strains and environmental isolates obtained from your site is recommended. Typical residual contaminants that can be important for cleaning validation studies include: Host-cell proteins Lipids DNA/host-cell nucleic acid Endotoxins Carbohydrates Membrane/chromatography matrix leachables Detergents Viruses TSEs Mycoplasmas, bacteria, fungi

Tables 13 list some of the most common agents BioReliance uses in these studies, but note that the final study design is developed with your specific process in mind. Studies typically involve 36 agents.

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A guide to planning your Cleaning Validation Study

Table 1Common bacteria and fungi used in cleaning validation studies.

Species* Pseudomonas aeruginosa Candida albicans Aspergillus niger Clostridium sporogenes Staphylococcus epidermidis Bacillus isolates Gram reaction/ cell morphology Gram negative/rod Yeast Mold Gram positive/ spore forming rod Gram positive/cocci Gram positive/rod 02 requirement Obligate aerobe Facultative anaerobe Aerobe Obligate anaerobe Facultative anaerobe Aerobe Bacteria or fungi Bacteria Fungi Fungi Bacteria Bacteria Bacteria Lab assay time 314 days 514 days 514 days 314 days 314 days 314 days Resistance to physical/chemical inactivation Moderate Low High Moderate Low High

*We also recommend using client-specific environmental isolates.

Table 2Common viruses used in cleaning validation studies.

Virus* Xenotropic murine leukemia virus (XMuLV) Murine minute virus (MMV) Porcine parvovirus (PPV) Pseudorabies virus (PRV) Bovine viral diarrhea virus (BVDV) Adenovirus (Adeno) Reovirus (Reo) Hepatitis A virus (HAV) Genome RNA DNA DNA DNA RNA DNA RNA RNA Envelope Yes No No Yes Yes No No No Family Retro Parvo Parvo Herpes Flavi Adeno Reo Picorna Size (nm) 80110 2025 2025 150250 4070 7090 6080 ~30 Lab assay time 79 days 1013 days 79 days 46 days 79 days 1214 days 79 days 1821 days Resistance to physical/ chemical inactivation Low High High Medium Medium High High High

*We also recommend using client-specific constructs/isolates.

Table 3Common species of mycoplasmas used in cleaning validation studies.

A. laidlawii M. gallisepticum M. hyorhinis M. orale M. pneumoniae M. synoviae

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A guide to planning your Cleaning Validation Study

Study process
Pre-study protocol
The first step in the process is a pre-study to assess the potential for bacteriostasis and/or fungistasis in the test system and to verify the suitability of the recovery methods and media selected for the test articles to be evaluated. This will establish the degree to which the process intermediates interfere (i.e., cause inhibition) with the accurate recovery of microbial contaminants. Although specific protocols can vary, the general approach is that individual samples of each process intermediate are spiked separately with less than 100 CFU of each challenge organism. After a four hour hold at 28C, the samples are filtered through a sterile microbiological membrane filter, rinsed three times with an appropriate sterile diluent, and plated on an appropriate growth medium. Upon the satisfactory completion of the interference study, an appropriate microbial recovery procedure will have been established.

Figure 1Example study design #1.

Title: Validation of Agent Elimination in Cleaning Procedures Process step: surface inactivation/removal studies (triplicate runs) Spiking agents: bulk virus, purified virus, virus and stabilizer Temperature: ambient

Recovery
Spot virus onto coupon Let dry

Experimental spray + wipe

Spot virus onto coupon Let dry

Spray only
Spot virus onto coupon Let dry

Wipe only
Spot virus onto coupon Let dry Using a sterile wipe wet with disinfectant, wipe up one time and down one time Incubate 10 min

Spray with medium until wet

Spray with disinfectant until wet

Spray with disinfectant until wet

Incubate 10 min Do NOT wipe

Incubate 10 min Using a sterile wipe wet with disinfectant, wipe up one time and down one time Immerse coupon in 5 ml medium and scrape Assay Assay

Incubate 10 min Do NOT wipe

Immerse coupon in 5 ml medium and scrape Assay

Immerse coupon in 5 ml medium and scrape Assay

Immerse coupon in 5 ml medium and scrape

Note: Samples will be treated immediately upon collection to quench the inactivation reaction. Samples will then be tested immediately.

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A guide to planning your Cleaning Validation Study

Study protocol including microbial spiking

Proper study design must take into account multiple variables, including the type, concentration, and preparation of the cleaning agents, as well as the contact time and temperature. Microbial spiking studies are performed in either duplicate or triplicate on coupons representative of surfaces found in your manufacturing facility. BioReli-

ance produces titered stocks of microbial strains needed, operates the coupon cleaning studies, and collects and neutralizes the samples as needed. Note that all challenge organisms will be characterized and identified prior to the start of the study to ensure that the organisms are verified to be the species intended. Several examples of different study designs are shown in Figures 14.

Figure 2Example study design #2.

Title: Validation of Spiking Agent Elimination by Surface Cleaning Procedures Process step: primary disinfectant efficacy testing Spiking agent: viruses and microbial agents Temperature: ambient

Experimental (spray + wipe) (2 runs per spiking agent)

Spot agent onto coupon* Let dry

Recovery controls (2 runs per spiking agent)

Spot agent onto coupon* Let dry

Spray primary disinfectant onto a clean room wiper until nearly soaked

Immerse coupon in 5 ml medium and scrape with a cell scraper

Wipe coupon (3 cycles of up and down) with wet wiper** Collect medium Let damp surface dry for 10 min Assay Immerse coupon in 5 ml medium and scrape with a cell scraper

Collect medium

Assay

* 2 inch square piece of 304 stainless steel. **Use long, straight, overlapping strokes and moderate pressure. For each stroke (up and down), fold the wiper so that it presents a clean face. Note: Samples will be treated immediately upon collection to quench the inactivation reaction. Samples will then be tested immediately.

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A guide to planning your Cleaning Validation Study

Figure 3Example study design #3.

Title: Validation of Virus Elimination Using Solid Surface Cleaning Procedures Process step: stainless steel cleaning Viruses: XMuLV, Ad2, and PRV Temperature: ambient

Recovery control
Spot and spread 1 ml virus onto coupon

Conflikt, Exspor, or Cavicide only

Spot and spread 1 ml virus onto coupon

70% IPA only

Spot and spread 1 ml virus onto coupon

Conflikt, Exspor, or Cavicide, plus 70% IPA

Spot and spread 1 ml virus onto coupon

Let dry

Let dry

Let dry

Let dry

Spray with medium until wet

Spray with agent until wet

Spray with 70% IPA until wet

Spray with agent until wet Incubate 10 min Spray with 70% IPA until wet Incubate 10 min Add 5 ml medium and scrape with rubber policeman Assay

Incubate 10 min

Incubate 10 min

Incubate 10 min

Add 5 ml medium and scrape with rubber policeman

Add 5 ml medium and scrape with rubber policeman

Add 5 ml medium and scrape with rubber policeman

Collect medium

Collect fluid

Collect fluid

Assay

Assay

Assay

Note: Samples will be treated immediately upon collection to quench the inactivation reaction. Samples will then be tested immediately.

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A guide to planning your Cleaning Validation Study

Figure 4Example study design #4.

Title: Steam in Place and Clean in Place for Bioreactors Process step: SIP/CIP bioreactor studies Viruses: client-specific viral construct

Bioreactor run
Drain reactor Assay harvest SIP Drain Assay fluid CIP Drain and rinse w/ USP H2O Assay rinse Disassemble piping and valve components Soak 3 pipe and 3 valve components for 55 min in 1% v/v CIP 100* Rinse w/ USP H2O Assay rinse Swab interior of each pipe and valve (6 separate swabs) Recover virus from each of the 6 swabs in assay medium 6 titrations *CIP 100 volume sufficient to cover pipe/valve components. Soak performed at RT, but solution preheated to 59C. Note: Samples will be neutralized to pH 68 and/or diluted as needed upon collection. Swab 3 defined areas of the reactor Swab 3 defined areas of the reactor Swab 3 defined areas of the reactor

Recover virus from each of the 3 swabs in assay medium 3 titrations Recover virus from each of the 3 swabs in assay medium 3 titrations Recover virus from each of the 3 swabs in assay medium 3 titrations

Cleaning technique
Coupons
While stainless steel coupons are commonly used for microbial spiking experiments, in some cases it is appropriate to evaluate a wider variety of surface materials. For example, in some manufacturing environments, wall, floor, curtain, glass, and linoleum surfaces are all tested. Furthermore, there can be important differences between new stainless steel and the older, pitted surface of well-used stainless steel. Coupon choice needs to reflect the conditions that are found in your actual manufacturing environment.

Swabbing validation

All steps in cleaning studies need to be carefully controlledeven the technique for swabbing a surface needs to be performed in a highly reproducible manner. For example, the pattern should be consistent (e.g., perpendicular, zigzag, or clockwise) if an operator is wiping a surface, and the cleaning validation studies need to reflect the procedures already established by your organization.

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A guide to planning your Cleaning Validation Study

Cleaning agents

The cleaning agents to be used in the studies depend upon your manufacturing process. Table 4 lists common cleaning agents. BioReliance has experience with virtually all commonly used cleaning agents.

Table 4Common cleaning agents tested.

Cavicide Conflikt Decon-Cycle Decon-Phene Decon-Spore 100 Decon-Spore 200 Plus Ethanol Exspor Hypochlorite IPA LpHse Septihol Spor-Klenz Vesphene II Vespore

TSE agents
Due to the risk of Transmissible Spongiform Encephalopathy (TSE) originating from infected bovine products, these agents represent potential contaminants for biologic products that deserve special attention. Study design for cleaning validation studies involving TSEs can be similar to the examples shown above, but note that direct testing methodologies do not currently exist for the detection of the TSE infectious agents. BioReliance has more experience with TSE studies than any other testing lab in the world, and we have developed a significant amount of technical knowledge to apply to these studies. As is the case with cleaning validation studies for the previously described microbial contaminants, the selection of appropriate model agents is an important part of the study design (Table 5). It is important that model agents can be grown in high-titer stocks and that the agents utilized can be safely handled by laboratory staff. The hamster-adapted 263K strain appears to be an especially good model for BSE, CJD, and other TSEs due to its wellknown incubation period and a well-characterized brain histopathology. Positive identification of TSE can be seen through vacuolized lesions from the brain tissue. There are two approaches for detecting TSEs: bioassays or western blots. BioReliance has an exclusive license on a unique TSE western blot assay that is fully validated and GLP compliant. This sensitive, specific assay is semi-quantitative over a 5.0 log10 range and can be used to rapidly screen and identify process steps. Table 6 gives an overview of the advantages and disadvantages of the two assays.

Table 5Model agents for TSE studies.

Model agent Mouse adapted scrapie strain ME7 Hamster adapted 263K strain Technique Intracranial injection into C57 BL mice Intracranial injection into Syrian golden hamsters Result Infected mice show symptoms from days 160 to 450 Infected hamsters show symptoms from days 70 to 200 Titer 107108 LD80 units/ml 108109 LD50 units/ml

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A guide to planning your Cleaning Validation Study

Table 6TSE assay systems.

Bioassay Advantages Disadvantages Measures both inactivation and removal Generally accepted by all regulatory agencies Good sensitivity Requires experimental animal use Involves greater time and expense Western blot Does not require the use of experimental animals Involves less time and expense Assay only measures removal Less likely to gain acceptance by regulatory agencies Lower sensitivity

Calculation methods: logarithmic reduction factor

The logarithmic reduction factor (RF) for each individual purification or inactivation step is calculated similar to the calculation methods employed in a viral inactivation study in accordance with statistical methods in the CPMP/BWP/268/95, Final, Note for Guidance on Virus Validation Studies: the Design, Contribution and Interpretation of Studies Validating the Inactivation and Removal of Viruses.

RF = log10

Input titer/Volume x Input volume Output titer/Volume x Output volume

An example: RF = 5.7 108 CFU/ml x 10 ml 102 CFU/ml x 20 ml

RF = log10

Cleaning validation study results

Data handling and record maintenance
In addition to high testing standards, BioReliance provides uncensored data reporting. The report will include raw data collation and calculation of the reduction factors for each cleaning agent. We have a dedicated scientific writing team with experience in cleaning validation studies. Unaudited results are faxed within 72 hours. The average turnaround time for a final, 100% audited report is 34 weeks. The final study report (one per test agent) will include results of the test for clearance of spiked microorganisms by the sponsors purification process material and a summary evaluation of the results. All important records are maintained including: microbial spiking records, sample records, media sterility and growth promotion records, process treatment records, batch records (where appropriate), microbial titration records, dilution records, inoculation records, and records of observations.

Delivering the study report

Customers have the services of a dedicated client liason/ project manager who will send biweekly status reports. Study directors also work directly with clients to discuss results and consult on scientific issues.

Conflikt, Decon-Cycle, Decon-Phene, Decon-Spore 100, and Decon-Spore 200 Plus are registered trademarks of Decon Laboratories; Cavicide is a registered trademark and Vespore is a trademark of Metrex Research Corporation; Exspor is a registered trademark of Alcide Corporation; LPH, Septihol and Spor-Klenz are registered trademarks of Steris, Inc.; Vesphene is a registered trademark of Vestal Laboratories.

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BioReliance Corp. 14920 Broschart Road Rockville, Maryland 20850 Tel: 800.553.5372 Fax: 301.610.2590 Email: [email protected] BioReliance Ltd. Todd Campus West of Scotland Science Park Glasgow, Scotland G20 0XA Tel: 44 (0) 141 946.9999 Fax: 44 (0) 141 946.0000 Email: [email protected] BioReliance Ltd. Innovation Park Hillfoots Road Stirling, Scotland FK9 4NF Tel: 44 (0) 141 946.9999 Fax: 44 (0) 141 946.0000 Email: [email protected] BioReliance, K.K. c/o Sigma-Aldrich Japan K.K. Tennoz Central Tower 4F 2-2-24 Higashi-Shinagawa Shinagawa-ku Tokyo 140-0002, Japan Tel: +81 (0)3 5796 7430 Fax: +81 (0)3 5796 7435 Email: [email protected] BioReliance Ltd. c/o Sigma-Aldrich Chemicals Pvt Ltd 102 Alpha Building Hiranandani Gardens Powai, Mumbai 400076 Tel: 91 22 40872364 Fax: +91 22 25797589 Email: [email protected]

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