6 Bioburden

Considerations in
Equipment-Cleaning
Validation

Equipment used in pharmaceutical and biopharmaceutical production is vul-

nerable to varying degrees of microbial contamination that originate from
many sources. An ineffective equipment-cleaning program can have many
negative consequences, including the risk to patient health and quality of prod-
uct manufactured that could in turn lead to product withdrawal from the mar-
ket. It is therefore not surprising that cleaning validation undergoes extensive
regulatory review in the pharmaceutical industry during inspections. In fact,
during the past few years, cleaning validation has ranked among the top 10
areas of concern in warning letters issued by the FDA. According to Kristen
Evans, leader of the Guidance and Policy Team in the Division of Manufactur-
ing and Product Quality in FDA’s Center for Drug Evaluation and Research,
equipment cleaning and maintenance ranked second in warning letters and
GMP citations for the fiscal year (FY) 2004–2005 [1]. Although not all clean-
ing issues relate to microbial contamination, controlling bioburden through
adequate cleaning processes is a regulatory expectation enforced by the FDA
as illustrated in the following excerpts from two regulatory citations:

• FDA 483 issued to Evans Vaccines (Merseyside, UK), an affiliate of Chi-

ron Corporation, regarding product sterility failure investigation in October
2004: “Cleaning validation for the CIP process for vessel … which is uti-
lized in the aseptic formulation of trivalent bulk influenza, did not include
an assessment of sprayball coverage for the vessel. In addition, the study
did not include swab sampling of the transfer lines used in the transfer
of monovalent blend pools into the mixing vessel … and for transferring
the aseptic trivalent formulated bulk back into a sterilized … liter tank in
formulation room.”
• FDA warning letter issued to MedImmune Inc. (Gaithersburg, MD) on
May 24, 2007, regarding bioburden deviations in the manufacture of bulk

139
140 Microbial Limit and Bioburden Tests

monovalent lots for FluMist vaccine during the 2006–2007 campaign: “Of
particular concern are your inadequate investigations into such excursions,
and your lack of implementation of appropriate corrective and preventative
actions, coupled with deficiencies in: aseptic practices by personnel, clean-
ing validation of equipment and effectiveness of the cleaning and disinfec-
tion processes used in your manufacturing facility and by your personnel.”

Microbial contamination is a multiparametric process, which is affected not

only by sources of contamination but also by the environmental conditions
and properties of contaminated materials (e.g., surface charge, hydrophobic-
ity, texture, etc.), which can contribute to microbial adhesion and proliferation.
Although sterilization and sanitization procedures for equipment are beyond
the scope of the FDA Guide to Inspections—Validation of Cleaning Processes,
the FDA does address in this document concerns regarding control of biobur-
den in process equipment. Indeed, the FDA expects companies to have written
procedures not only to prevent ingress of contamination but also to eradicate
or reduce bioburden through validated sterilization and cleaning procedures.
Ideally, production equipment used in the manufacture of drugs that will be
rendered sterile should be sterilized (e.g., steamed in place or autoclaved) to
prevent product contamination.

However, due to the equipment design and materials used in construction,

many large systems such as chromatography skids and columns cannot be
sterilized, and therefore must be cleaned in place using chemical sanitizer
solutions. In addition, although equipment used in nonsterile manufacturing
is not required to be sterile, bioload on equipment surfaces must be reduced to
acceptable levels that will ensure product quality and safety. In the Guide to
Inspections—Validation of Cleaning Processes, the FDA states that whether
or not clean-in-place (CIP) systems are used for cleaning of processing equip-
ment, microbiological considerations should be given to equipment-cleaning
protocols through either preventative measures or removal of contaminants.
The FDA also indicates that companies should provide evidence that routine
cleaning and storage of equipment do not allow microbial proliferation, issues
also addressed in the regulatory guidance documents: Good Manufacturing
Practices Guidelines # 02-122102-681 [2] and the Pharmaceutical Inspection
Convention/Pharmaceutical Inspection Cooperation Scheme (PIC/S) docu-
ment PE 009-1 [3].

In this chapter, the main regulatory concerns and expectations in terms of

bioburden and endotoxin as it relates to equipment-cleaning validation are
discussed. Although the topics of sterilization and chemical residues (e.g., to-
tal organic carbon [TOC]) are beyond the scope of this book, readers are
encouraged to become familiar with sterilization processes for pharmaceuti-
cal manufacturing equipment and TOC testing for verification of equipment
cleaning along with the applicable regulatory requirements and guidance
documents.
Bioburden Considerations in Equipment-Cleaning Validation 141

Equipment Used in
Manufacturing

Clean or Sterile Dirty Hold Time*

Hold Time*
(Ready for Use)

Cleaning/Sanitization*
(nonsterile equipment)

Sterilization*
(sterile equipment)

Cleaning*
(sterile equipment)

*Validated Process

Figure 6.1  Clean-and-use cycle of manufacturing equipment.

Biocontamination Control
Contamination can be defined as the presence of a substance (e.g., solid, liquid, gas-
eous, biological, or chemical) that is likely to negatively affect the safety or quality
of the product manufactured. Regarding microbial contaminants, risk considerations
include not only viable organisms detected by bioburden recovery methods but also
their residues, such as endotoxins, enterotoxins, and proteases. For equipment that
are not required to be sterile, cleanliness is not absolute. Acceptable levels of biobur-
den and bacterial endotoxins (e.g., applicable to equipment used in the manufacture
of parenteral and sterile nonpyrogenic inhalation products) are established by the
manufacturer based on the types of processes (e.g., dry formulation, bioprocess, ster-
ile), step in the process (e.g., upstream, downstream), and whether equipment can be
steamed in place or is chemically sanitized.
The clean-and-use cycle of pharmaceutical manufacturing equipment has vari-
ous stages that include validated processes for cleaning/sterilization of equipment and
validated hold steps (Figure 6.1). For a risk-based approach to validation of cleaning
processes, the greatest risks to microbial contamination of production equipment
are from initial ingress and storage (clean-hold time). Clean hold is defined as a
state in which the equipment is ready for use. Consideration should also be given to
the possibility of microbial proliferation during equipment dirty-hold time, which
is defined as the maximum qualified amount of time that the equipment can remain
soiled prior to cleaning.
Microbial contamination can be introduced into a piece of equipment (initial
ingress) via raw materials and excipients, during open connections as well as dur-
ing changeover procedures. In most cases, effective cleaning prior to use ensures
microbial control through the elimination or reduction of bioburden introduced into
142 Microbial Limit and Bioburden Tests

the equipment. However, bioburden can proliferate and form biofilms if the sanitiza-
tion and equipment storage solutions are not effective, and whenever the equipment
is not of sanitary design (e.g., with dead legs, inappropriate finishing materials, not
free draining, and with crevices). Indeed, microbiological cleanliness is attained not
only through control of bioburden ingress and equipment sanitization procedures
but also through

• Assurance of antimicrobial effectiveness of sanitization and storage solutions

• Use of sanitary equipment as specified in the 3-A Sanitary Standards
(www.3-a.org)
• Maintenance of clean conditions during clean-hold time
• Storage of equipment in dry conditions after cleaning whenever possible
• Use of cold storage for chromatography resins or packed columns and other
similar equipment/materials
• Extensive operator training and supervision to ensure strict adherence to
cGMPs procedures
• Cleaning the equipment as soon as possible following use
• Sanitizing cleaned equipment prior to use

Disposable and Single-Use Equipment

In the 21st century, many single-use and disposable pieces of equipment and materi-
als have been introduced in pharmaceutical manufacturing. In biopharmaceutical
production, disposable flasks, filtration systems, spinner bottles, and even bioreactors
have been used. The main advantages to using disposable equipment are of course
prevention of microbial contamination and elimination of the need for cleaning vali-
dation and its associated costs. Indeed, the use of disposable/single-use equipment
and materials is a trend in the pharmaceutical industry, and the regulatory agencies
are openly endorsing these technologies. However, there are still some issues that
must be addressed if a company chooses to employ single-use equipment, such as
scalability, compatibility with raw materials and products, the need for preuse clean-
ing/flushing, and concern with extractables (compounds that migrate from material
into solvents under extreme temperature and time exposure) and leachables (com-
pounds that migrate into the drug product under normal processing conditions).
Despite some of these concerns and, perhaps, some challenges posed by the pos-
sible unfamiliarity of regulatory inspectors with single-use technologies, the use of
disposable materials and equipment does and will provide great benefits to phar-
maceutical and biopharmaceutical companies in terms of biocontamination control
and prevention. Impact, if any, of both leachables and extractables from disposable
equipment can be addressed during process validation, and when carefully evalu-
ated, it is found that they rarely have a negative impact on the quality and safety of
products, and so their use is easily defendable.

Equipment-Cleaning Methods
Equipment-cleaning methods use chemical and/or physical means and they fall into
two main categories: clean-in-place (CIP) or steam-in-place (SIP), and clean-out-of-
Bioburden Considerations in Equipment-Cleaning Validation 143

place (manual). For chemical cleaning, CIP is preferred because it can be automated
and the equipment need not be disassembled. CIP systems, which can either be hard
piped to the production vessels or used as a mobile unit, are able to clean equipment
in situ. For sterilization of large pieces of equipment, such as production vessels,
companies use SIP cycles, whereas small pieces of equipment and materials are
autoclaved. Regardless of the type of method used, a validation study must be per-
formed to ensure the effectiveness and reproducibility of the cleaning/sterilization
procedures. This chapter will focus on the cleaning and sanitization of equipment
that are not required to be sterilized.
Chemical cleaning can be achieved via oxidation, hydrolysis, and enzymatic
action. Effective cleaning and sanitization procedures include, but are not limited
to, the following:

• Treatment with acids and bases

• High-velocity hot water, steam, solvent rinses
• Use of sanitizers and detergents
• Drying at high temperatures

The quality of the water used in the preparation of cleaning solutions and as a pre-
rinse or postrinse is critical. In general, for manufacturing nonsterile products, the
use of purified water to clean and rinse production equipment is suitable. For clean-
ing and rinsing equipment used in the production of sterile drugs and in downstream
processing (purification steps in biopharmaceutical production), the water used must
meet the water for injection (WFI) quality attributes.
In biopharmaceutical manufacturing, maintaining nonsterile equipment (e.g.,
packed columns) under microbial control is a challenge. Alkaline solutions, such as
sodium hydroxide (from 0.1 to 1.0 M solutions), are widely used as cleaning agents
and for regeneration and storage of chromatography media (carbohydrate and poly-
meric matrices) because alkaline solutions are particularly effective in removing
proteins and fermentation process residues. A 1 M sodium hydroxide (NaOH) solu-
tion is a good sanitizer and capable of reducing, but not totally inactivating, spores
of Bacillus subtilis when used at 22°C [4]. NaOH is also very effective in removing
fungi and endotoxins. Some cleaning solutions are supplemented with sodium hypo-
chlorite (about 400 ppm), salt (e.g., sodium chloride), or alcohol to combine cleaning
with sanitization. Although sodium hypochlorite is a well-known sanitizer, its use is
somewhat limited due to risk of damage to materials, the possibility of generating
toxic chlorinated by-products, and its impact on the chemical stability of chroma-
tography resins.
Peracetic acid, a strong oxidizer with sporicidal properties, offers some advan-
tages over sodium hypochlorite for chromatography column cleaning. Studies per-
formed indicated that a 22°C solution of peracetic acid (1500 ppm) supplemented
with 30% ethanol and in 0.5 M acetate buffer, at pH 5, showed a 90% reduction
(D10 value) in a population of B. subtilis spores within 0.4 min compared to a D10
value of about 860 min obtained for a 0.5 M NaOH solution at 4°C [5]. In addition,
there are safety benefits to the use of peracetic acid as this compound decomposes
to acetic acid, oxygen, and water. Acid–cleaner formulations combine at least one
144 Microbial Limit and Bioburden Tests

acid and a surfactant. These chemicals are effective in removing alkaline salt, some
sugars, and particulate matter residues. Enzymatic cleaners are substrate specific
and accelerate the breakdown of certain organic residues. Hydrolysis renders some
types of chemical residue soluble in water and helps their removal from surfaces via
a chemical reaction of an aqueous alkaline or acidic solution with an ester, ether, or
amide compound.
When selecting a cleaning agent, a company must evaluate its chemical compat-
ibility with the equipment and materials surfaces, ecological compatibility, chemical
toxicity, and contact time required. Other factors that must be taken into account
while selecting a cleaning method include

• Static immersion versus dynamic flow

• Need for mechanical cleaning
• Temperature of the cleaning agent
• Volume of the cleaning solution (e.g., from 1 to 10 column volumes)

Selection of a cleaning agent is critical because the chemical it contains is consid-

ered a contaminant to the process, and its removal and possible interference with
bioburden recovery must also be demonstrated during cleaning validation studies.

Validation of Cleaning Methods

Validation of cleaning methods is required to ensure that the equipment-cleaning
cycle consistently provides results that meet acceptable levels of cleanliness. Guide-
lines for cleaning requirements are provided to the pharmaceutical industry in the
cGMP documents in the United States, Europe, and other countries. Equipment-
cleaning requirements are also addressed in the PIC recommendations and the PDA
Technical Report No. 29, Points to Consider for Cleaning Validation [6]. Clean-
ing validation for microbial contamination and endotoxin residues involves testing
equipment surfaces to ensure that the cleaning methods reduce contamination to
preestablished acceptable levels. For bioburden analysis, validation is also needed
to establish an adequate storage condition for the dirty (dirty-hold time) and cleaned
(clean-hold time) equipment.
The first step in establishing a cleaning validation program is to define the
strategy to be used to ensure the efficacy and reproducibility of the cleaning pro-
cedures. Typically, a company starts the qualification studies with selected neu-
tral agents (e.g., hot water at high velocity) or with chemicals known to be least
harmful to the equipment. If initial test results fail, then other cleaning agents are
evaluated.
Cleaning validation should be implemented via a validation master plan that
can be drafted using guidelines provided in various regulatory documents for phar-
maceutical production, such as the ICH Q7, Good Manufacturing Practice Guid-
ance for Active Pharmaceutical Ingredients. As part of a cleaning validation master
plan, a company selects the sampling and detection methods, sets the acceptable
levels of contamination prior to protocol approval, executes the protocol, collects
and analyzes data, and writes procedures for cleaning verification and the training
Bioburden Considerations in Equipment-Cleaning Validation 145

of operators who clean the equipment. For manual cleaning methods, qualification/
validation of personnel must also be performed to demonstrate reproducibility in
cleaning results.
Some of the topics that address cleaning validation and are highlighted in the
ICH Q7 document include

• Cleaning validation should be performed for process steps in which con-

tamination or material carryover poses the greatest risk to product quality.
• Cleaning validation should reflect actual patterns for equipment usage.
• A company should use validated methods that have the sensitivity to detect
residues and contaminants.
• Equipment cleaning/sanitization studies should address microbiological
and endotoxin contaminations, as appropriate.
• Cleaning validation should include monitoring of equipment at appropri-
ate intervals to ensure that cleaning procedures are effective during rou-
tine production.
• Sampling should include swabbing, rinsing, or alternate methods, as
appropriate.

Sampling Recovery Methods

One of the main steps in equipment-cleaning validation is selecting the best residue
detection method. There are two primary types of sampling techniques widely used
in cleaning validation studies and during the routine monitoring of pharmaceutical
equipment and surfaces: direct surface sampling (swabbing) and rinsing (diluent or
placebo). There are advantages and disadvantages to both techniques as described in
Table 6.1. Many cleaning verification protocols combine both techniques, depending
on the type of equipment that needs to be sampled.
For bioburden recovery in cleaning validation studies, the focus is on recovery of
mesophilic aerobic microbes. For this purpose, TSA medium incubated at 30–35°C
is suitable. However, alternate media and incubation conditions may be required if
the detection of a specified microbial species is a concern.
Bacterial endotoxins are typically detected from swab and rinse samples using
the Limulus amebocyte lysate (LAL) method. If swabs are used, extraction methods
must be developed prior to processing the samples.

Swabbing of Equipment
Equipment swabbing must be performed by qualified personnel, and sterile swabs
made from materials that do not interfere with the test should be used. There
are various types of swabs used to monitor flat or hard-to-reach surfaces such as
the bottom of a tank, O-rings, traps, transfer lines, and U-bends. As mentioned
in Chapter 5, the QUANTISWAB® (bioMérieux Inc., Hazelwood, MO) has been
proven an excellent choice for bioburden recovery from surfaces. Swab sampling
should be carried out wearing sterile gloves to minimize adventitious contamina-
tion. For bioburden recovery, after swabbing is complete, the swab may be streaked
onto an agar medium or broken into a neutralizing diluent, vortexed for about 30
146 Microbial Limit and Bioburden Tests

Table 6.1
Comparison between the Two Primary Types of Sampling Techniques:
Swabbing and Rinsing
Swabbing Rinsing
Advantages Disadvantages Advantages Disadvantages
Sampling of hard-to- Some swabs may trap Ability to sample large Rinse solvent may not
reach areas microorganisms and surface area dislodge bioburden
Sampling of defined reduce recovery Sampling of areas not that has adhered to the
surface area Technique/operator accessible by swabs surface
Adds capability of dependent Ability to automate Water can lead to cell
physical removal May lead to (online monitoring) lysis and reduced
Economical adventitious and contain (less microbial recovery
Widely available contamination during exposure to Large dilution may
sampling environment) lower test sensitivity
Invasive technique Less intrusive/no need Inability to determine
Results subject to site to disassemble the exact location of
selection/assumes equipment contamination
uniform contamination Technique independent Difficult to control the
as compared to swabs area sampled

s, and the liquid sample preparation tested by pour-plate or membrane filtration

method. The incubation conditions for the recovery media vary depending on com-
pany protocols. However, in general, swab preparations are plated with TSA and
plates incubated at 30–35°C for 3–5 d. Results are reported as number of CFU
per swab or area sampled. If swabs are to be transported to the testing laboratory,
they need to be stored in a manner that preserves the samples collected as well as
prevents adventitious contamination.

Rinsing of Equipment
Equipment rinse is performed using a solvent that will not interfere with recovery
of residues. Sometimes placebo is used, although this approach is not typical for
collection of bioburden or bacterial endotoxin. Most rinse samples are collected
using purified water or WFI. For bioburden recovery, after the rinse sample is
collected, it is processed via the membrane filtration technique, the filter plated
onto TSA, and the agar plate incubated at 30–35°C for 3–5 d. The use of water for
equipment rinse may interfere with microbial recovery due to cell lysis. Although
this topic is not addressed in cleaning validation articles and reference docu-
ments, a company should include test controls in the validation recovery studies
to assess the possibility of low bioburden recovery due to loss of microbial viabil-
ity. Also, rinsing and swabbing (to a certain extent) are only partially effective
in removing cells from a multilayer biofilm. Companies must consider this fact
when analyzing equipment-cleaning data because microbial recovery methods
may only provide a semiquantitative indication of the microbial contamination
on equipment surfaces.
Bioburden Considerations in Equipment-Cleaning Validation 147

Qualification of Sampling Methods

Part of a cleaning validation study and typically the first protocol step executed is
the actual qualification of the test methodologies chosen to assess the microbiolog-
ical quality of equipment surfaces. One must demonstrate that the chosen method
is able to recover viable organisms and that any product or cleaning agent residue
will not interfere with their detection. One key point to consider is that the limit
of quantitation (LOQ) for the method developed must be equal to or below the
established cleaning validation limit for the given piece of equipment. Although
not discussed in detail in this chapter, the same principles apply to qualification
of methods to detect bacterial endotoxin on surfaces. However, due to difficulties
in recovering bacterial endotoxin from surfaces using swabs, rinsing is often the
method of choice.
Qualification of recovery methods is performed using clean and sterilized/sani-
tized coupons, which are small pieces of material representing the equipment to be
sampled (e.g., stainless steel, polypropylene, glass, silicone, etc.). The use of coupons
avoids the contamination of the actual equipment or manufacturing environment.
Bioburden recovery is determined as a percentage of the original amount of
microbial inoculum added to the coupons. This value can be affected by many fac-
tors such as the inherent variability of microbial recovery methods, desiccation of
vegetative cells, the type of swab used, the sampling technique, and the type of
equipment surface (regular versus irregular). Therefore, in order to ensure the cor-
rectness of test results during assessment of equipment cleaning, a study to deter-
mine the accuracy and reproducibility of the chosen recovery methods is required.
A bioburden recovery test is performed by inoculating sterile/sanitized coupons
with a known level of microorganisms (typically less than 100 CFU per coupon) and
sampling the surface as proposed in the cleaning validation protocol. The number
of recovered organisms is compared to the number of inoculated organisms and a
percent recovery rate is calculated. For chemical residues (including endotoxin), a
recovery greater than 80% is considered good, a recovery greater than 50% is con-
sidered reasonable, and a recovery value less than 50% is considered questionable
[7]. However, for bioburden, there is no set standard for an acceptable recovery from
surface sampling; the USP Chapter <1227> Validation of Microbial Recovery from
Pharmacopeial Articles is designed to show adequate neutralization for testing of
microbial limits in pharmaceutical products and raw materials, and does not apply to
bioburden surface recovery methods. In general, a standard practice in the industry,
and one recommended by the author, is to establish a minimum bioburden recovery
of 50%, a value that has been found acceptable by most regulatory investigators.
The test organisms often used to inoculate the coupons represent a wide spec-
trum of microbial flora typically found in a manufacturing environment and that
may be detrimental to pharmaceutical equipment and products (see Table 6.2). Other
isolates, including environmental strains, can be added to the test organism panel or
as a substitute for the American Type Culture Collection (ATCC) species at the dis-
cretion of the manufacturer. Organism suspensions can be prepared as described in
Chapter 7, or used directly from commercial preparations that have a predetermined
population range.
148 Microbial Limit and Bioburden Tests

Table 6.2
List of Test Organisms
Organism Name ATCC Number Organism Type
Staphylococcus aureus 6538 Gram-positive coccus
Bacillus subtilis 6633 Gram-positive spore-forming rod
Pseudomonas aeruginosa 9027 Nonfermenting, Gram-negative rod
Escherichia coli 8739 Fermenting, Gram-negative rod
Candida albicans 10231 Yeast
Aspergillus niger 16404 Filamentous fungus
Environmental isolates (company specific)
Others as needed (e.g., specified microbial
species)

Recovery Study Using the Wet Method

This procedure is performed for vegetative cells (bacteria and fungi) to prevent loss
of viability due to desiccation (Figures 6.2 and 6.3). Inoculate each type of coupon
with less than 100 CFU of the inoculum suspension prepared, for example, in 0.1%
peptone water. Ideally, the inoculum level should be between 25–100 CFU. An inoc-
ulum with less than 25 CFU may lead to greater than normal plate count variability,

Streak onto TSA

or Disperse in
diluent and plate
Add < 100 CFU of Contact time < 1 min. Remove inoculum w/TSA
vegetative cells w/dry swab
or Disperse in
diluent, filter, and
plate w/TSA
Incubate TSA plates at 30–35°C for 3–5 days
Compare recovery to average inoculum plate count

Figure 6.2  Swab recovery qualification using the wet method.

Rinse inoculum Collect rinse and filter

Add < 100 CFU of Contact time < 1 min. from coupon or plate w/TSA
vegetative cells

Incubate TSA plates at 30–35°C for 3–5 days

Compare recovery to average inoculum plate count

Figure 6.3  Rinse recovery qualification using the wet method.

Bioburden Considerations in Equipment-Cleaning Validation 149

thereby reducing the accuracy of the test. An inoculum count greater than 100 CFU
may not be considered a low-level inoculum; besides, it may lead to reduced accu-
racy during plate counting due to crowding effects.
Verify the inoculum level by the pour-plate or streak-plate method, in duplicate
or triplicate, using TSA medium and incubating test plates at 30–35°C for a mini-
mum of 2–3 d. After the liquid inoculum is added to the coupon surface, allow a
contact time of less than 1 min. Use either the swab or rinse method to recover the
test organisms:

• Swab method: Using a dry swab, remove the liquid inoculum from the sur-
face of the coupon. Streak the swab onto an agar medium or break it into
a neutralizing diluent, vortex for about 30 s, and process the liquid sample
preparation by either pour-plate or membrane filtration method. Use TSA
with incubation conditions at 30–35°C for 3–5 d. Results are reported as
number of CFU per swab.
• Rinse method: Place the inoculated coupon in an aliquot of sterile diluent
(e.g., purified water) contained in a sterile vessel, which will be used to
rinse the given piece of equipment. Shake well and then remove the cou-
pon aseptically. Attempts should be made to best mimic the rinse solution
temperature, contact times, and cleaning conditions (e.g., dynamic flow
versus static contact). Filter the rinse solution through a 0.45-μm mem-
brane filter. Then, aseptically remove the membrane filter and place it onto
a solidified TSA agar plate. Incubate the prepared plates at 30–35°C for
3–5 d. At the end of the incubation period, enumerate the recovered colo-
nies and report results.

Perform this procedure (swab and/or rinse sampling), in triplicate, for each chal-
lenge organism. Perform a test-negative control for each swab and rinse set to verify
aseptic manipulations by carrying out the procedure just described but with unin-
oculated coupons.
Average the counts obtained for the three swab and/or rinse sample preparations
and compare results to the average result obtained for respective inoculum count
plates. Calculate the percent recovery for each test organism. For example,

Average count for inoculated coupons: 35 CFU

Average count for inoculum level: 46 CFU
Percent recovery: (35 CFU/46 CFU) × 100 = 76%

Test acceptance criteria

• If values are below the established acceptance criterion (e.g., less than 50%),
the cause for the less-than-adequate recovery should be investigated and
eliminated prior to performing any retests. Modifications to the swab/rinse
technique to improve microbial recovery may include the use of an alter-
nate type of swab, or use of an alternate recovery method and/or medium.
150 Microbial Limit and Bioburden Tests

• If any of the inoculum level counts exceeds 100 CFU, the test should
be repeated.
• If microbial growth is recovered from the negative control samples, the test
should be repeated.

Recovery Studies Using the Dry Method

The dry method is to be used for spore-forming bacteria only because vegetative cells
suffer desiccation and, therefore, would not be viable on dry surfaces (Figures 6.4
and 6.5). Inoculate each type of coupon with less than 100 CFU of the spore prepara-
tion inoculum suspension. Allow the inoculum to evaporate to dryness under lami-
nar flow conditions.
Verify the spore inoculum level used by the pour-plate or streak-plate method, in
duplicate or triplicate, using TSA medium and incubating test plates at 30–35°C for
a minimum of 2–3 d. After the inoculum is added to the coupon surface and dried,
use either the swab or rinse method to recover the test organisms:

• Swab method: Using a wet swab, remove the dried inoculum from the sur-
face of the coupon. Streak the swab onto an agar medium or break it into
a neutralizing diluent, vortex for about 30 s, and process the liquid sample
preparation by either pour-plate or membrane filtration method. Use TSA
with incubation conditions at 30–35°C for 3–5 d. Results are reported as
number of CFU per swab.

Streak onto TSA

or Disperse in
diluent and plate
Add < 100 CFU of Allow to air dry Remove inoculum w/TSA
spore suspension w/wet swab
or Disperse in
diluent, filter and
plate w/TSA
Incubate TSA plates at 30–35°C for 3–5 days
Compare recovery to average inoculum plate count

Figure 6.4  Swab recovery qualification using the dry method.

Rinse inoculum Collect rinse and filter

Add < 100 CFU of Allow to air dry from coupon or plate w/TSA
spore suspension

Incubate TSA plates at 30–35°C for 3–5 days

Compare recovery to average inoculum plate count

Figure 6.5  Rinse recovery qualification using the dry method.

Bioburden Considerations in Equipment-Cleaning Validation 151

• Rinse method: Place the inoculated coupon in an aliquot of sterile diluent

(e.g., purified water) contained in a sterile vessel, which will be used to
rinse the given piece of equipment. Shake well and then remove the coupon
aseptically. Attempts should be made to best mimic the rinse solution tem-
perature, contact times, and cleaning conditions (e.g., dynamic flow versus
static contact). Filter the rinse solution through a 0.45-μm membrane filter.
Then, aseptically remove the filter and place it onto a TSA agar plate. Incu-
bate the prepared TSA plates at 30–35°C for 3–5 d. At the end of the incuba-
tion period, enumerate the recovered colonies and report results.

Perform this procedure (swab and/or rinse sampling), in triplicate, for each challenge
spore-forming bacterium. Perform a test-negative control for each swab and rinse set
to verify aseptic manipulations by carrying out the procedure just described but with
uninoculated coupons.
Average the counts obtained for the three swab and rinse sample preparations
and compare the results to the average of the result obtained for the respective inocu-
lum count plates. Calculate the percent recovery for each test organism as described
earlier in this chapter.
Test acceptance criteria

• If values are below the established acceptance criterion (e.g., less than
50%), the cause for the less-than-adequate recovery should be investi-
gated and eliminated prior to performing any retests. Modifications to
the swab/rinse technique to improve microbial recovery may include the
use of an alternate type of swab, or use of an alternate recovery method
and/or medium.
• If any of the inoculum level counts exceeds 100 CFU, the test should
be repeated.
• If microbial growth is recovered from the negative control samples, the test
should be repeated.

Effects of Product and/or Cleaning Agent Residue

on the Recovery of Microorganisms

The purpose of this qualification study is to evaluate any interference from prod-
uct and/or cleaning agent residue on the recovery of microorganisms (Figures 6.6
and 6.7). As such, this study demonstrates that the chosen medium/diluent ensures
adequate neutralization of any chemical inhibitory effects. This study should be per-
formed prior to the execution of the cleaning validation protocol and after the deter-
mination of a suitable recovery method.
In order to determine whether product residue might interfere with the recovery
of test organisms, a solution of the product is prepared (in an appropriate sterile dilu-
ent) at the maximum allowable concentration level for a residue left on the equipment
surface after the cleaning procedure. Coupons are immersed in and coated with the
prepared product dilution, aseptically removed, and allowed to dry under laminar
flow conditions. In order to evaluate any interference from the cleaning agent on
152 Microbial Limit and Bioburden Tests

Vegetative cells;
contact time < 1 min.
Streak onto TSA
Remove
inoculum or Disperse in
w/dry diluent and plate
Coat coupon with Allow to air dry Add < 100 CFU swab w/TSA
product/cleaning of inoculum
agent (max. residue
concentration) or Disperse in
Spore-formers, Remove diluent, filter and
allow to air dry inoculum plate w/TSA
w/wet
swab
Incubate TSA plates at 30–35°C for 3–5 days
Compare recovery from test samples (coated coupons) to average
recovery from positive control (same procedure but using uncoated
coupons)
Compare recovery from positive controls to average inocula counts

Figure 6.6  Product/cleaning agent residue interference study using the swab method.

Vegetative cells;
contact time < 1 min.

Rinse inoculum from

coupon
Coat coupon with Allow to air dry Add < 100 CFU
product/cleaning of inoculum Collect rinse and filter
agent (max. residue or plate w/TSA
concentration)
Spore-formers,
allow to air dry

Incubate TSA plates at 30–35°C for 3–5 days

Compare recovery from test samples (coated coupons) to average
recovery from positive control (same procedure but using uncoated
coupons)
Compare recovery from positive controls to average inocula counts

Figure 6.7  Product/cleaning agent residue interference study using the rinse method.

microbial recovery, a separate set of coupons is immersed and coated with a solution
of the cleaning/sanitizer agent prepared at the maximum allowable concentration
level for a residue left on the equipment surface after the cleaning procedure. In case
it is known or suspected that the combined product/cleaning agent residue may have
an additional adverse effect in the recovery of microorganisms, it is recommended
that the test be performed using a mixture of the two solutions.
After the coupons are coated with product or cleaning solution and dried, each
coupon is inoculated with less than 100 CFU of each of the test organisms. Perform
the procedure, in triplicate, for each type of coupon and challenge organism. For
vegetative cells, process the test preparations within 1 min of inoculation. For spore-
forming bacteria, allow inocula to dry under laminar flow conditions.
Prepare test-positive controls by inoculating untreated (uncoated) coupons with
less than 100 CFU of each of the test organisms, as described earlier in this chapter.
Verify the inoculum level by the pour-plate or streak-plate method, in duplicate or
triplicate, using the TSA medium and incubating test plates at 30–35°C for a mini-
mum of 2–3 d. Perform a test-negative control for each swab and rinse set to verify
aseptic manipulations by carrying out the procedure just described but using unin-
oculated and uncoated coupons.
Bioburden Considerations in Equipment-Cleaning Validation 153

To recover the microorganisms from the test coupons (coated) and positive con-
trol coupons (uncoated), use the swab and/or rinse methods by either the wet method
or dry method approaches, as applicable. Depending on the type of product and
cleaning agent used, addition of appropriate and specific chemical neutralizers to the
recovery medium and/or rinse solution may be required. Test plates are incubated
at 30–35°C for 3–5 d. At the end of the incubation period, enumerate the recovered
colonies and report results. Average the counts obtained for the three swab and rinse
product/cleaning agent–coated coupon preparations, and compare the results to the
average counts obtained for the respective test-positive controls (uncoated coupons).
Calculate the percent recovery for each test organism.
Test acceptance criteria

• If values are below the established acceptance criterion (e.g., less than
50%), the cause for the less-than-adequate recovery should be investigated
and eliminated prior to performing any retests. The use of an alternate
recovery medium, to include the use of appropriate chemical neutralizers,
and/or incubation conditions may be required to reduce product/cleaning
agent interference in microbial recovery.
• If any of the inoculum level counts exceeds 100 CFU, the test should
be repeated.
• If microbial growth is recovered from the negative control samples, the test
should be repeated.

Establishing Limits
As discussed earlier in this chapter, acceptance criteria for bioburden and endotoxin
should be calculated and justified as a function of the nature of the product manufac-
tured and the stage of the process in which equipment is used (e.g., upstream versus
downstream processing). For bioburden, in addition to numerical values, require-
ment for absence of given microbial species may be needed based on the type of
product that comes in contact with the equipment. According to the FDA Guide to
Inspections—Validation of Cleaning Processes, it is impractical for the FDA to set
acceptance specifications or methods for determining whether a cleaning process is
valid or not. However, the FDA states that limits for cleaning validation should be
logical, practical, achievable, verifiable, and should be based on scientifically sound
methodologies. There are a few guidance documents that provide recommendations
for establishing limits for chemical residues. The Cleaning Validation Guidelines
published by Health Canada (last revised June 18, 2002) is one such document. As
far as chemical residue is concerned, it is standard industry practice to accept the
following limits as proof of validation of a cleaning process:

• Not more than (NMT) 10 ppm detected by analytical methods such as TOC.
• NMT 1/1000 of the normal therapeutic dose.
• No visible residue.
• For certain allergens (e.g., penicillins, steroids, and cytotoxic materials),
the set limit is often established below the limit of detection of the best
available method.
154 Microbial Limit and Bioburden Tests

The acceptable limit for bacterial endotoxins is generally set only for equipment
used in the manufacture of parenteral and inhalation products. For rinse samples,
limits are often set based on the WFI specification (i.e., <0.25 EU/mL) or based
on safety requirements for the product to be manufactured. Bacterial endotoxin is
difficult to measure from surfaces using swabs and, therefore, there is typically no
general limit guideline for this type of test.
There are a few approaches used by companies to establish bioburden limits
for equipment cleaning, all acceptable by the regulatory agencies as long as there
is good scientific rationale to justify the chosen values. For rinse samples, the most
common approach is to use the recommended bioburden limits for pharmaceuti-
cal-grade waters as the minimum requirement. For example, for equipment cleaned
with purified water, the limit is set at NMT 100 CFU/mL, and for equipment cleaned
with WFI the limit is set at NMT 10 CFU/100 mL or <1 CFU/10 mL. For surface
sampling of nonsterile equipment, it is an industry practice to accept a limit in the
range of 0–10 CFU/25 cm2.
Another approach used for nonsterile product manufacturing is to apply the
“next product” bioburden specification or the proposed compendial bioburden lev-
els for nonsterile pharmaceuticals for the “next product” (see USP Chapter <1111>
Microbiological Examination of Nonsterile Products: Acceptance Criteria for Phar-
maceutical Preparations and Substances for Pharmaceutical Use) to calculate the
surface bioburden limit for cleaned equipment used in the manufacture of the “prior
product.” The following is an example calculation to illustrate this approach.

Example

Next product: Topical Formulation

Bioburden specification: NMT 100 CFU/g
Equipment surface area: 200,000 cm2
Batch size: 100 kg
Rinse volume: 1,000 L

Calculations for CFU/cm2 (swab):

100 CFU/g × 100 kg × 1000 g (conversion factor)

200,000 cm 2

Acceptable number of CFU/cm2 of equipment = 50 CFU/cm2

Calculations for CFU/mL (rinse):

surface limit (swab) × total surface area

Rinse Volume

Where rinse volume is determined by volume used and not collected rinse
volume
Bioburden Considerations in Equipment-Cleaning Validation 155

Calculations for CFU/mL (rinse):

50 CFU/cm 2 × 200, 000 cm 2

1, 000 L × 1, 000 mL (conversion factor)

Acceptable number of CFU/mL (rinse): 10 CFU/mL

Execution of Equipment-Cleaning Validation Protocol

Once acceptance criteria have been established, recovery studies completed, and a
cleaning validation protocol created and approved, a company is ready to execute the
validation protocol for equipment cleaning. Sampling of equipment for verification
of cleaning is performed after the soiled equipment (e.g., after production or soiled
with product or another material for the study) has undergone the cleaning cycle.
Typically, three runs are performed to show reproducibility of the cleaning methods,
but results should be evaluated separately and not averaged. Some studies include
sampling of equipment before and after cleaning/sanitization for an evaluation of
cleaning effectiveness. During validation studies, it is also important to identify rep-
resentative microorganisms isolated to establish a baseline for trending purposes
and to assist during investigations into future events of bioburden recoveries from
manufacturing equipment.
Results obtained from rinse and/or swab samples from all the three runs are
evaluated against established limits. If the cleaning method fails to meet protocol
acceptance criteria, modifications to the cleaning procedure are needed, and the
cleaning validation study must be repeated.

Validation of Cleaned Equipment Hold Time

A cleaning validation protocol should include validation studies to establish a cleaned
equipment hold time (CEHT) defined as the elapsed time from end of equipment
cleaning until the time equipment is used again. Surfaces that have been cleaned and
sanitized are always at risk of being recontaminated if the appropriate precautions
and protocols are not followed. Therefore, this type of study will focus on the condi-
tion of the equipment at the beginning of storage (e.g., dry or wet), how, where, and
for how long the equipment will be stored. In addition, the protocol must address the
sampling locations based on potential routes of contamination during storage. These
sampling sites may or may not be the same ones sampled during the original equip-
ment-cleaning validation process.
A typical protocol for CEHT study includes sampling of clean equipment
(rinse and/or swab) at the beginning of storage (baseline) and again at the end of
the proposed storage period, using validated recovery methods. This study can be
performed as part of the equipment-cleaning validation protocol or as a separate
protocol, depending on the manufacturing schedule flexibility, to avoid delays in
completing the cleaning validation protocol. Acceptance criteria for bioburden and
bacterial endotoxin levels at the end of the storage period must meet established lim-
156 Microbial Limit and Bioburden Tests

its for equipment cleaning. Further, for bioburden levels, most protocols require that,
at the end of the storage period, the bioload does not increase by more than 0.5 log
(0.3 log harmonized) from the initial bioburden (baseline).

Example Calculation (using 0.5 log variability)

Recovery for initial bioburden (or control): 63 CFU

Log10 63 = 1.7993
Upper limit = antilog10 (1.7993 + 0.5) = 199
Lower limit = antilog10 (1.7993 − 0.5) = 19.9, rounds to 20
Log variability: 20 to 199 CFU

The recovery for the test sample at the end of the hold time is 57 CFU. Therefore,
the test passes as 57 CFU is less than 199 CFU. Data generated from a CEHT study
provide supporting documentation for the storage of cleaned equipment under the
evaluated environmental conditions and for the given maximum time period.

Validation of Dirty Equipment Hold Time

Another typical study performed as part of equipment-cleaning validation estab-
lishes the maximum allowed time that a piece of equipment can remain soiled prior
to being cleaned. It is critical that, during this time frame, the soil remain in a phys-
ical condition that allows easy removal and no additional bioburden or bacterial
endotoxin be generated above and beyond the capabilities of the cleaning and sani-
tization methods.
A typical protocol for dirty equipment hold time (DEHT) study includes sam-
pling of cleaned equipment (rinse and/or swab) following the proposed storage
period/dirty hold time. As a test control, sampling is also performed after the equip-
ment is cleaned immediately following production or soiling. As with a CEHT study,
the DEHT study can be performed as part of the equipment-cleaning validation pro-
tocol or as a separate protocol, depending on the manufacturing schedule flexibil-
ity, to prevent delays in completing the validation protocol. Acceptance criteria for
bioburden and bacterial endotoxin levels for equipment cleaned at the end of the
dirty hold period must meet established limits for cleaned equipment. Data gener-
ated from a DEHT study provide supporting documentation for the storage of soiled
equipment under the given environmental conditions and for the given maximum
time period prior to cleaning.

OnGoing Verification of Cleaning

Once validation efforts are complete, and results have established the effectiveness
of the cleaning and sanitization procedures, a microbiological control program for
manufacturing equipment must be implemented along with a program for clean-
ing validation maintenance. Key considerations to ensure continued validity of the
cleaning procedures include
Bioburden Considerations in Equipment-Cleaning Validation 157

• Need for additional validation work when new products are introduced in
the plant
• Retraining of cleaning and equipment sampling personnel
• Need for revalidation whenever changes to the cleaning procedures are
implemented
• Need for ongoing verification of cleaning

The cGMPs regulations require that companies perform some type of verification
of cleaning prior to equipment use. In most cases, this type of cleaning verification
includes visual inspection and limited chemical testing. However, depending on the
type of equipment or process, additional testing including bioburden and endotoxin
testing may be warranted. One such example is following area changeover that takes
place between campaigns of different products. During a changeover procedure,
equipment soft parts are changed and the reassembled equipment is cleaned and
sanitized. Because this type of activity is conducive to introduction of bioburden into
the equipment, most companies choose to collect bioburden and endotoxin (if appli-
cable) samples after the cleaning cycle. The FDA also recommends taking biobur-
den rinse samples of chromatography columns prior to use to monitor for potential
biofilm formation.

Validation of Holding Time/Shipping Conditions

The purpose of this study is to check that microorganisms that are viable at the time
of swabbing/rinsing remain viable and stable until testing occurs in the laboratory.
This study is necessary only if there is a significant lag time (e.g., more than 8 h
under refrigeration) between sampling and processing of swabs or rinse aliquots.
This test is performed by inoculating the tip of the chosen type of swab,
prewetted in the chosen holding/transport medium, with less than 100 CFU of one
of the challenge organisms. Then, the swab is placed in a sterile tube containing
the chosen sterile holding/transport medium. If rinse technique is the method of
choice, inoculate 10-mL aliquots of rinse diluent contained in a sterile container
with less than 100 CFU of one of the challenge organisms. Prepare three repli-
cates for each of the challenge organisms. Include one uninoculated swab plus
holding/transport medium or aliquot of rinse diluent, to serve as a negative con-
trol. Prepare triplicate test-positive controls for each test organism by inoculating
replicate test tubes containing 10 mL of 0.1% peptone water or sterile saline solu-
tion with less than 100 CFU of the test inocula. Hold all sample preparations in a
controlled environment that simulates the proposed holding/shipping conditions,
such as the following:

• Refrigerated (2–8°C) for 8, 24, 36, or 48 h

• Room temperature (15–30°C) for 8, 24, 36, or 48 h

After the desired holding time is achieved, test the inoculated swabs and/or rinse
sample preparations (as described earlier in this chapter), by the pour-plate, streak-
plate, or membrane filtration method.
158 Microbial Limit and Bioburden Tests

Perform inocula counts by plating the same inocula used in the test to verify that
the initial microbial challenge was within the target range; plate selected dilutions of
the inocula, in duplicate or triplicate, with TSA and incubate at 30–35°C for 2–3 d.
At the end of the incubation period, enumerate the recovered microbial colonies and
calculate the average number of CFU. Compare the average number of recovered
colonies from the inoculated test samples with the average number of recovered col-
onies obtained for the test-positive controls. In addition, compare the average micro-
bial recovery from the test-positive controls to the average inoculum plate count for
the given test organism. The study acceptance criteria are as follows:

• No microbial growth is obtained for the negative control preparations.

• The average number of microorganisms recovered from the inoculated
swabs or rinse samples should be within 0.5 log (0.3 log harmonized) of the
average counts obtained for the test controls.
• The average number of microorganisms recovered from the test-positive
control preparations should be within 0.5 log (0.3 log harmonized) of the
inocula plate counts for the given test organism.

Refer to page 156 for an example calculation to determine if result is within 0.5 log
variability of control. If the sample preparation and/or test control recoveries fail to
meet the proposed test acceptance criteria, there is a possibility that the microbial
populations are not stable and that the challenge organisms may be multiplying or
losing viability under the holding/shipping conditions. In such cases, a laboratory
investigation should be performed in order to improve the proposed sample hold
conditions to ensure accuracy of the test data. A different combination of swab/hold-
ing medium or transport system for the rinse samples may be needed to overcome
loss of microbial viability or to prevent microbial proliferation under the given sam-
ple holding/shipping conditions.

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(PIC/S), Geneva.
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