See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/320161667

Microbiological Aspects of Cleaning Validation

Article · September 2017

CITATION READS

1 11,698

1 author:

Tim Sandle
The University of Manchester
755 PUBLICATIONS 1,535 CITATIONS

SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Endotoxin View project

Microbiological testing View project

All content following this page was uploaded by Tim Sandle on 12 November 2019.

The user has requested enhancement of the downloaded file.

Microbiological Aspects of
Cleaning Validation
By
Tim Sandle

Sep 26, 2017 7:00 am PDT

Introduction

Cleaning validation refers to the methodology applied to give the assurance that a cleaning
process has removed residues and contaminants from a piece of equipment or machinery (1).
Residues may be microorganisms; active pharmaceutical ingredients; other process chemicals,
such as buffers; cleaning agents themselves (such as detergents); or microbiological culture
media (in relation to aseptic process simulations). Assurance relating to cleaning validation
refers to both the effectiveness of the cleaning process and the consistency at which cleaning is
applied. As with any other type of validation, the ‘validation’ aspect refers to providing
documented evidence that that the acceptance criteria have been met.

Cleaning is assessed based on the level of residues that remain, either those directly found on
the equipment or those indirectly contained within the final rinse after water has passed through
or over the equipment. Whether the residues remaining have been reduced to a satisfactory low
level is based on predetermined acceptance criteria (2). The levels need to be sufficient low to
ensure that the next product manufactured is not compromised by waste or contamination from
the previous product (that is in ensuring that cross-contamination does not occur). An additional
concern is with the microbial bioburden.

With equipment cleaning, this can be undertaken using an automated process (such as CIP -
Clean-in-Place technologies) or manually (which often involves the physical removal of the
equipment and its transport to a wash-bay; sometimes called COP - Clean-Out-of-Place). In
general manual cleaning should be avoided due to process and operator variability. Where
manual cleaning cannot be avoided, due to technical limitations, robust controls need to be in
place to ensure consistency.

The chemical verification of cleaning validation is relatively well described. What is often less
clear, and some contentious in terms of whether it needs to always be included, is the
microbiological aspect. Important to the microbiological aspect are microorganisms themselves
(a direct hazard) and the presence of residues that potentially provide a microbial growth source,
should contamination be present or contamination occur during the hold period (an indirect
hazard).

To evaluate these microbiological risks a sound microbiological sampling plan is required. The
emphasis on sampling is important since microorganisms cannot be introduced into the process.
This is unlike a chemical assessment where equipment can be deliberately soiled with a residue
to test out cleaning efficacy. Microbial controls should not be introduced into the cleaning
process. This article assesses the risks from microorganisms and the cleaning requirements
necessary to achieve microbial control.

GMPs for Cleaning Validation

 Cleaning validation is an essential requirement for the manufacture of pharmaceutical products.
In the U.S. the necessity is set out in the Code of Federal Regulations (CFR). In particular:

• PART 111--CURRENT GOOD MANUFACTURING PRACTICE INMANUFACTURING,

PACKAGING, LABELING, OR HOLDING OPERATIONS FOR DIETARY SUPPLEMENTS

§ 111.27(d) You must maintain, clean, and sanitize, as necessary, all equipment, utensils, and
any other contact surfaces used to manufacture, package, label, or hold
components or dietary supplements.

• PART 211 -- CURRENT GOOD MANUFACTURING PRACTICE FOR FINISHED

PHARMACEUTICALS

§ 211.67 Equipment cleaning and maintenance

• PART 820—QUALITY SYSTEM REGULATION

§ 820.70(e) Contamination control. Each manufacturer shall establish and maintain procedures
to prevent contamination of equipment or product by substances that could reasonably
be expected to have an adverse effect on product quality

Both the U.S. Food and Drug Administration (3) and European Medicines Agency, together with
PIC/S (4), have guidances in place for cleaning validation. These are designed to prevent cross-
contamination; to ensure product quality is maintained and to minimize patient risk. The
guidances begin with the need to have written procedures describing how the validation will be
approached as well as clarifying who is responsible for undertaking the exercise (5). Within the
protocol the methods of evaluation; the acceptance criteria; and number of runs must be
specified. There is no regulatory guidance relating to the number of runs; however, by
convention, three runs are normally performed in order to demonstrate that the obtained results
are not due to chance. Performing three runs assumes that the process is consistent; other
variables, such as different types of product, might require greater than three runs to be
performed. The methods selected for each stage of the evaluation must themselves be validated
(6).

A note should also be added, to the documentation, about when revalidation is required and the
time interval between the initial validation and the revalidation exercise. With acceptance criteria
the major international regulators do not set the acceptance criteria and instead expect the user
to define these based on risk assessment. Microbiological acceptance criteria are discussed
below.

When new equipment is considered for purchasing, as part of the evaluation into the equipment
the cleaning validation requirements must be considered and the expected outcomes from
cleaning validation included in the User Requirement Specification (URS).

Types of Cleaning Validation

The processes that form part of cleaning validation have an impact upon microorganisms
remaining post-cleaning and on microbial survival. The microbiologist should have input into and
some control over the cleaning agents used and the suitability of the utilities (such as water and
compressed air, as well as the state of the controlled environment or cleanroom within which
equipment is held or processed). With cleanrooms, humidity control is recommended for areas
where equipment is held and stored in order to reduce the level of moisture (given that moist
environments support the survival and growth of many microorganisms and there is a particular
association with Gram-negative bacteria and fungi).
 Cleaning Chemicals

The types of cleaning agents selected will have different affects upon microorganisms
depending on the nature of chemical and the species (and numbers) of microorganisms present
on a given surface. These cleaning agents, which are typically one caustic based agent (sodium
hydroxide) and one acid based reagent (such as hydrochloric acid), should be included as part of
the cleaning validation. Such agents are not typically subject to disinfectant efficacy studies,
which means that the inclusion in the cleaning validation is the primary way to evaluate their
effectiveness.

In general, the following agents will exert some level of microbial inactivation or aid microbial
disassociation and removal; however, in situ effectiveness is dependent upon factors like
concentration, temperature and exposure time:

• Caustic

Caustic is a general term relating to the corrosive nature of a range of chemicals. The term is
best avoided and reference should be made to the specific chemical. Typically sodium hydroxide
is used in cleaning validation. An alternative chemical for cleaning validation is potassium
hydroxide. Sodium hydroxide is an alkylating agent (an alkali of sodium). In processing, sodium
hydroxide is added to water, heated, and then used to clean process equipment, storage tanks,
etc. It can dissolve grease, oils, fats and protein-based deposits. Potassium hydroxide is a
colorless solid is a prototypical strong base; it is similar to sodium hydroxide, ceteris paribus.

These chemicals can destroy microorganisms through oxidation and they can exhibit a
depyrogenating effect by destroying bacterial lipopolysaccharide provide that there is sufficient
contact time.

• Acids

Acids used in cleaning validation include hydrochloric acid and citric acid. These acids are used
for decaling and form removing minerals. At the right concentrations and with a sufficient
exposure, acids can destroy microorganisms.

• Chelants such as EDTA

These agents help to prevent deposition from ionic substances found in scale (like calcium
deposits from hard water). Here a chelant is a specialized molecule designed to bind to positively
charged metal ions, most commonly calcium and magnesium, in solution and thereby prevent
these ions from forming insoluble precipitates with other ions that may be present. An example is
ethylenediaminetetraacetic acid (EDTA).

• Surfactants

Surfactants used in cleaning include sodium lauryl sulfate and sodium dodecyl benzene sulfate.
Such agents increase dispersion and emulsifying soil. They can help to disassociate
microorganisms from surfaces (the complexities of microbial attachment are discussed below).

• Solvents

Solvents help to remove organic deposits. As organic deposits are removed, microorganisms can
be displaced from surfaces.
 Many commercial cleaning agents are formulated cleaning (in addition to water) with a
surfactant, an alkalinity source (such as sodium hydroxide), and a chelant (7).

Importantly the prime use of many cleaning agents is to remove chemical residues; that some
have antimicrobial properties is useful, but the chemicals should not be seen as disinfecting in
the sense that disinfection is defined as the known reduction of a population of microorganisms
as demonstrated through controlled laboratory studies.

As well as the chemicals themselves other aspects of the cleaning process can prove hostile to
microbial survival. These include the temperature of the water used for cleaning,s
(especially where temperatures are above 60oC) and pH ranges below 4 and above 11.

Water Rinses

Water rinses will remove cleaning chemicals and will siphon away any microorganisms in the
planktonic state (free-floating organisms). The final water rinse, due to the chemical and
microbiological 'purity' of the water, is Water of Injections (this is for all types of pharmaceutical
products). An important point following rinsing is that equipment should not be left with residual
water after cleaning. The last step of the cleaning procedure involve drying, perhaps with the
addition of a solvent (such as 70% sterile isopropyl alcohol) or flushing with sterile compressed
air; hence ensuring that there is no opportunity for microbial growth.

Critical process parameters and critical quality attributes

Based on the above, suitable critical process parameters for cleaning validation are:

• Time (dirty and clean hold times;, process run rime).

• Activity (chemical exposure, number of water rinses etc).
• Chemical (concentration).
• Temperature (cleaning temperature).

Critical process parameters are key variables affecting the production process. The parameters
are attributes that are monitored to detect deviations in standardized production operations and
product output quality or changes in critical quality attributes.

And appropriate critical quality attributes arecan include:

• Water quality.
• Type of soil.
• Nature of the soil.
• Surface material and surface quality.

In addition, for manual cleaning processes checks should be made in relation to number of rinses
or rinse times and periodic assessments of how operators are performing manual cleaning steps,
such as wiping or brushing.

Critical quality attributes are chemical, physical, biological and microbiological attributes that can
be defined, measuredassessed, and controlled. Some, such as water quality and the types of
products that are run on the equipment (and which produce soil) can be continually monitored to
ensure final product outputs remain within acceptable quality limits. Others, such as surface
material and quality should form part of regular equipment inspections. The attributes are an
essential aspect of a manufacturing control strategy and should be identified early in process
validation and design.

These factors should be accounted for in the validation exercise.

Risk Assessment

The level of cleaning required relates to the stage in the process that the equipment is used for
and the acceptable level of remaining microbial contamination (if any). With stages of
manufacture, equipment used with products at early stage in the process chain generally
requires lower levels of cleaning (compared with equipment used for product that is at an
intermediate or final stage). Moreover, levels of cleaning need to greater if the cleaning is to be
followed by sanitization (by chemicals with proven disinfectant properties, like sodium
hypochlorite; or by steam) or sterilization (as would be required in relation to sterile products
manufacture). This draw in a sterile products manufacturing and non-sterile products
manufacturing divide and the extent of remaining microorganisms. The permitted levels for
equipment used in non-sterile processing require a separate risk assessment and limits setting
compared with equipment used for sterile processing. These nuances are drawn out in this
section.

Another factor to assess is the type of soil and whether it will encourage microbial proliferation or
not. Product residues where the product contains a preservative (such as the manufacture of a
non-sterile ointment) will probably be a lower risk than a proteinaceous product.

An associated risk is what happens if microorganisms remain when the equipment is used for the
next stage of processing. Moreover, different microorganisms will interact with different residues
in terms of surface attachment. Here such a risk is difficult to determine, leading to the broad
aim of reducing microbial contamination down as far as possible.
Cleaning validation should be approached using a risk assessment schema whereby hazards are
identified and risk assessed, based on the severity of the hazard and the likelihood that the
hazard will occur (8). Microorganisms represent a hazard. Microbial hazards can be broken down
into:

a) Microbial contamination on the equipment post-use (and pre-cleaning);

b) The effects of hold time prior to cleaning (in relation to microbial proliferation and the release
of endotoxin);

c) Additional microbial challenges from the storage environment;

d) The efficacy of the cleaning process to remove microorganisms and endotoxin;

e) Storage of the equipment post-cleaning prior to use or a subsequent sanitization or

sterilization step (in relation to recontamination from the environment).

Each of these scenarios should be subject to a risk assessment, to which needs to be added is
the degree of severity should a level of microorganisms be present and the likelihood of
microorganisms still being present after a cleaning or storage step. Likelihood is affected by the
equipment design and easiness of cleaning. Factors that might help to inform such a risk
assessment are presented in this article.

In terms of risk assessment methodology, Hazard Analysis and Critical Control Points (HACCP)
is perhaps the most useful. A general schematic for HACCP, and which will be useful when
reviewing cleaning validation, is:
Where:

Figure 1: HACCP approach for assessing microbial risks

Microbiological Tests to Assess Cleaning Effectiveness

With microbiological testing a combination approach is often best given that different methods
will detect different types of contamination. The two board method groupings are direct sampling
methods, which will indicate the level of contamination remaining on surfaces. These are
particularly useful when orientated to areas that are hardest to clean. The second grouping is
indirect sampling methods in the form of rinse samples. Indirect methods show that the final rinse
applied (normally Water-for-Injections) is not eluting contamination, so that the final rinse signals
that a sufficient number of rinses have been conducted so that contamination is no longer being
drawn away from the surface. Where bacterial endotoxins are a concern, testing rinse samples
remains the only practical way to measure endotoxin content.
 When samples are taken, the person carrying out the sampling should be appropriately trained
and gowned appropriately according to the area within which the samples are taken (for
example, within a cleanroom the sampler should wear a cleanroom suit, gloves and a facemask).

Microbiological tests fall into the following broad areas (3):

1. Direct Surface Sampling.

This is either undertaken using swabs or contact plates. Of the two methods, contact plates tend
to achieve higher microbial recoveries (9). Contact plates can also be directly quantifiable to a
set surface area (based on the surface area of the agar, which is 24 to 25cm2). A swab can be
given the same connection to surface area should a template be used; however, if a square
template can be fitted onto a flat surface then why not use the superior contact plate? Swabs
also have more variability in that the wetness of the swab tip is hard to control and a the
standardization in terms of the number of strokes is less easier to control than the use of a
contact plate held within an applicator that can control time and pressure.

Where swabs have an advantage is with sampling equipment that cannot be sampled with a
contact plate due to the design of the surface, such being curved or cylindrical. With the contact
plate and the swab, the results obtained from the sampling provide an assessment of the
contamination per area of surface sampled.

Both the contact plate and swab methods selected should be validated in advance of the study to
show that the methods are capable of recovering a significant proportion of the contamination
from the surface . With this there is no industry standard acceptance criteria; what is important is
to establish the levels of organisms that can be consistently recovered from a representative
range of different surfaces. There are measures that can be taken to maximize recovery, such as
the use of contact plate applicators (which control time and pressure) and with types of swabs
(where flocked swabs give a superior recovery to plain tipped swabs).

Here no method will recover all of the contamination; nevertheless, the microbiologist should be
aware of the limitations of the method and understand what proportion is being recovered. In
addition, the culture media used and incubation temperatures and times should be qualified in
terms of promoting optimal recovery. Studies should be run in house due to each facility being
different; for comparison, studies should be run using reference cultures in order to allow inter-
laboratory qualifications to be compared.

Given that both methods are technique dependent, only operators trained in environmental
monitoring should undertake the sampling. Furthermore, in the event that any cleaning chemicals
remain on the surface, agars used for contact plates and for swabbing out should contain an
appropriate broad spectrum disinfectant neutralizer in order to ensure the validity of the test. A
full assessment of the neutralizer will require a second study to examine the effect of any
cleaning validation residue.

With direct surface testing the locations selected are of great importance. Many approaches to
cleaning validation assume that the chemical residues are distributed evenly across the
equipment surface. While homogeneity may sometimes apply to chemical residues it is unlikely
to apply to microorganisms. The locations selected should be a mix of those where microbial
contamination is likely to exits combined with areas that are potentially the most difficult to clean.
The selected locations should be justified and sufficient in number to provide an adequate
assessment of the item. When setting assessment criteria, each individual location must meet
the required limit and averaging should not be undertaken. This is something commonly checked
for by regulators.

2. Rinse Samples.

Unlike direct sampling, rinse samples are an indirect measure of cleanliness. Rinse samples
provide an assessment as to the number of rinses required to leave the final rinse water at the
same quality of the supply water. Thus, if Water-for-Injections is used and this has a bioburden
limit of 10 CFU/100mL for the supply water then similar low level of microbial contamination
should be obtained from the final rinse water. When assessing the rinse water it is also useful to
assess the supply water at the same time to assess differences in bioburden.

As well as assessing the final rinse water quality, the use of rinse samples allows for a that a
larger surface area to be sampled than is possible with the direct sampling methods, and for
inaccessible systems or parts of the equipment that cannot be routinely disassembled to be
sampled and evaluated assuming that the rinse gets into contact with these equipment parts
(10).

However, rinse samples alone are not sufficient to verify the microbiological status of cleaned
equipment. This is because, as indicated earlier, microorganisms may remain attached to
surfaces and because some surfaces may be occluded and therefore not sufficiently treated by
the chemicals or water rinses.

Rinse samples are typically tested for bioburden using a membrane filtration method where a
minimum of 100mL is tested using a 0.45 µm filter. This volume is sufficient, in terms of meeting
pharmacopeia requirements, providing that the sample is representative. It could be argued that
a larger sample should be taken since any microorganisms present are unlikely to be
homogenously distributed; a counter argument is that if the final rinse has been appropriately
timed then the recovery of microorganisms would not be expected and thus a 100mL volume is
sufficiently representative. The agar selected should be one applicable to the water (for instance,
R2A agar may be suitable for Water for Injections) (11). As microbial samples are tested, any
issues with associated chemical tests should be reported. If, for example a Total Organic Carbon
level was high this may signal that the rinse contains chemical residues and this may render the
microbial test invalid.

As well as bioburden endotoxin testing may be required. The endotoxin test requirement should
always be used for equipment that will be sterilized and used in sterile products manufacture.
Here an assessment for endotoxin is important because the presence of high levels of endotoxin
may prove resistant to later processing of the cleaned equipment (such as sterilization). Bacterial
endotoxin is resistant to standard sterilization temperatures.

For non-sterile processing, the need to assess the rinse water for bacterial endotoxin should be
determined by risk assessment. The most common method to assess endotoxin in water is the
Limulus Amebocyte Lysate (LAL) test (12).

3. Microbial characterization

With both the direct contact test and test of the final rinse water it is important to establish the
natural bioburden as well as the numbers of organisms recovered. The types of microorganisms
recovered provides information about the challenge to the cleaning process and can also help to
assess whether survivors pose any challenge to the subsequent sanitization or sterilization
processes that the cleaned equipment will be subject to. Identifying organisms to genus level
should be sufficient to determine the potential points of origin.
 4. Routine production in-process control

The cleaning assessment can also consider what happens when the equipment is used for
subsequent processing, that is following the completion of all cleaning methods (and any
subsequent sanitization or sterilization processing). Such testing is also based on direct
bioburden testing of a sample of the product, using a Total Microbial Aerobic Count method
(such as a pour plate or membrane filtration test). It should be noted that such testing is an
indirect measure and should only be used to support cleaning validation efficacy and not in lieu
of it.

5. Environmental monitoring

To ensure that equipment is stored in a suitable environment, either prior to processing or post-
processing the type of environment is important (for example, cleaned equipment should be fully
segregated from dirty equipment and cleaned equipment should not be stored in a wet area such
as a wash-bay). Information about the suitability of areas can be gathered from environmental
monitoring data derived from monitoring performed within the equipment holding areas. This will
only provide an indirect measure, although it will indicate as to the overall suitability of an area in
terms of the controlled environment or cleanroom maintaining control. In addition to
environmental assessments, the establishment of the clean hold time is a critical part of the
cleaning validation activity.

Of the different methods described above, direct sampling is arguably the most important
provided that the correct sample locations are selected. This is because the final rinse water may
indicate that the number of rinses (and the chemical treatment before that) has been sufficient,
however contamination may remain adhered to a surface and which is not detectable in the rinse
water.

Setting Microbiological Limits

Regulatory agencies do not provide any direct guidance about suitable microbiological test limits.
This is because acceptable levels can only be decided through risk assessment and it stands
that the needs of non-sterile processing (where cleaned and sanitized equipment is acceptable)
differs to sterile processing (where the equipment is subject to a sterilization step). With sterile
processing, an acceptable number of survivors will depend on the challenge these survivors may
pose to sanitization or sterilization method. This is a factor dependent upon the absolute
numbers seen and the species (for instance, spore forming bacteria may pose a greater
challenge).

As a general indicator, for rinse water samples Water of Injection limits are often applied for the
final rinse water. While these are not defined in the United States Pharmacopoeia the European
Pharmacopoeia limits provide a useful indicator, namely:

• For bioburden: Not more than 10 CFU/100mL;

• For endotoxin: Not more than 0.25 EU/mL.

With surface sampling an equivalent value to the bioburden test may be suitable, that is not more
than 10 CFU per cm2 (for a contact plate) or per swab.

For non-sterile equipment a formula devised by Docherty in 1999 has been widely used (13).
This is based on the permitted microbial levels in the finished product and then working
backwards to determine what might be permitted on the surface of an item of equipment (as
colony forming units per square centimeter). Docherty's process became adjusted to a 'universal'
 figure of 25, e.g. 25 CFU per cm2. Using a similar approach LeBlanc outlines a formula for
setting a bioburden limit for the direct contact test (14):

• Bioburden limit in the subsequent product multiplied by the minimum batch size (A)
• Value of A divided by the product contact surface area.

Le Blanc's example:

Product limit: 70 CFU/g

Minimum batch size: 200,000g

Product contact surface area: 260,000 cm2

Where:

70 x 200,000 = 14,000,000

Then:

14,000,000 / 260,000 = 54 CFU/ cm2

The approach is dependent upon making an appropriate assessment of the bioburden in the
product and with assigning suitable acceptance criteria (15).

These approaches is less common today and the limit is more often based on a review of
historical data (16), reviewing what is typically recovered from surfaces from a range of cleaning
validation studies (taking note of product quality and the typical bioburden levels recovered from
finished products). Historical reviews tend to lead to lower limits being set and this, in this
author’s experience, tends to be preferred by regulatory assessors.

Documentation

With cleaning validation being a documented process the microbiological aspects of cleaning
validation need to be recorded along with other aspects of the validation. Points to cover in
documentation include:

• The microbiological test samples required: types and number of samples e.g. rinses, swabs,
contact plates.
• A sample diagram, showing where samples for microbiological testing are.
• Reference to sampling SOPs.
• Verifying that the person who took the samples was appropriately trained.
• Check-list to record when samples are taken.
• Sample transfer conditions.
• A receipt section for the arrival of samples in the microbiology laboratory.
• Test details, including verification that subsequent testing has been conducted in the laboratory
e.g. the membrane filtration testing of rinse samples.
• List of all consumables, culture media and lot numbers (e.g. batch number of swabs used).
• A results section.
• Test limits.
• Note of any deviations from procedure.
• Laboratory management sign-off.

Many of these points will feature in a validation plan and protocol.

Concern with Chemical Residues

While this article is concerned with microbial recoveries from cleaned equipment it is important
that the microbiologist understands the risks from any remaining residues (where the
assessment of soiling forms the substantive part of cleaning validation). Should some residues
remain, such as protein from product or culture media (such as from a media simulation trial run
on a filling machine), these residues can provide nutrients and growth factors for any
microorganisms that may survive the cleaning process, or microorganisms that subsequently
contaminate the equipment post-clean during storage. With nutrients, and favorable
temperatures, microbial proliferation is more likely.

With cleaning validation often one residue is selected as 'worst case'. The selection is often
based on a factor like viscosity.

Attention should also be paid to the ability of any residues to support and to promote microbial
growth. A viscous substance that does not readily support microbial growth may sometimes not
be as great a risk as a growth-promoting substance like broth media residues. In assessing risk,
the dirty-hold time is an important factor for consideration since the longer the period a growth
promoting material remains in contact with the item of equipment for, the greater the chance
there is of microbial growth.

When to Conduct Testing

Cleaning validation needs to be tested at the end of the cleaning process. This should happen
immediately at the end of cleaning (for testing the final rinse) and as soon as applicable after the
cleaning once the equipment has been dried (for the direct surface testing). There is also a case
to assess the status of the equipment post-use and to determine how long an item of equipment
can be left for before it is cleaned. This latter point carries the risk of bioburden increasing as a
result of microbial proliferation (17).

With this latter point there are two key concepts in relation to equipment - the "clean-hold time"
and "dirty-hold time". Clean hold time is the time between the completion of cleaning and the
initiation of the subsequent manufacturing operation (this is discussed later). With the dirty hold
time, this can begin when the clean equipment is initially soiled, but more often is defined as the
 time between the end of manufacturing and the beginning of the cleaning process. It is important
that the cleaning validation assesses the maximum hold time since dirty equipment is harder to
clean (soil becomes more difficult to remove), and carries a greater chance for any microbial
contamination to build-up, the longer the hold time lasts for (18).

If a dirty hold time occurs in practice for an item of equipment that has been through cleaning
validation there is arguably a case for repeating the cleaning validation, depending on the
outcome of a risk assessment and the extent of the overage.

Reasons for Cicrobiological Test Failures

Microorganisms can be recovered following what was, theoretically at least, intended to be a

satisfactory cleaning validation exercise. Where microorganisms are detected in final rinse water
this is often easier to address than with the detection of microorganisms from surfaces. The
recovery of organisms in the rinse water may signal that either the chemical treatment was
insufficient or that the number of rinses was inadequate. This can be resolved through altering
the chemical treatment or by increasing the number of rinses. When microorganisms are
recovered from a surface this signals that surface attachment continues and that a different
approach to cleaning may be required in order to remove the organisms from a surface.

Sometimes, when equipment cleaning validation is run over a number of test cycles an
occasional failure is reported in terms of microbial recoveries. While this could be due to
sampling error it may also indicate a biofilm, where the release of microorganisms is following an
unpredictable pattern. A biofilm is when a group of microorganisms where the cells stick to each
other and adhere to a surface. Many different bacteria form biofilms, including Gram-positive
(e.g. Bacillus spp and Staphylococcus spp) and Gram-negative species (e.g. Escherichia coli, or
Pseudomonas aeruginosa). In general, Gram-negative bacteria form biofilms more readily.
Biofilms are rarely made up of one species (19). A biofilm will not always occur when a microbial
cell affixes to a surface. This is dependent upon the type of surface, the condition of the surface
(such as presence of crevices or abrasions), time, the type of organism and the number of
organisms; hence, owing to the interactions that occur upon contact between the two entities (the
surface and the bacterium), the cell may adhere to the surface either "reversibly" (that is,
temporarily) or "irreversibly" (that is, permanently).

The group becomes a community with its own unique characteristics in that microorganisms in a
biofilm are physiologically different to the same microorganisms in the planktonic state. The
adherent cells become embedded within a self-produced matrix of extracellular polymeric
substance (and which has a 'slime'-like appearance) formed from a polymeric conglomeration of
extracellular DNA, proteins, and polysaccharides (known as glycocalyx - hydrated polymeric
slimy matrices) (20). The steps involved with biofilm formation are (21):

1. Individual cells populate the surface (initial attachment).

2. Extrapolymeric substances (EPS) are produced and attachment becomes irreversible.
3. Maturation.
4. Biofilm architecture develops and matures.
5. Single cells are released from the biofilm.

Biofilms are often resistant to chemical treatments and to rinsing, which reflects one of the
differences that cells within the biofilm acquire: a greater resistance to extreme environments.
Rising alone is unlikely to affect a biofilm and will fail to remove in from a surface in the way that
rinsing can remove lower levels of microbial populations loosely bound to surfaces. The
effectiveness of cleaning and disinfection will partly relate to how bacteria are adhered to a
surface. The process is made easier where there is reversible attachment and more difficult
where there is irreversible attachment. Disinfectants work more effectively against bacteria in
suspension. Effectiveness is increased through contact time and the additional use of heat.

Thus during the washing process, biofilms can often resist sanitization and the survival allows
bacteria to spread. With cleaning validation the main focus is on preventing a biofilm from
forming, which is mostly the product of good design and avoiding poor design features like dead-
legs which can allow biofilm communities to become established (22).

With these scenarios considered there are two primary reasons for microbiological test failures
are: equipment design and the inadequacy of the cleaning method (and any subsequent
sanitization or disinfection step).

Equipment Design

With equipment design, there may be aspects of the equipment that are not being cleaned
adequately due to design limitations. An example is piping where ball valves are used. A second
example is with piping, especially long piping as might be found in transfer lines. The
assessment should include dead-legs and the associated risk of biofilm formation.

Linked to design is training. An assessment should be made as to whether the operators are
adequately trained to operate the item of equipment and if all cycles are being run correctly.
Related to training, the protocol followed by the operators should be assessed to determine if it
provides adequate instruction.

Cleaning Method

With the second main area, the cleaning method either the chemicals selected may be
insufficiently antimicrobial or the number of rinses may not be enough. Alternatively, the chemical
may be suitable but the contact time or operational temperature may not be suitable. With time, it
is important that the cleaning validation has assessed the worst-case time point between the end
of processing and the beginning of the cleaning validation step. Variations in the drying of
residues can affect the penetrative ability of a cleaning or sanitizing agent to reach any
microorganisms affixed to the equipment surface.

Sampling Error

To these a third area can be added: sampling error. Sampling error can occur if, for example,
there is a separate sampling valve and this is not subject to the cleaning process (in full or in
part). Errors in sampling can also arise if aseptic technique is not followed.

Equipment Storage
As well as cleaning validation, control measures need to be in place to prevent recontamination.
The main microbial risk arises from equipment either not being dry or becoming wet post-
cleaning. A considerable risk arises from stagnant water remaining inside the equipment. Water
remaining provides a growth source for many microorganisms in the vegetative state. If
equipment is left dry, opportunities for proliferation are much reduced; while many organisms will
survive dry conditions for prolonged periods (and with endospores, indifferently) the conditions
often suboptimal for growth.

Where equipment undergoes later sanitization or sterilization it is important that such controls are
in place to guard against the risk of any residual bioburden presenting a resistance risk to the
subsequent decontamination step. Having a fixed time in place is important because clean
equipment has a greater chance of becoming soiled as hold time increases and clean equipment
will not stay clean indefinitely despite using appropriate storage conditions.

Summary

This article has reviewed cleaning validation from the microbiological perspective, giving this
important (and often neglected) area of pharmaceutical manufacturing attention. While the article
has considered many variables that impact upon the success of microbiological cleaning
validation perhaps the most important are the control of time and preventing re-contamination of
cleaned equipment, which relates to the suitability of post-clean storage. With each factor
considered microbial growth will always be a variable factor and the validation should strive to
replicate worst-case condition.

The importance of microbiological control demonstrates why it is important that a microbiologist

is included from the outset of any cleaning validation project and is involved with the
development of the cleaning validation plan. One reason for this is because microorganisms
cannot be introduced into the cleaning validation process (due to the inherent contamination
risks), therefore someone cognizant of the potential origins of microbial contamination and routes
of transfer is required in order to set the biological contamination context.

As well as the initial validation, monitoring should be on-going. This means regular reviews of
process parameters and periodic sampling (which can take the form of re-validation exercises).
This is necessary to ensure that cleaning remains consistent over time (accounting for variables
like changes in personnel, training; and damage to equipment, surface abrasions and so on;
some of these variables will be greater with manual cleaning steps).

It seems odd that regular reviews are not always undertaken. Where a new disinfectant is
introduce for the sanitization of a cleanroom environmental monitoring conducted as part of a
field trial is continually reassessed as part of an on-going environmental monitoring program and
yet cleaning validation is too often seen as a stand-alone activity without much in the way of on-
going checks.

In summary, microbiological aspects of cleaning validation can readily be captured within the
broad cleaning validation approach. However, in doing so, the variables that might lead to
microorganisms surviving and the approaches need to remove or inactivate microorganisms
need to be fully considered and embedded into the cleaning validation strategy.

References

1. Agalloco, J. (1992). Points to consider' in the validation of equipment cleaning procedures.

PDA Journal of Pharmaceutical Science and Technology. 46 (5): 163–8
2. Fourman, G., and Mullin, M. (1993) Determining Cleaning Validation Acceptance Limits for
Pharmaceutical Manufacturing Operations, Pharmaceutical Technology, April 1993, pp54-60

3. FDA, Guide to Inspections Validation of Cleaning Processes. (Silver Spring, MD, 1993).

4. Pharmaceutical Inspection Convention/Pharmaceutical Inspection Co-Operation Scheme

(PIC/S). Validation Master Plan Installation And Operational Qualification Non-Sterile Process
Validation Cleaning Validation. (Geneva, Switzerland, Sept., 2009).

5. Jenkins, KM; Vanderwielen, AJ; Armstrong, JA; Leonard, LM; Murphy, GP; Piros, NA (1996).
"Application of total organic carbon analysis to cleaning validation. PDA Journal of
Pharmaceutical Science and Technology. 50 (1): 6–15

6. Leblanc, D. A (1998). Establishing scientifically justified acceptance criteria for cleaning

validation of finished drug products. Pharmaceutical Technology. 22 (10): 136–48

7. Verghese, G. and Kaiser, N. (2009) “Cleaning Agents and Cleaning Chemistry, Chapter 7” in
Pluta, P. (Ed.) Cleaning and Cleaning Validation Volume I, Davis Healthcare International and
Parenteral Drug Association, 2009 pp 103-121.

8. Sandle, T. (2013). Contamination Control Risk Assessment in Masden, R.E. and

Moldenhauer, J. (Eds.) Contamination Control in Healthcare Product Manufacturing, Volume 1,
DHI Publishing, River Grove: USA, pp423-474

9. Sandle, T. (20212). Environmental Monitoring: a practical approach In Moldenhauer, J.

Environmental Monitoring: a comprehensive handbook, Volume 6, PDA/DHI: River Grove, USA,
pp29-54

10. Harder, S. W. (1984) The Validation of Cleaning Procedures, Pharm. Technol. 8 (5), 29-34

11. Sandle, T. (2014) Assessment of the suitability of R3A agar for the subculture of
microorganisms isolated from pharmaceutical water systems, European Journal of Parenteral
and Pharmaceutical Sciences, 19 (3): 85-94

12. Upton, A. and Sandle, T. (2012). Best Practices for the Bacterial Endotoxin Test: A Guide to
the LAL Assay, Pharmaceutical Microbiology Interest Group: Stanstead Abbotts, UK

13. Docherty, S. (1999) Establishing microbial cleaning limits for non-sterile manufacturing
equipment, Pharmaceutical Engineering, 19 (3): 36-40

14. LeBlanc, D. (2002) Equipment Cleaning Validation: Microbial Control Issues, Journal of
Validation Technology, 8 (4): 40-46

15. LeBlanc, D. A. (1998) Establishing Scientifically Justified Acceptance Criteria of Finished

Drug Products. Pharmaceutical Technology. 19 (5): 136-148

16. Walsh, A. (2011) Microbial aspects in cleaning validation. In Saghee, M. R, Sandel, T. and
Tidswell, E. C. (Eds.) Microbiology and Sterility Assurance in Pharmaceutical Industry, Business
Horizons, New Dehli, pp.1-14

17. Sandle, T. (2015) Assessing Process Hold Times for Microbial Risks: Bioburden and
Endotoxin, Journal of GXP Compliance, 19 (3): 1-9
 18. Fugate, T. (2007) Hold Time Studies: A Lost Parameter for Cleaning Validation, J. Val.
Technol. 13 (3), 206–209

19. Donlan, R. M. (2002) Biofilms: Microbial Life on Surfaces, Emerg Infect Dis. 8(9): 881–890.

20. Wingender, J., Neu, T. R., and Flemming, H. C. (1999) What are bacterial extracellular
polymeric substances? In: Microbial Extracellular Polymeric Substances (Springer-Verlag Berlin
Heidelberg) pp. 1-19

21. Percival, S. L. and Walker, J. T. Potable water and biofilms: a review of the public health
implications, Biofouling 42, 99–115, 1999

22. Sandle, T. (2013). Bacterial Adhesion: an Introduction, Journal of Validation Technology, 19

(2): 1-10

View publication stats