Microbiological Aspects of Cleaning Validation

Dr. Tim Sandle

Pharmaceutical Microbiology Resources: www.pharmamicroresources.com
Introduction
• Cleaning
• Cleaning validation & GMPs
• Microbiological concerns
• Microbiological risks
• Risk assessment
• Microbiological tests and acceptance criteria
• Why failures happen
 • 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.
• An additional concern is with
the microbial bioburden.
What is cleaning?
 Cleaning validation - methodology applied to give the
assurance that a cleaning process has removed residues and
contaminants from a piece of equipment or machinery.

Residues:
Cleaning • Microorganisms
validation •
•
Active pharmaceutical ingredients
Other process chemicals, such as buffers
• Cleaning agents themselves (detergents)
• Microbiological culture media

The ‘validation’ aspect refers to providing documented

evidence that that the acceptance criteria have been met.
Relative risks – manual
vs automated cleaning
• Manual cleaning is inherently high-risk compared
with automated cleaning.
• There will be variations between each cleaner.
• Variations of time.
• Variations with quantities of cleaning chemicals
used.
• Manual cleaning procedures may need to be
monitored and maintained.
GMPs #1
• Code of Federal Regulations (CFR):
• 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
GMPs #2
• FDA, Guide to Inspections Validation of
Cleaning Processes. (Silver Spring, MD,
1993).
• 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).
Number of runs?
• No direct guidance relating to the number of runs.
• By convention, three runs are normally performed in
order to demonstrate that the obtained results are
not due to chance.
Microbial Aspects
Microbial aspects #1
• The chemical verification of cleaning validation is
relatively well described. What is often less clear is
the microbiological aspect.
• Divided into:
• The microorganisms themselves (a direct
hazard)
• The presence of residues that potentially
provide a microbial growth source (an indirect
hazard).
• To evaluate these microbiological risks a sound
microbiological sampling plan is required.
Microbial aspects
#2
Microbial aspects #3

• The processes that form part of cleaning validation have an impact upon
microorganisms remaining post-cleaning and on microbial survival.
• 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).
• The microbiologist should input.
Cleaning chemicals and microbial survival #1

The types of cleaning agents

These cleaning agents, which are
selected will have different affects Many commercial cleaning agents
typically one caustic based agent
upon microorganisms depending on are formulated cleaning (in addition
(sodium hydroxide) and one acid
the nature of chemical and the to water) with a surfactant, an
based reagent (such as hydrochloric
species (and numbers) of alkalinity source (such as sodium
acid), should be included as part of
microorganisms present on a given hydroxide), and a chelant.
the cleaning validation.
surface.
Acid and caustic?

There is a recurrent discussion about the need for both an acid and a caustic for
cleaning validation.

Alkaline detergents are good for removing organic soils, i.e. oils, fats, proteins,
starches, and carbohydrates. They hydrolyze peptide bonds and breaking down
large, insoluble proteins into small, more easily soluble polypeptides.

Theoretically microbiological contamination, including viral residues,

can be overcome by using sodium hydroxide in the cleaning process.
Alkaline reagents are good for microbial Sodium hydroxide dissolves proteinaceous soils and fatty oils by
kill. saphonification.
Sodium hydroxide effectively dissolves protein and organic matter.
 Acid and caustic?
The use of an acid depends on
which impurities are of a
concern, and whether these
will act as a barrier for the
alkaline agent and protect
microorganisms.
Acid detergents do not work
well on heavy soils, oils, and
glucans.
Phosphoric acid is useful in removing
protein residues
Acid detergents are effective
for the prevention or removal
of water scale (calcium and
magnesium carbonates), and
aluminium oxide.
They are also good at bacteria
kill, but they are rarely used
without alkaline detergents.
Cleaning chemicals and
microbial survival #2
• Cleaning agents 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.
• Other aspects of the cleaning process can prove
hostile to microbial survival:
• Temperature of the water used for cleaning
(above 60oC)
• pH ranges below 4 and above 11.
Effect of water rinses
#1
• Water is a key part of cleaning validation.
• Water rinses will remove cleaning chemicals and will
siphon away any microorganisms in the planktonic state.
• Water is:
• Capable of wetting surface to penetrate the soil
deposit
• Has the capacity to break the soil into fine particles
• Water holds small fine particles into suspension
• Water can prevent residues from redepositing onto a
cleaned surface
Effect of water rinses #2
• Final water rinse is Water of Injections (this is for all types of pharmaceutical products).
• Water must meet the required microbiological specification e.g. with WFI – NMT 10
CFU/100mL for bioburden and NMT 0.25 EU/mL for bacterial endotoxin.
• Following rinsing 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.
Critical process and quality parameters

• Critical process parameters for cleaning validation

are:
• Time (dirty and clean hold times and process
run time).
• Activity (chemical exposure, number of water
rinses etc).
• Chemical (concentration).
• Temperature (cleaning temperature).
• Critical quality attributes include:
• Water quality.
• Type of soil.
• Nature of the soil.
• Surface material and surface quality.
Risk assessment #1
• Need to assess worst-case factors:
• Stage of manufacture
• Type of soil
• Equipment design
• Ease of equipment drying
• Manual processing (as opposed to automated
processing)
• Surface materials
• Equipment age
• Equipment damage
• Dirty storage areas
• Dirty hold time
Risk assessment #2
• 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).
• Levels of cleaning need to greater if the cleaning is to be followed by sanitization or sterilization.
• The permitted microbial levels for equipment used in non-sterile processing require a separate
risk assessment and limits setting compared with equipment used for sterile processing.
Risk assessment #3

• Hazards need to be identified and risk assessed, based on the severity of the hazard and the likelihood that the hazard
will occur.
• Microorganisms represent a hazard:
• Microbial contamination on the equipment post-use (and pre-cleaning);
• The effects of hold time prior to cleaning (in relation to microbial proliferation and the release of endotoxin);
• Additional microbial challenges from the storage environment;
• The efficacy of the cleaning process to remove microorganisms and endotoxin;
• Storage of the equipment post-cleaning prior to use or a subsequent sanitization or sterilization step (in relation
to recontamination from the environment).
• Plus 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.
Risk assessment #4
Worst-case
conditions for testing
• Residues
• The ability of any residues to support and to promote
microbial growth should be assessed.
• 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.
• Time of testing
• The dirty-hold time is an important factor for
consideration.
• Cleaning validation needs to be tested at the end of
the hold time and the cleaning process.
Microbial tests
• Direct tests • Follow-up testing
• Surface sampling • On-going in-process controls e.g.
• Contact plate intermediate product bioburden testing
• Swab • Microorganism characterisation
• Indirect tests • Hold time related testing
• Final rinse tests • Environmental monitoring
• Bioburden by membrane filtration
• Endotoxin testing
Locations for monitoring

• Locations need to be representative and of worst-case locations.

• Be aware of non-uniform contamination transfer.
• Equipment needs to be categorized between uniform contamination
equipment and non-uniform contamination equipment.
• Define the most difficult-to-clean locations in equipment.
• Sites should be in a protocol.
• Assessment for location needs to be provided and justified.
• Sampling locations chosen arbitrarily or easiest-to-clean locations should
be avoided.
Swabbing vs rinsing

• Rinse and swab measure two different things

• They will not necessarily correlate
• Area:
• Swabs focus on small area
• Rinses focus on larger area
• Representativeness:
• Swab measures worst case
• With appropriate locations
• Rinse measures average bioburden
• Failures:
• If a swab fails, a rinse may pass
• If a rinse fails, it is likely a swab will also fail
Limitations of
microbial methods
• Methods are limited:
• In terms of culture media
• Incubation time
• Incubation temperature
• Suitability of the medium
• In terms of the method, in general
• Swabs recovery 30-60%
• In terms of type of bacterial attachment to surfaces
• In terms of whether the organisms can be cultured
• Not capable of growing on the culture medium
• Too stressed to recover on the medium
Qualification of microbiological media
• Validate environmental monitoring media
• Confidence that residues of cleaning agents or disinfectants are not influencing the recovery
of organisms on media used to perform routine environmental monitoring.
• Enables pharmaceutical and healthcare facilities to implement the use of adequate contact
plates and swabs containing the appropriate neutralizers in the environmental monitoring
process.
Microbial attachment
to surfaces #1
• Bacteria attach to surfaces in different ways and according to different
circumstances.
• Bacterial adhesion is a consequence of the balance of attractive and
repulsive physicochemical interactions between bacteria and surfaces.
• Bacteria prefer to grow on available surfaces (sessile form) rather
than existing in planktonic form within an aqueous environment.
• The process of bacterial adhesion to a surface comprises of three stages:
• Transport.
• Initial adhesion (bioattachment).
• The cell may adhere to the surface either "reversibly" (that
is, temporarily) or "irreversibly" (that is, permanently).
• Colonization.
 Irreversible attachment is harder to remove
• Irreversible bacterial adhesion occurs because of short-range
molecular interactions like hydrogen, ionic, and covalent
bonding, interactions involving extracellular structures, and
secretions.

Microbial • Connected with biofilm formation.

Bioattachment occurs relatively slowly.

attachment to • Affected by the type of bacterium involved, the size of the
bacterial population in the environment, and the duration of its
surfaces #2 growth phase.

Gram negative bacteria form biofilms more readily.

• This is because EPS and LPS are found in greater abundance.
• Important in relation to water, Gram negative bacteria
constitute the majority of the bacterial populations found in
aquatic environments.
 Control is improtant
Microbial control is important, due to
limitations of methods and surface attachment
challenges.
This means:
• Observing time.
• Sticking with validated parameters
• Avoiding cross-contamination.
 Regulatory agencies do • Acceptable levels need to be decided through risk
not provide any direct assessment and it stands that the needs of non-
sterile processing (where cleaned and sanitized
guidance about suitable equipment is acceptable) differs to sterile
microbiological test processing (where the equipment is subject to a
limits. sterilization step).

As a general indicator,
Microbial for rinse water samples
Water of Injection limits • For bioburden: Not more than 10 CFU/100mL;
are often applied for the
limits #1 final rinse water,
namely:
• For endotoxin: Not more than 0.25 EU/mL.

With surface sampling

an equivalent value to • That is not more than 10 CFU per cm2 (for a
the bioburden test may contact plate) or per swab.
be suitable
Microbial limits #2
• For non-sterile equipment a formula devised by Docherty is also widely used.
• 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.

Ref: Docherty, S. (1999) Establishing microbial cleaning limits for non-sterile manufacturing
equipment, Pharmaceutical Engineering, 19 (3): 36-40
Documentation
• Types and number of samples e.g. rinses, swabs, contact plates.
• A sample diagram, showing sample locations.
• 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 of testing.
• List of all consumables, culture media and lot numbers.
• A results section.
• Test limits.
• Note of any deviations from procedure.
• Laboratory management sign-off.
Cleaning validation challenges #1

• Reproducibility of manual cleaning of small parts and non-clean-in-place (CIP)

systems.
• Effective design of fully automated CIP systems to avoid dead legs and lack of
drainability.
• Build-up of physical protein residue on the walls of equipment cleaned by CIP.
• Failure of CIP systems over time due to blocked steam traps and poor maintenance
practices.
Cleaning validation challenges #2
• Developing acceptance criteria and validation strategies.
• Achieving degradation or deactivation of proteins and their breakdown products
• Using inappropriate risk-assessment methodologies.
• Such as a weakness with a matrix approach.
• Addressing air-liquid interface issues.
• Ensuring suitable a high-level of cleanliness to prevent potential for microbial excursions and
biofilms.
• Avoiding rouge formation on surface cleanliness.
Rouging
• Rouge formation is a steady chemical process that is underway in
all metallic piping systems in contact with water and all stainless
steels corrode over time.
• What varies is the pace of the reaction.
• Exacerbated by high temperature and particular metal
compositions.
• The risk is with product contamination, through the presence of
particulates, could occur.
• Plus an impact on water quality.
• Plus buildups of rouge byproducts can lead to blockages in filters.
• Localized corrosion (pitting) provides increased opportunities for
microbial attachment.
Why failures happen

• Recovery of organisms in the rinse water may signal

that either the chemical treatment was insufficient or
that the number of rinses was inadequate.
• Resolved through altering the chemical
treatment or by increasing the number of rinses.
• Insufficient chemical contact times
• Biofilms
• Poor equipment design
• Ball valves.
• Long piping with dead-legs.
• Sampling error
• Poor training
Equipment storage,
post-cleaning
• Control measures are required 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.
Microbial risks with
wet equipment
• Some Gram-negative growth examples, for doubling
times:
• Escherichia species: 19-20 minutes
• Klebsiella species: 38-40 minutes
• Acinetobacter species: 48 -55 minutes
• Pseudomonas species: 100 minutes
• Plus, they become harder to remove over time.
References
• Sandle, T. (2013). Bacterial Adhesion: an Introduction, Journal of Validation Technology, Vol. 19,
Issue 2
• Sandle, T. (2015) The Rouging Effect in Pharmaceutical Water Systems: Causes and Strategies for
Prevention, Journal of GXP Compliance, Vol. 19, Issue 1
• Sandle, T. (2017) Microbiological Aspects of Cleaning Validation, Journal of GxP Compliance, Vol.
21, Issue 5
Summary

The variables that might lead

Microbiological aspects of
to microorganisms surviving
cleaning validation can This needs to be captured in
and the approaches need to
readily be captured within the cleaning validation
remove or inactivate
the broad cleaning validation strategy.
microorganisms need to be
approach.
fully considered.
 Thank you

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