Measuring Bioburden for

Cleaning Validation Protocols

Destin A. LeBlanc
Cleaning Validation Technologies
destin@cleaningvalidation.com

November 2004 A3P Canada

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 Why address?
z For process equipment
z Initial regulatory focus: drug active and
cleaning agent
z FDA guidance not apply to micro residues
z But do address micro concerns on storage
z Practical matter
z Microbial residues are related to cleaning
process
z Microbial residues are potential
contaminants of next product

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 Issues?
z Probably not a problem except…
z Little or no documentation
z Approaches to control
z Setting limits
z Analysis & sampling
z Documentation strategies
z New validation
z Already validated process
z CEHT (expiry)

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 Control measures

z Bioburden of raw materials

z “Cleaning” itself
z Sanitizing agents
z Drying of equipment

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 Good cleaning
z Factors hostile to microbes --
z High temperature
z pH extreme
z Oxidizer (biocidal)
z Surfactant (wetting, physical removal)
z Removal of chemical residues --
z Microbe “trap”
z Nutrient
z In most cases, effective cleaning can
meet microbial control objectives

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 Separate sanitizing step
z Possible agents
z Hypochlorite
z Quats
z Alcohol
z Hydrogen peroxide
z Peracetic acid
z May require final rinse (exc. alcohol &
peroxide)
z Another residue concern

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 Final rinse

z Rinse to remove sanitizing agent

residues
z Unless rinse with sterile water, will
reintroduce organisms into system

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 Sanitizing option
z Sanitize after storage (as opposed
to after cleaning)
z Address rinsing
z Address worst case & demonstrate
effective sanitation under those
conditions
z Storage time & conditions
z Worst case “starting conditions”

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 Is sanitizing necessary?
z Major issue: microbes in equipment at
beginning of subsequent manufacturing
process
z How demonstrate – data is critical
z Scale-up studies (after clean/after
sanitize comparison)
z Monitoring data on just cleaning

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 Drying
z Heat
z Alcohol
z Time
z Issue addressed in FDA Guideline
for stored equipment
z Preparation for storage and storage
conditions should be in an SOP

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 Limit for microbes
z For chemical residues, can predict level
in next product and estimate effects
z Microbes are living organisms –
z Must consider proliferation in next product
z Consider
z Species
z Further processing of subsequent product
z Preservative in subsequent product
z Level based on past practices, baseline
data, and/or industry standards
z May also affect endotoxin levels
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 Effects in next product

z Water activity <0.6%, microbes not

grow
z Neutral pH, aqueous products --
can expect proliferation unless
preserved or until sterilized

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Calculating acceptable limits

z Use same principles as for chemical

residues
z Limit (CFU) in next product
z Calculate CFU per surface area
(CFU/cm2) based on –
z Equipment surface area
z Batch size

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 Limit in non-sterile product
z Use the “next product” specification
for bioburden
z Or, start with proposed U.S.P.
<1111>, “Microbial content of Non-
sterile Pharmaceuticals”
z Solid oral: <1,000 CFU/g
z Liquid oral: <100 CFU/g
z Topicals: <100 CFU/g

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 Other microbial sources
z Sources of “microbial content”
z Equipment surfaces
z Raw materials
z Components

z Apply factor to account for that

allowed from cleaned surfaces (0.1
as starting point)

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 Limit per surface area
z Calculate limit in CFU per surface
area as:
(Spec.) X (factor) X (wt. product)
(shared surface area)

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 Example
z Dry oral dosage, spec. limit is 500 CFU/g
z Limit from cleaned surfaces in next
product is 100 CFU/g
z Batch size is 200 kg & surface area is
250,000 cm2
z Therefore, equipment CFU/cm2 is:
50 X 200 kg X 103 = 40 CFU/cm2
250,000
Or 1,000 CFU per contact plate (25
cm2)

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 Rinse limit
z Calculate as:
Surface limit X total surface area
Rinse volume
z Surface area is area sampled
z Use rinse sampling principles to
determine rinse volume (DO NOT use
collected sample volume)
z Rinse limit may vary for different
equipment items in a train
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 Limit - sterilized product
z Key issue is limit in product at time of
sterilization
z Bioburden level is assumed for
sterilization validation
z Limit (CFU/g) is based on assumed worst
case for sterilized product validation
z Calculate surface limit as for non-sterile
product
z Also consider effect on endotoxin

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 Limit - aseptic production
z Difference is that equipment (not
product) is sterilized
z Key issue is bioburden for sterilization
validation
z Do not consider limit in next product,
only limit per surface area
z Limit set on expected bioburden and
assumes worst case for sterilization
validation
z Also evaluate endotoxin effects

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 Measurement tools
z Conventional micro tools
z Rinse water (membrane filtration)
z Swab, with desorption and pour plate
count
z Contact plate

z Focus is aerobic bacteria, but may

have to consider molds/yeasts

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 Recovery?
z From surfaces –
z Swab: 25-50%
z Contact plate: 50%
z Rinse: <25%
z Generally not necessary to include
recovery factors as for chemical
residues
z Do not confuse 70% recovery in USP
<1227>
z <1227> is not surface recovery, but
recovery to show adequate neutralization

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 Why no recovery?
z Bioburden enumeration is highly variable:
5 CFU and 8 CFU are not significantly
different
z When spike vegetative organism onto
surfaces and allow to dry, significant
percentage may die
z Which organism do you perform recovery
on?
z Practical limits well below limits based on
scientific principles

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 Endotoxin limits
z Generally only set limits for
finished drug manufacture for IV
and inhalation products
z Limits are set at “industry
standard” (or arbitrary limit) of
WFI spec in rinse water (0.25
E.U./mL)
z Difficult to measure on surfaces

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 Validation strategies

z For new process

z Design cleaning process
z Determine if sanitizing step is needed
z Determine change in bioburden during
storage
z Set micro limits acceptance criteria
z Measure as part of protocol

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 Validation strategies
z For validated process, either –
z Establish micro limit
z Perform PQ for micro only

Or
z Establish micro limit
z Begin monitoring program to establish
routine levels

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 Validation strategies (2)
z If impetus for data is due to lack of
data --
z Avoid immediate reaction to add sanitizer
z Focus on data to show sufficiency of
validated process
z If impetus is due to observed high micro
levels --
z Due to cleaning process or to storage issues?
z Separate sanitizer step only if data supports
it

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 CEHT
z Additional concern for bioburden:
Cleaned Equipment Hold Time
(CEHT)
z Focus is maintenance of “clean”
state during storage
z Addressed in FDA, PIC/S and
Canadian guidance documents

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 CEHT concerns
z Condition of equipment at
beginning of storage
z Storage conditions
z Time
z Environment
z Change during storage

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 Establishing CEHT

z Negotiate with production

z Maximum time equipment will be stored
z Establish protocol
z Residues to measure
z Sampling location
z Analytical techniques
z Acceptance criteria

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 CEHT protocol
z Residues
z Bioburden / endotoxin
z “Dirt”
z Analytical procedures
z Standard micro counting procedures
z TOC (optional)
z Sampling locations
z Not same as in CV protocol
z Consider how equipment recontaminated

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 CEHT acceptance criteria
z Visually clean
z Bioburden – the greater of
z NMT 25 CFU/25 cm2, and
z NMT 1 log change from baseline
z Option: TOC –
z NMT 2X TOC of baseline TOC (net of
blank)
z Must perform baseline testing

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 Separate protocol?
z Option 1: Part of CV protocol
z Option 2: Separate protocol
z Advantages of Option 2
z Allows closeout of CV protocol without
possible delays from CEHT protocol
z One CEHT protocol may cover several
CV protocols
z Flexibility in CEHT determination
depending on scheduling

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 Summary
z More regulatory attention to microbial
control data in CV
z Issue is more lack of data than data
showing problems
z High acceptance limits based on
scientific principles
z Demonstrate through added PQ
testing or through monitoring data
z CEHT data
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