CLEANING VALIDATION

Microbial Monitoring
Anthony M. Cundell Ph. D.
Associate Director, QA
Microbiological Development &
Statistics
Wyeth-Ayerst Pharmaceuticals
Pearl River, New York

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What are the the current Good

Manufacturing Practices
requirements for cleaning
validation?

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21 CFR 211.67 Equipment cleaning
and maintenance
a) Equipment and utensils shall be
cleaned, maintained and sanitized
at appropriate intervals to prevent
contamination that would alter the
safety, identity, strength, quality or
purity of the drug product.
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21 CFR 211.113 Control of

microbiological contamination
(a) Appropriate written procedures,
designed to prevent objectionable
microorganisms in drug products
not required to be sterile, shall be
established and followed.

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21 CFR 211.113
Control of microbiological
contamination
(b) Appropriate written procedures,
designed to prevent objectionable
microorganisms in drug products
required to be sterile, shall be
established and followed. Such
procedures shall include
validation of any sterilization
process.
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What is the accepted industry

practice for microbial monitoring
during cleaning validation?

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FDA Mid-Atlantic Region Inspection

Guide on Cleaning Validation
(July, 1992) does not address
microbial monitoring.

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FDA Guide to Inspection of
validation of Cleaning processes
(July, 1993) states that
microbiological aspects of
equipment cleaning should be
considered. There should be some
evidence that routine cleaning and
storage does not allow microbial
proliferation.
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PhRMA report on microbiological
monitoring in nonsterile
pharmaceutical manufacturing
areas (March, 1997)
recommended that depending on
the product, e.g. inhalation
products,oral aqueous liquids,
vaginal creams, etc, cleaning
validation should include
microbial sampling to ensure
microbiological quality.
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PhRMA Guidelines for the Validation

of Cleaning Procedures for Bulk
Pharmaceutical Chemicals
published in September, 1997
declared that microbial issues was
not within the scope of the report.

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Microbial contamination can be
prevented by:
Selection of suitable equipment
Sound cleaning programs
Cleaning equipment directly after
use
Dry storage of equipment

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CLEANING VALIDATION
Microbial Monitoring Methods
Swab Method
Surface Rinse Method
RODAC Plate Method
Limulus Amoebocyte Lysate
Method
ATP Bioluminescence Method

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What are the Advantages &

Disadvantages of the the Available
Microbial Monitoring Methods?

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Swab Method
Advantages: Most common
method used with selective media
to isolate directly different
microbial populations.
Disadvantages: Recovery may not
be reproducible & quantitative.

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Surface Rinse Method
Advantages: Higher counts
obtained than swab method &
better overall assessment
possible.
Disadvantages: Entire surface
evaluated, microbial population
must be detached & membrane
filtration necessary to obtain
countable numbers.
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RODAC Plate Method
Advantages: Direct growth on
media in contact plate is
convenient, neutralizers may be
included in media & different
media may be used.
Disadvantages: Only applicable to
surfaces that are smooth & have
low counts.
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Limulus Amoebocyte Lysate Method
Advantages: Rapid, sensitive,
quantitative measure of bacterial
endotoxin levels.
Disadvantages: Indirect
measurement of high numbers of
gram-negative bacteria only.

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Bioluminescence Method
Advantages: Rapid, highly
accurate & reliable method.
Disadvantages: Suitable for
microbial counts in the range of
104 to 108 organisms as
insufficiently sensitive for low
microbial counts.
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CLEANING VALIDATION

Microbial requirements for nonsterile pharmaceutical drug

products: Control of the total
bioburden, elimination of USP
indicator and objectionable
microorganisms.

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Microbial Limit for a oral solid dosage form,
i.e., Tablet or capsule.
Total Aerobic Microbial Count NMT 1000
cfu/g
Total Combined Yeast & Mold Count NMT100
cfu/g
Absence of USP Indicator organisms, i.e., E.
coli, S. aureus and Salmonella spp.
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Case history: What should be the

acceptance criteria for a blender
used to process a wet granulation
tablet?

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Approach: The microbial limit in

terms of cfu per 25 cm2 can be
determined from a knowledge of
the internal surface area of the
blender, the quantity of
granulation processed and the
Microbial Limit for the product
(oral solid dosage form)
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Example: A Gemco 20 blender has
an internal surface area of 46,128
cm2 & a capacity of 220 kg of
granulation.
With a surface bioburden of 100 cfu/
25cm2 there is potential to transfer
<1 cfu/g to the granulation. cf.
Microbial Limit for an oral solid
dosage form NMT 1000 cfu/g.
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Example: A Gemco 75 blender has
an internal surface area of 100,944
cm. sq. & a capacity of 500 kg of
granulation.
With a surface bioburden of 100
cfu/25cm2 there is potential to
transfer <1 cfu/g to the
granulation. c.f. Microbial Limit for
an oral solid dosage form NMT
1000 cfu/g.
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Example: A Littleford blender has an
internal surface area of 22,718 cm.
sq. & a capacity of 100 kg of
granulation.
With a surface bioburden of 100
cfu/25 cm2 there is potential to
transfer <1 cfu/g to the
granulation. c.f. Microbial Limit for
an oral solid dosage form NMT
1000 cfu/g.
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Recommended Acceptance Criteria

for Blenders:
NMT 100 cfu per 25 cm2 &
Absence of USP Indicator
Organisms.
Recommended Monitoring Method:
Swab Method

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Microbial requirements for sterile

pharmaceutical drug products:
Control of total bioburden to
maintain the Sterility Assurance
Level & elimination of endotoxin
to prevent pyrogenic reactions.

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Case history: What should be the

acceptance criteria for a presterile bulk solution tank used to
aseptically fill a parenteral
product?

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Approach: The microbial limit in
terms of cfu per 25 cm2 can be
determined from a knowledge of
the internal surface area of the
bulk tank, the volume of the bulk
solution processed and the
square footage of the cartridge
filter used to sterile filter the
product.
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Calculations:
Challenge cfu per sq. cm of Filter =
Fill Volume(Liters) X Bioburden
(cfu/l)/Surface Area of the Filter
(sq.cm)
Conversion factor: 10 sq. ft. = 9290
sq. cm
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Example:
A 1000 Liter bulk tank with a
bioburden of 10 cfu per 100 mL
will represent a total bacterial
challenge of 105 to a 10 sq. ft.
cartridge filter, i.e., 10 cfu per cm2
of filter surface , which well below
the filter retention rating of 107 per
sq. cm.
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Acceptance criteria (Sterile

products): Based on pre-sterile
bulk solution bioburden limit, i.e.
NMT 10 cfu/100 mL (asepticallyfilled products), heat resistance of
bacterial spores (terminallysterilized products or equipment)
& endotoxin contribution.
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Example: 125 Liter pre-sterile
filtration bulk tanks (Precision
Stainless, Inc.) with a product
contact surface area of 2356 sq.
cm. With a surface bioburden of
33 cfu/25 cm2 there is potential to
transfer 4 X 103 organisms or 1
cfu/100 mL to the bulk solution.

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Endotoxin considerations:
Endotoxin limits for parenteral
products are set using the
maximum human dose for the
individual products.
Since a bacterial cell weights 10-13
grams, 105 cells in the bulk
solution represents 10-8 gram of
cellular material (10 ng).
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Endotoxin considerations:
If endotoxin represents 10% of
the cell weight then a bulk
solution of 125 Liters will
contain less 0.00001 ng/mL of
endotoxin.

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Example: 125 Liter pre-sterile
filtration bulk tanks (Precision
Stainless, Inc.) with a product
contact surface area of 2356 sq.
cm. With a surface endotoxin level
of 0.5 EU/25 cm2 there is potential
to transfer up to 20 EU or 0.002
EU/mL to the bulk solution.
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Recommended Acceptance Criteria

for pre-sterile filtration bulk tanks
NMT 33 cfu per 25 cm2 & NMT 0.2 EU
per sq. cm
Recommended Monitoring Method:
RODAC Plate Method &/or Rinse
sampling.
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Conclusions: Based on the desired

Microbial Limit for a non-sterile
product or a pre-sterile filtration
bulk solution, given a certain
equipment surface to material
quantity ratio, it is possible to set
appropriate surface bioburden
limits for cleaning validations.
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