In-House Microbial Isolates in Compendial Testing: Regulatory Requirements

In-House Microbial
Isolates in Compendial
Testing
Regulatory Requirements
1

In-House Microbial Isolates in Compendial
Testing: Regulatory Requirements
2
Agenda
 Current Industry Debate Over the Use of In-
House Isolates in Compendial Testing
 Disinfectant Efficacy Testing
 Media Growth Promotion Testing
 Test Method Suitability/Qualification
 Rapid Microbiological Methods
 Selection of In-House Isolates for Use in
Compendial Testing

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Current Industry Debate Over the Use
of In-House Isolates in Compendial
Testing

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"Good practice includes the periodic
challenge of prepared media with low levels
of organisms. This includes USP indicator
organisms as well as normal flora."
U.S. Food and Drug Administration: “Guide to Inspections of Microbiological
Pharmaceutical Quality Control Laboratories” (1993)

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PMF List (2010)
 “As soon as an environmental isolate is put onto growth media, it
is no longer an ‘environmental’ isolate’.”
 “EM [environmental monitoring] organisms will stop to behave as
such after a few transfers.”
 “Environmental isolates are often mismanaged by the
microbiology lab.”
 “Many companies simply don't have the qualified equipment and
personnel to preserve and maintain stock cultures of their
environmental bugs.”
 “This is one of the problems with using 483 observations as
‘cGMP’. They are not. They are the observations of one inspector
who may or may not be knowledgeable in microbiology.”

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The arguments against the inclusion of in-house
isolates fall into three categories:
1. An “environmental” isolate, once cultured in the
Microbiology laboratory, loses its “wild-type” traits.
2. Preparing an in-house microbial isolate to create the
organism concentration necessary for compendial
testing is too problematic to be practical.
3. The regulatory expectation that in-house microbial
isolates be included in compendial testing is
represented by only a few “rogue” FDA inspectors,
and is not an Agency-wide expectation.

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Does an “environmental” isolate lose its “wild-type” traits
after being cultured?
 There has been no published empirical evidence that an
in-house microbial isolate either does or does not lose its
“wild-type” traits upon culturing.
 United States Pharmacopeia (USP <61>): “Seed-lot
culture maintenance techniques (seed-lot systems) are
used so that the viable microorganisms used for
inoculation are not more than five passages removed
from the original master seed-lot”.
 Although in most cases an in-house isolate likely
becomes physiologically more “robust” upon culturing, it
is reasonable to assume that a limited number of
passages from the source culture allows the
microorganism to retain its “wild-type” genetic traits.

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“It’s too difficult to be practical” does not address the scientific
merit of including in-house isolates in compendial testing.
 The basic process includes preparation of a suspension of
unknown concentration, performing serial dilutions and plate
counts to determine the concentration, and then standardizing
the suspension to the desired concentration.
 During the incubation period for serial dilution plates, the
suspension is typically held under refrigerated conditions for
several days.
 During this time, the concentration of the organism in suspension
may change, resulting in either a decrease or increase.
 Standardization and final use of this suspension may therefore
result in a “miss” in terms of its assumed concentration,
discovered only after testing is complete.

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Are 483 observations and Warning Letters
limited to just a few inspectors?
 “Compliance versus science”
 The increasing trend in citing firms for not
meeting this requirement demonstrates that
the Agency’s SMEs recognize the scientific
merit of including in-house isolates in
compendial testing.
 Defending against this expectation is a
challenge!

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Disinfectant Efficacy Testing
Methodology

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Point of clarification …
 “Disinfectant”: A chemical agent used on
inanimate objects (e.g., floors, walls, sinks)
to destroy virtually all recognized pathogenic
microorganisms, but not necessarily all
microbial forms (e.g., bacterial endospores).
[Centers for Disease Control and Prevention]
 “Sanitizer”: A chemical agent that
simultaneously cleans and disinfects.

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 Step 1: Determine the test method
◦ Tube method: Inoculate dilutions of the
disinfectant and determine the microbial
reduction. Typically referred to as “use-
dilution” testing.
◦ Coupon method: Inoculate the surfaces of
coupons representing facility materials of
construction. Expose them to disinfectant.
Remove the inoculum. Determine log
reduction. "Six Steps to Qualifying Disinfectants“, Deborah
Ensign, Microtest Laboratories, Inc., 2011

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 Step 2: Determine the Challenge
Organisms
◦ American Type Culture Collection (ATCC)
organisms (gram-negative, gram-positive,
spore-former, fungi)
◦ Environmental isolates

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 Step 3: Determine the Surface Types to
be Tested
◦ Examples: stainless steel, glass, plastic, tile,
plexiglas
◦ 2” x 2” coupons
◦ Sterilize or disinfect before using.

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 Step 4: Determine Expiration of
Disinfectants
◦ Use the same concentration and contact
exposure time used in the facility.
◦ Use the worst-case expiration date for each
disinfectant.

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 Step 5: Method Validation
◦ Critical!
◦ Typically called the “recovery” part of the test
◦ Must demonstrate adequate recovery of the
challenge organisms in the presence of the
disinfectants.
◦ Inoculate the test system (i.e., the medium
exposed to disinfectant) with a low-level challenge
of test organism. Include a control that has not
been exposed.
◦ Recovery: 50-200% of control

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 Step 6: Efficacy Testing
◦ USP <1072>, “Disinfectants and Antiseptics”
◦ Typically called the “challenge” part of the test.
◦ Inoculate tube/coupon.
◦ Expose to the desired concentration of the
disinfectant for the desired contact time.
◦ Demonstrate 2-log reduction for spore-formers.
◦ Demonstrate 3-log reduction for vegetative
bacteria.

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Compliance Deficiencies

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 “Disinfectant effectiveness studies
against representative microorganisms
and/or specific in-house isolates were not
conducted for cleaning agents used in
your facility to disinfect production areas,
including aseptic areas.” [FDA Warning
Letter, March 28, 2008]
 “No rationale was provided for the ATCC
[American Type Culture Collection]
organisms used nor was [sic] actual EM
isolates used for the study.” [FDA
Memorandum, Biologics License
Application, 2009]

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 “The firm was unable to provide scientific rationale for
the use of the selected organisms used in the
Disinfectant Efficacy study. These organisms were not
representative of organisms isolated from the facility
nor were they representative of the USP guidelines.”
[FDA 483 Observation, May 25, 2011]
 “Your response states that a supplemental
disinfectant efficacy study, using mold spores of in
house isolates on various surfaces, will be performed
and completed by [redacted]. We suggest that this
proposed study be conducted as soon as possible.”
[FDA Warning Letter, July 12, 2012]

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Industry Guides

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 “Routinely used disinfectants should be effective against the
normal microbial vegetative flora recovered from the facility... If
indicated, microorganisms associated with adverse trends can be
investigated as to their sensitivity to the disinfectants employed
in the cleanroom in which the organisms were isolated.” [FDA
Guidance for Industry: Sterile Drug Products Produced by Aseptic
Processing - Current Good Manufacturing Practice, September
2004]
 “If using an Association of Official Analytical Chemists (AOAC)
method, microorganisms specified in the reference which are
most likely to be found in the manufacturing environment should
be used and tests performed on microbial isolates frequently
found in the environment can provide additional information on
the effectiveness of disinfectants.” [“Regulatory Aspects
Concerning the Quality Controls of Microbiological and Nonviable
Particulate Contamination in Pharmaceutical Manufacturing”, Oji,
(FDA), American Pharmaceutical Review, January/February 2004]

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United States Pharmacopeia, <1072> “Disinfectant and Antiseptics”
 “… the most frequently isolated microorganisms from an environmental
monitoring program may be periodically subjected to use-dilution testing with
the agents used in the disinfection program to confirm their susceptibility, as
there are real differences among different species in resistance to the lethal
effects of different sanitizers.”
 “To demonstrate the efficacy of a disinfectant within a pharmaceutical
manufacturing environment, it may be deemed necessary to conduct the
following tests: (1) use-dilution tests (screening disinfectants for their efficacy
at various concentrations and contact times against a wide range of standard
test organisms and environmental isolates); (2) surface challenge tests (using
standard test microorganisms and microorganisms that are typical
environmental isolates, applying disinfectants to surfaces at the selected use
concentration with a specified contact time, and determining the log reduction
of the challenge microorganisms); and (3) a statistical comparison of the
frequency of isolation and numbers of microorganisms isolated prior to and
after the implementation of a new disinfectant.”

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 “It is a sound practice to perform challenge
testing of the selected sanitizers/disinfectants
with isolates routinely recovered by the
environmental monitoring program. This
establishes the practical effectiveness of the
disinfectants.” [Parenteral Drug Association
Technical Report No. 13, “Fundamentals of an
Environmental Monitoring Program”,
September/October 2001]
 “Regulatory authorities now expect evidence
of the efficacy of disinfection agents against
environmental isolates.” [ASTM E2614 – 08,
Standard Guide for Evaluation of Cleanroom
Disinfectants]

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Industry Literature

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 “Validation of sanitising agents for effectiveness against
isolated organisms found in cleanroom environments is
increasingly becoming an area of concern to both
manufacturers and regulatory agencies.” [“Surface
Disinfectant Validations”, Nelson Laboratories, Inc., 2004]
 “It is generally recommended to use fungal spore
suspensions of both reference cultures and environmental
isolates... Regulatory authorities also expect to see the
specific environmental isolates most frequently found in
the facility included in the disinfectant effectiveness
testing.” [“Control Strategies for Fungal Contamination in
Cleanrooms”, Lopolito et al., Controlled Environments
Magazine, September 2007]

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 “… users are expected to challenge disinfectants not only
with standard test organisms but also with facility
environmental isolates because commercially available
microorganisms behave quite differently from their ‘wild’
counterparts. Selection of the test organisms is crucial and
an important issue to the regulatory agencies, especially
when it comes to environmental isolates.” [“Microbial Limit
and Bioburden Tests: Validation Approaches and Global
Requirements”, Clontz, October 14, 2008]
 “...it is crucial to make an educated investment in
antimicrobial products that are effective against your
specific mold isolates.” [“Evaluating the Activity of
Disinfectants Against Fungi”, Polarine et al., CleanRooms,
Volume 23, No. 2, February 2009]

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 “…scientifically-based references and
conventional wisdom used to select these
products does not alleviate drug manufacturers
of the requirement to validate disinfectants,
sporicides, and even isopropyl alcohol for use
in facilities under actual use conditions against
environmental isolates.” [“Prevention of
Microbial Contamination“, Kopis Sartain and
Polarine, Pharmaceutical Technology, June 2,
2011
 “Disinfectant Efficacy Studies should be
considered as a fundamental requirement
during the development of qualifications of
disinfectants on various surfaces with both
ATCC and in-house isolates.” [Friedman, Blog,
August 15, 2011]

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 “Disinfectant testing involves challenging the disinfectant solution
(as a suspension test) and challenging different surface materials
with disinfectant and a range of different microorganisms
including isolates from the facility.” [ “Cleanroom Cleaning and
Disinfection: Eight Steps for Success”, Sandle, Controlled
Environments Magazine, March 2012]
 “This study clearly demonstrates that the most frequently isolated
microorganisms from an environmental monitoring program may
be periodically subjected to microbroth dilution testing with
cleanroom disinfectant agents used in the disinfection program to
confirm their sensitivity profile.” [“In vitro Antifungal Efficacy of
Biguanides and Quaternary Ammonium Compounds against
Cleanroom Fungal Isolates”, Vijayakumar et al., PDA Journal of
Pharmaceutical Science and Technology, May/June 2012]

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Media Growth Promotion Testing
Methodology

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 USP <61>, “Microbiological Examination
of Nonsterile Products: Microbial
Enumeration Tests”
 USP <62>, “Microbiological Examination
of Nonsterile Products: Tests for Specified
Organisms”
 USP <71>, “Sterility Tests”

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 Challenge the medium with a low-level
inoculum of test organism: <100 cfu
 Control = Previously qualified medium
 ATCC organisms
 In-house isolates
 Agar: 50-200% recovery
 Broth: Turbidity

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 Not limited to aseptic manufacturers!
Manufacturers of non-sterile products
have been subject to regulatory
enforcement.

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 “… the growth promotion tests for media
used during environmental monitoring did
not include the use of the normal microbial
flora commonly recovered and isolated from
the various production and support areas.”
[FDA Warning Letter, February 24, 1997]
 “Growth promotion qualification of the media
used for environmental monitoring does not
include a challenge with mold isolates.” [FDA
Warning Letter, July 10, 1998]

38
 “Growth promotion testing performed on media
fill vials does not include evidence the media is
capable of detecting environmental isolates
found in class 100 filling areas. For example,
mold organisms are not used to challenge media,
even though mold isolates have been identified
in filling room 1.” [FDA Warning Letter, January
11, 2001]
 “… your firm does not perform challenge testing
to the sterility media with environmental isolates
from the environmental monitoring program.”
[FDA Warning Letter, October 29, 2010]

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 Growth promotion studies were deficient because
there was “no inclusion of environmental isolates
in the growth promotions that are conducted,
including growth promotion studies for aseptic
media simulations.” [FDA 483 Observation, May
2008]
 “… the firm does not test TSA … during growth
promotion tests for microorganisms to include for
example, molds, yeasts and other potential
known environmental contaminants found in the
manufacturing facility and/or raw materials.”
[FDA 483 Observation, April 30, 2010]

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 The FDA expressed its satisfaction that
the firm complied with its expectation:
“Growth promotion studies were
conducted successfully using indicator
microorganisms per USP as well as local
isolates.” [FDA Memorandum, BLA
Recommendation, January 16, 2009]

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Industry Guides

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 “The QC laboratory should determine if USP indicator
organisms sufficiently represent production-related
isolates. Environmental monitoring and sterility test
isolates can be substituted (as appropriate) or added
to the growth promotion challenge.” [FDA Guidance
for Industry: Sterile Drug Products Produced by
Aseptic Processing - Current Good Manufacturing
Practice, September 2004]
 “… for the Growth Promotion test, representative
microflora isolated from the controlled environment or
ATCC strain preparations of these isolates may also
be used to test media.” [USP <1116>,
“Microbiological Evaluation of Clean Rooms and Other
Controlled Environments”]

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 “… microorganisms used in growth-
promotion testing may be based on
the manufacturer's recommendation
for a particular medium, or may
include representative environmental
isolates.” [USP <1117>,
“Microbiological Best Laboratory
Practices”]
 USP clarifies parenthetically that
“these latter are not to be construed
as compendial requirements.”

44
From PDA Technical Report No. 22, “Process Simulation
for Aseptically Filled Products” (2011):
 “The growth promotion properties of the incubation
media should be evaluated using pharmacopeial
methods. The inclusions of tests for environmental
organisms or those isolated from sterility test
positives are recommended.”
 “Growth promotion testing ... is performed using
pharmacopeial methods. Consideration should be
given to testing with other microorganisms found in
the aseptic processing area environment, such as
those isolated during environmental and personnel
monitoring and sterility test contaminants.”

45
From the Pharmaceutical Inspection
Convention/Pharmaceutical Inspection Cooperation
Scheme (PIC/S):
 “The medium selected should be capable of
supporting a wide range of microorganisms, which
might reasonably be encountered and be based also
on the in house flora (e.g. isolates from monitoring
etc.).” [Recommendation on the Validation of Aseptic
Processes. PI-007-6, 1 January 2011]
 “Environmental or fastidious organisms may be used
if alternative non-selective enrichment media have
been selected for the sterility test.” [Recommendation
on Sterility Testing. PI-012-3, 25 September 2007]

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Industry Literature

47
 “Growth promotion should be demonstrated using
organisms listed in USP General Chapter <71> as well as
environmental, personnel, and sterility test failure isolates
[at the] <100-cfu [colony-forming unit] challenge.”
[“Microbial Testing in Support of Aseptic Processing”,
Cundell, Pharmaceutical Technology, June 2004]
 “In general, a microbial growth medium such as soybean
casein digest medium should be used. This media selected
should be demonstrated to promote growth of United
States Pharmacopoeia (USP) <71> indicated organisms as
well as representative isolates identified from
environmental monitoring, personal monitoring, and
positive sterility test results.” [Environmental Monitoring for
Cleanrooms and Controlled Environments, Dixon (Ed.),
November 2, 2006.]

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 “In addition to the compendial organisms required in the [media
growth promotion] tests, addition of specific microorganisms of
interest could be useful if they have been recovered from past
tests (e.g., a Sterility Test contaminant or a frequent
environmental monitoring isolate).” [“Quality Control of
Microbiological Culture Media”, Sutton, PMF Newsletter, January
2006]
 “The media must be challenged with the organisms listed in the
current version of the compendia as well as one or more in-house
isolates… The in-house cultures used must be qualified as well,
with regards to identity and population size. The challenge should
be with <100 CFU and the population size must be verified and
recorded.” [“Introduction to Sterility Testing: Control of the
Environment, Test Limitations, and Investigating Manufacturing
Systems”, Mentel, American Pharmaceutical Review, Jan/Feb
2006]

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 “In addition to type cultures, environmental
isolates are commonly used in media
testing regimes. These organisms are
designed to demonstrate that a particular
lot of culture media will grow
microorganisms which are representative of
the types that are found in the
manufacturing environment... The use of
such isolates is increasingly becoming a
regulatory expectation.” [“Assessment of
Culture Media in Pharmaceutical
Microbiology”, Sandle, Pharmaceutical
Microbiology Blog, April 3, 2012]

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Test Method Suitability/Qualification
Industry Guides

51
 There has been no documented evidence that
the FDA has cited a manufacturer for lack of
inclusion of in-house isolates in microbiological
test method suitability/qualification.
 “The test organisms selected should reflect
organisms that could be found in the product,
process, or manufacturing environment.” [FDA
Final Rule on “Amendments to Sterility Test
Requirements for Biological Products”, June 4,
2012]
 Recent Rule… more regulatory scrutiny in the
near future!

52
Test Method Suitability/Qualification
Industry Literature

53
 “There is a strong argument that environmental isolates
are the best challenge to media and for validation studies
like sterility test validation. They are the most sensitive
micro-organisms, having been exposed recently to
disinfectants, particular soils etc.” [“The Use of
Environmental Isolates”, Sandle, Pharmaceutical
Microbiology Blog, January 10, 2010]
 “The author highly suggests performing test method
suitability studies using wild-type isolates from production
surfaces instead of laboratory-adapted strains of bacteria.
Wild-type strains are a better representation of the
organisms encountered on production areas than those
strains that lack wild characteristics.” [“Bioburden Method
Suitability for Cleaning and Sanitation Monitoring: How Far
Do We Have to Go?”, Salaman-Byron, Pharmaceutical
Technology, August 2, 2010]

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Rapid Microbiological Methods
Industry Guides

55
 Rapid microbiological methods (RMMs)
have become a growing area of
advancement over the past several years.
 There has been no documented evidence
that the FDA has cited a manufacturer for
lack of inclusion of in-house isolates in
rapid microbiological methods.

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 “You should develop a panel of microorganisms
relevant to the product and process to challenge
the performance of your RMM. We recommend
that you include in your panel microorganisms
which represent the following categories: …
◦ isolates detected in starting materials,
◦ isolates detected by in-process testing or during
preliminary product testing,
◦ isolates detected by environmental monitoring of your
manufacturing facility,
◦ isolates from your production areas which represent low
nutrient and high stress environments …”
FDA Guidance for Industry: Validation of Growth-Based Rapid
Microbiological Methods for Sterility Testing of Cellular and
Gene Therapy Products (Draft), February 2008

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Rapid Microbiological Methods
Industry Literature

58
 “To demonstrate the [RMM’s] ability to detect
a range of microorganisms, at least six
replicate samples of drug product were
spiked with 10 to 100 cfu of microorganisms
from site-specific isolates and … compendial
microorganisms … ” [“The Introduction of
Qualitative Rapid Microbiological Methods for
Drug-Product Testing”, Newby et al.,
Pharmaceutical Technology, Process
Analytical Technology, 2004]

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 “Validation studies evaluated sterility test samples from
primary culture, expansion culture, and final product
culture with a microbial challenge comprised of 10
microbial species. These species included a mix of
commercial reference strains and stressed cells from frozen
archives of environmental isolates and sterility test failures.
During the validation process, FDA recommended challenge
microorganisms … ” [“Automated Rapid Microbiological
Methods for the Biopharmaceutical Industry: Selection,
Validation, and Implementation for an Autologous Cell
Therapy Product”, Duguid & du Moulin, American
Pharmaceutical Review, June 2009]
 “Additional considerations [for RMM validation] may be
provided with regard to ... environmental isolates.”
[“Revision of PDA Technical Report Number 33”, Miller and
Moldenhauer, American Pharmaceutical Review, February 1,
2010] PDA Technical Report 33, Evaluation, Validation and
Implementation of New Microbiological Testing Methods, May 2000

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Selection of In-House Isolates for Use
in Compendial Testing
Risk

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 Depends upon the test itself, and upon
the nature of the manufacturing process
and final product.
 Risk
◦ Assessment
◦ Management
◦ Mitigation

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 21 CFR 211.113(a) : “Appropriate written
procedures, designed to prevent
objectionable microorganisms in drug
products not required to be sterile, shall be
established and followed.”
 21 CFR 211.113(b): “Appropriate written
procedures, designed to prevent
microbiological contamination of drug
products purporting to be sterile, shall be
established and followed. Such procedures
shall include validation of all aseptic and
sterilization processes.”

63
“Case Studies of Microbial Contamination in Biologic Product
Manufacturing”, Suvarna et al., American Pharmaceutical
Review, 2011.

64
 “HACCP [Hazard Analysis Critical Control Point] might be
used to identify and manage risks associated with physical,
chemical, and biological hazards (including microbiological
contamination).” ICH Q9, “Quality Risk Management”,
2005]
 “Where warranted, a risk-based assessment of the relevant
factors [regarding recovery of unspecified microorganisms]
is conducted by personnel with specialized training in
microbiology and in the interpretation of microbiological
data. For raw materials, the assessment takes account of
the processing to which the product is subjected, the
current technology of testing, and the availability of
materials of the desired quality.” [USP <1111>
“Microbiological Examination of Nonsterile Products:
Acceptance Criteria for Pharmaceutical Preparations and
Substances for Pharmaceutical Use”]

65
 “Since the microbial quality of pharmaceutical
products is a function of microorganisms
introduced through excipients, the significance of
microorganisms recovered from excipients should
be evaluated in terms of the number and type of
organisms, whether or not they will survive the
manufacturing process, their ability to grow in
the product formulation, the use of the product
and the potential hazard to the user.”
[“Microbiological Considerations When Selecting
Excipients During Product Development”,
Montgomery and Manu-Tawiah, American
Pharmaceutical Review, 2004]

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 Design of the manufacturing facility
and environmental monitoring (EM)
program
 “Where microbiological specifications
have been established for the
intermediate or API, facilities should
also be designed to limit exposure to
objectionable microbiological
contaminants, as appropriate.” [ICH
Q7A, “Good Manufacturing Practice
Guidance for Active Pharmaceutical
Ingredients”, 2000]

67
 Environmental monitoring program
◦ Characterization of normal facility microflora
◦ Identifying shifts in microflora, seasonal trends
 PDA Technical Report No. 13, “Fundamentals
of an Environmental Monitoring Program”,
September/October 2001
◦ “Characterizing microorganisms recovered from
environmental and personnel monitoring is an
important part of surveillances programs”
◦ “… routine characterization should continue to
determine whether isolates are part of the normal
microbial flora or represent something different.”

68
 PDA Technical Report No. 13,
“Fundamentals of an Environmental
Monitoring Program”,
September/October 2001
◦ “A change in the microbial flora or the
introduction of a previously undetected
species might signify a change in a
system that should be investigated”
◦ “Characterizations can be useful clues as
to the possible source of isolates.”

69
 “A well-established microbial EM program must
identify, at least at the genus level, microorganisms
isolated from samples that reach the alert and action
limits established. Evaluation of the isolates is
necessary and would be evaluated for their ability to
grow in the non-sterile product present at the time of
sampling. It is necessary to make a list of
microorganisms isolated and identified at least at the
genus level during the first year of implementation.
This list is crucial to identify the profile of
microorganism species found in production areas, its
possible origin, trends, and seasonal peaks.”
[“Bioburden Method Suitability for Cleaning and
Sanitation Monitoring: How Far Do We Have to Go?”,
Salaman-Byron, Pharmaceutical Technology, 2010]

70
 “Although drugmakers have begun to perform
environmental monitoring in their nonsterile
manufacturing facilities, many companies are
uncertain about how to interpret the data and
develop alert and action levels … The industry should
record the types and numbers of microorganisms that
it identifies in its environmental-monitoring programs
and evaluate the information on a quarterly basis …
This strategy could provide guidance, based on
scientific rationale, for assessing and controlling the
risk of contamination.” [“Manufacturers Recalibrate
Microbial Control for Nonsterile Drugs”, Greb,
Pharmaceutical Technology, Equipment and
Processing Report, February 16, 2011]

71
Selection of In-House Isolates for Use
in Compendial Testing
Strategies

72
 Two bases for selection of isolates
◦ Demonstrating adequate reduction of a high-
level presence of an organism
 Disinfectant efficacy testing (“challenge”)
◦ Demonstrating adequate recovery of a low-
level presence of an organism
 Disinfectant efficacy testing (“recovery”)
 Media growth promotion testing
 Test method suitability/qualification
 Rapid Microbiological Methods

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 Disinfectant efficacy testing
◦ Microflora of the manufacturing environment
◦ Typically evaluated in EM trend reports
◦ Undesirable trends (e.g., seasonal)
◦ Recovery of organisms considered
“objectionable” or “of concern”

74
Example …
 EM trend reports describe historical and consistent
seasonal “blooms” of filamentous fungi (mold) during the
autumn season.
 Corrective action = frequent sanitization with a sporicidal
agent
 Characterize (ID) the species of mold(s).
 Include the mold(s) in disinfectant efficacy testing.
 Importantly, the early recovery of the species (detected
through EM) signals the need to proactively implement
frequent sanitization with a sporicidal agent.
 Don’t react to the “bloom” … Risk mitigation!

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Example …
 Introduction of an “objectionable” organism into
the facility
◦ Recurring incident?
◦ Ingress into more stringently classified areas as a result
of material, personnel or equipment flow?
 Conclusion
◦ Adverse trend?
◦ Shift in the facility’s normal microflora?
 Include the organism in disinfectant efficacy
testing.

76
Example …
 EM excursion
 Data trend analysis
 Conclusion = adverse trend
 Include the organism(s) in disinfectant
efficacy testing.

77
 Subjecting the organisms to disinfectant
efficacy testing …
◦ Lack of efficacy demonstrates the need to
change disinfectant
◦ Demonstrating efficacy implicates inadequate
cleaning procedures/practices
 Include the organisms in EM media growth
promotion testing.

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 Inclusion of in-house isolates in media
growth promotion testing
◦ Media used for EM and clean utility monitoring
◦ Media used for raw material, component, in-
process product and finished product testing

79
 Media used for raw material and
component testing
◦ Previously recovered “objectionable” or
fastidious organisms from non-sterile materials
◦ Previously recovered contaminants from sterile
materials

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 Media used for in-process product testing
◦ Review of historical data
◦ Critical control points
 Media used for finished product testing
◦ For non-sterile products, the approach is
similar to that of selecting isolates from non-
sterile raw materials and components.
◦ For sterile products, include isolates recovered
from previous sterility test or media fill failures.

81
 Test method suitability/qualification
◦ Employ the same strategy as that used for
selection of isolates for growth promotion of
media used for raw material, component, in-
process product and finished product testing.
 “Objectionable” and fastidious organisms
 Sterility failures
 Media fill failures

82
 Sources of information useful for selecting
in-house isolates
◦ Annual product reviews
◦ EM and clean utility trend reports
◦ Process validation
◦ Cleaning validation
◦ Media fills
 Organisms representing risk

83
Review
 Current Industry Debate Over the Use of In-
House Isolates in Compendial Testing
 Disinfectant Efficacy Testing
 Media Growth Promotion Testing
 Test Method Suitability/Qualification
 Rapid Microbiological Methods
 Selection of In-House Isolates for Use in
Compendial Testing

84
Robert Westney
Director of Quality and Operations
Cryologics, Inc.
(610) 847-8781
rwestney@cryologics.com
www.Cryologics.com
Links to references:
http://www.cryologics.com/resources/bibliography

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