Microbiology Testing: USP Requirements For Sterile and Nonsterile Preparations

Microbiology Testing:
USP Requirements for Sterile

and Nonsterile Preparations
Thomas C. Kupiec, PhD

CEO / President
tkupiec@arlok.com
© 2017 Quality Compounding Summit

Disclosures
Thomas Kupiec declare(s) no conflicts of

interest, real or apparent, and no financial
interests in any company, product, or service
mentioned in this program, including grants,
employment, gifts, stock holdings, and
honoraria.”
The American College of Apothecaries is accredited by the Accreditation
Council for Pharmacy Education as a provider of continuing pharmacy
education.

Agenda
• Review of USP Microbiology General Chapters

• Microbiological Tests
• Biological Tests and Assays
• Informational Chapters
• USP Nonsterile Drug Product Testing Requirements
• Environmental Monitoring
• Investigations and Microbial Identification

Learning Objectives
At the conclusion of this program, the participating
pharmacist or technician will be able to:
1. Explain testing requirements based on USP microbiology test
general chapters
2. Identify microbiological tests used in managing the production of

nonsterile drug products
3. Interpret sterility test results and assess sterility test limitations
4. Identify areas of pharmacy operations that require environmental

monitoring

EXPLAIN TESTING REQUIREMENTS
BASED ON USP MICROBIOLOGY
TEST GENERAL CHAPTERS

United States Pharmacopeia
• Scientific nonprofit organization that sets standards for

the quality, purity, identity, and strength of medicines
• USP’s drug standards are enforceable in the United

States by the Food and Drug Administration
• The U.S. Federal Food, Drug, and Cosmetics Act

designates the USP–NF as the official compendia for
drugs marketed in the United States.

United States Pharmacopeia
Monographs and General Chapters:
– A monograph consists of a series of tests, procedures

for the tests, and acceptance criteria.
– General chapters less than 1000 are enforceable.
– General information chapters greater than 1000 are

not enforceable.

USP General Chapters
for Sterile and Nonsterile Products
Microbiological Testing Requirements
General Tests and Assays – Microbiological Tests Chapter
Antimicrobial Effectiveness Testing <51>
Biological Indicators-Resistance Performance Tests <55>
Microbiological Examination of Nonsterile Products: Microbial <61>
Enumeration Tests
Microbiological Examination of Nonsterile Products: Tests for <62>
Specified Organisms
Sterility Tests <71>
General Tests and Assays – Biological Tests and Assays Chapter

Bacterial Endotoxins Test <85>

USP General Chapters
for Sterile and Nonsterile Products
Microbiological Testing Requirements
General Information Chapter
Microbiological Examination of Nonsterile Products: Acceptance Criteria <1111>
Microbial Characterization, Identification, and Strain Typing <1113>
Bioburden Control of Nonsterile Drug Substances and Products <1115>
Microbiological Control and Monitoring of Aseptic Processing Environments <1116>
Microbiological Best Laboratory Practices <1117>
Sterilization and Sterility Assurance of Compendial Articles <1211>
Validation of Microbial Recovery from Pharmacopeial Articles <1227>
Monitoring of Bioburden <1229.3>

USP <51>
Antimicrobial Effectiveness Testing
• Demonstrates preservative effectiveness for all injections,
topicals, oral products and antacids packaged in multiple-dose
containers.
• Challenge organisms are generally based on likely contaminants

to a drug product.
• The sample scheme and acceptance criteria are based on its

physical attributes, formulation, and intended use.

USP <51>
• The procedures and acceptance criteria for effectiveness apply to a
product in the original, sealed container in which it was distributed.
• The ability of the procedure to detect challenge microorganisms in

the presence of a suitably neutralized product to be tested must be
established. (method suitability)
• The growth-promoting capabilities of media used in this procedure

must be established. (growth promotion)

USP <51>
• The sample is divided into 5 parts and each is individually inoculated
with a challenge microorganism. This represents inadvertent
introduction of organisms during, or subsequent to, the
manufacturing process, as well as introduction from repeatedly
withdrawing individual doses from a multiple-dose container.
• The inoculated product is tested throughout a 28 day incubation

period where the population’s log reduction is calculated.

USP <51>
Table 1. Compendial Product Categories
For Category 1 Products - Parenterals, Nasal, and Opthalmic
NLT 1.0 log reduction from the initial calculated count at 7 days, NLT
3.0 log reduction from the initial count at 14 days, and no increase
Bacteria from the 14 days' count at 28 days
Yeast and molds No increase from the initial calculated count at 7, 14, and 28 days
For Category 2 Products - Topicals
NLT 2.0 log reduction from the initial count at 14 days, and no
Bacteria increase from the 14 days' count at 28 days
Yeast and molds No increase from the initial calculated count at 14 and 28 days
For Category 3 Products - Oral Products
NLT 1.0 log reduction from the initial count at 14 days, and no
Bacteria increase from the 14 days' count at 28 days
Yeast and molds No increase from the initial calculated count at 14 and 28 days
For Category 4 Products - Antacids
Bacteria, yeast, and molds No increase from the initial calculated count at 14 and 28 days

USP 55
Biological Indicators-Resistance Performance Tests
• Provides confirmation of the spore count on or in a biological

indicator.
• Biological indicators are spore containing strips or ampules that are

used during development, validation, and control of sterilization
processes.
• Viable spore count test is performed by subjecting the

microorganisms contained in the biological indicator to conditions
that encourage their growth, followed by subsequently incubating
and counting the recovered colonies.

USP 55
Biological Indicators-Resistance Performance Tests
• Verifies the resistance of the microorganisms contained within

a biological indicator to sterilization measures.
• D-value, or resistance testing, is performed by subjecting the

biological indicators to at least 5 different conditions that are
consistent with those intended for its use, then incubating and
counting the recovered colonies.
• Examples:
• Geobacillus stearothermophilus is used during verification of steam sterilization
• Bacillus subtilis is commonly used for verification of dry heat sterilization

IDENTIFY MICROBIOLOGICAL TESTS
USED IN MANAGING THE PRODUCTION
OF NONSTERILE DRUG PRODUCTS

USP <61>
Microbiological Examination of Nonsterile
Products: Microbial Enumeration Tests
• Determines the total population of aerobic bacteria and yeast
and molds in the product.
• Used for bioburden determination in raw materials, during

production, and in the finished product.
• Bioburden monitoring is an important aspect of process control.

USP <61>
• The ability of the test to detect microorganisms in the presence of

product to be tested must be established. (method suitability)
• The growth-promoting capabilities of media used in this procedure

must be established. (growth promotion)

USP <61>
• The test requires not less than 10 g or 10 mL of the product to be
tested (see USP <61> for small batch exceptions)
• Product is diluted to overcome antimicrobial properties and tested by

membrane filtration or plate count method
• At the completion of incubation, colonies are counted and results are

calculated based on the dilution factor for the total aerobic microbial
count and the total yeast and mold count
• USP <1111> provides guidance on bioburden limits

USP <62>
Products: Tests for Specified Organisms
• Provides testing for common microorganisms where absence
is required based on product type that may be detected under
the conditions of the test.
• The ability of the test to detect microorganisms in the presence of

product to be tested must be established. (method suitability)
• The growth-promoting capabilities as well as indicative properties

and inhibitory properties of media used in this procedure must be
established. (growth promotion)

USP <62>
Products: Tests for Specified Organisms
• The tests require either 1 g or 1 mL or 10 g or 10 mL of the product to be
tested, depending on the specified organism.
• The product is diluted in growth media and incubated to encourage growth

of microorganism populations.
• Next, dilutions and incubations in selective and indicative growth medias are
used to isolate and show the presence of the specified microorganism.
• Isolated organisms must be identified to verify the presence of the

specified organism
• USP <1111> provides guidance on organisms to test based on drug’s route

of administration

USP <61> and <62>
Table 1. Acceptance Criteria for Microbiological Quality of Nonsterile Dosage Forms
Total Aerobic Total Combined

Microbial Count Yeasts/Molds
(cfu/g or Count (cfu/g or
Route of Administration cfu/mL) cfu/mL) Specified Microorganism(s)
Nonaqueous preparations 103 102 Absence of Escherichia
for oral use coli (1 g or 1 mL)
Aqueous preparations for 102 101 Absence of Escherichia
oral use coli (1 g or 1 mL)
Rectal use 103 102 —
Oromucosal use 102 101 Absence of Staphylococcus
aureus (1 g or 1 mL)
Absence of Pseudomonas
aeruginosa (1 g or1 mL)
Gingival use 102 101 Absence of Staphylococcus
aeruginosa (1 g or 1 mL)
Cutaneous use 102 101 Absence of Staphylococcus
Nasal use 102 101 Absence of Staphylococcus
USP <61> and <62>
Table 1. Acceptance Criteria for Microbiological Quality of Nonsterile Dosage Forms
Auricular use 102 101 Absence of Staphylococcus

Vaginal use 102 101 Absence of Pseudomonas
Absence of Staphylococcus
Absence of Candida
albicans (1 g or 1 mL)
Transdermal patches (limits 102 101 Absence of Staphylococcus
for one patch including aureus (1 patch)
adhesive layer and backing) Absence of Pseudomonas
aeruginosa (1 patch)
Inhalation use (special 102 101 Absence of Staphylococcus
requirements apply to liquid aureus (1 g or 1 mL)
preparations for Absence of Pseudomonas
nebulization) aeruginosa (1 g or 1 mL)
Absence of bile-tolerant
Gram-negative bacteria (1 g
or 1 mL)

USP <61> and <62>
Table 2. Acceptance Criteria for Microbiological Quality of Nonsterile Substances for
Pharmaceutical Use
Total Aerobic Total Combined

Microbial Count Yeasts/Molds Count
(cfu/g or cfu/mL) (cfu/g or cfu/mL)
Substances for pharmaceutical 103 102
use

INTERPRET STERILITY TEST RESULTS
AND ASSESS STERILITY TEST
LIMITATIONS

USP <71>
Sterility Tests
• Applies to substances, preparations, or articles which,
according to the Pharmacopeia, are required to be sterile.
• Pharmacopeial procedures are not by themselves designed to

ensure that a batch of product is sterile or has been sterilized. This
is accomplished primarily by validation of the sterilization
process or of the aseptic procedures.
• A satisfactory result only indicates that no contaminating

microorganism has been found in the sample examined under
the conditions of the test.

USP <71>
Sterility Tests - Growth Promotion
Growth promotion confirms that media to be used is appropriate for that test. The
media is suitable if a clearly visible growth of the microorganisms occurs.
Tryptic Soy Broth (TSB):
• Candida albicans
• Bacillus subtilis
• Aspergillus brasiliensis
Fluid Thioglycollate Medium (FTG):
• Clostridium sporogenes
• Pseudomonas aeruginosa
• Staphylococcus aureus

USP <71>
Sterility Tests - Method Suitability Test
• Method suitability demonstrates the validity of the
sterility test by eliminating the antimicrobial
activity in the formulation, and establishing that
contamination, if present, will be detected.
• Once method suitability has been demonstrated, it

applies to that specific formulation and does not need
to be repeated unless there is a change in formulation,
production method, or test procedures.

USP <71>
Sterility Tests
Table 2. Minimum Quantity to be Used for Each Medium

USP <71>
Sterility Tests
Table 3. Minimum Number of Articles to be Tested
in Relation to the Number of Articles in the Batch
Sampling Across the Batch – Beginning, Middle and End

USP <71>
Sterility Tests - Interpretation of Results
• The sample test media is checked for macroscopic evidence of

growth at intervals during a 14 to 18 day incubation period.
• If, at the conclusion of the incubation, no evidence of growth is

observed, the product is said to comply with the test.

USP <71> Sterility Tests
Interpretation of Results Out of Specification
• If evidence of growth is found, the product does not comply with the
test for sterility.
• In order to declare a not-sterile test invalid, it must be clearly

demonstrated that the failure of the test was unrelated to the product
being examined.

Possible reasons to declare a test invalid:
• The data of the microbiological monitoring of the sterility testing facility

show a fault.
• A review of the testing procedure used during the test in question

reveals a fault.
• Microbial growth is found in the negative controls.
• After determination of the identity of the microorganisms isolated from

the test, the growth of this species (or these species) may be ascribed
unequivocally to faults with respect to the material and or the technique
used in conducting the sterility test procedure.

• If the test is declared invalid, the test is repeated with the same
number of units as in the original test.
• If no evidence of microbial growth is found in the repeat test, the

product examined meets the test requirements for sterility.
• If microbial growth is found in the repeat test, the product examined

does not meet the test requirements for sterility.

What are the chances a USP <71> test will
actually “catch” a contaminated sample?
• If only a few articles are contaminated, it is unlikely a sterility test will detect them
• There is a statistical limitation to the likelihood of finding a contaminated article

USP <85>
Bacterial Endotoxins Test
• Tests the finished product for the presence of bacterial endotoxins and
ensures that the level of endotoxins meets the criteria for the product.
• Endotoxins are components of the cell walls of gram-negative bacteria and may
be present due to microorganisms in the bulk product.
• Endotoxin testing is another indicator of process control.
• Endotoxin may be present even if the sample meets the requirements of the
sterility test.

USP <85>
Bacterial Endotoxins Test
• The absence of interference factors must be demonstrated.
Common causes of interference are:
• pH of the sample dilution

– Endotoxin samples must fall within the pH range specified by the lysate
manufacturer, typically 6.0-8.0.
• Presence of β-glucans
• Endotoxin is calculated based on the reaction times of the standard

concentrations and the concentration of the sample after dilution.

IDENTIFY AREAS OF PHARMACY
OPERATIONS THAT REQUIRE
ENVIRONMENTAL MONITORING

Environmental Monitoring
• Provides environmental data and confirms the effectiveness of
microbial controls present in the manufacturing and testing
areas.
• Some examples of microbiological controls are:

• Sanitization procedures
• HEPA filtration and air flow
• Gowning procedures
• Aseptic technique



• This is critical for areas where sterile products are manufactured.
• This should include establishing a robust monitoring program,

sampling of critical zones and high traffic areas, and trending of
microbial counts and recoveries.
• The isolates should be identified for trending related to the type of

microorganism, the level of control in the compounding environment,
and the potential source of the microorganism.
• Nonsterile product manufacturers can monitor the environment with

reduced frequency and with expectations of higher recovery.

Investigations and Microbial Identification
USP <1113> Microbial Characterization,
Identification and Strain Typing
• Provides information regarding the level of identification that is
appropriate for microorganisms that have been isolated during
different phases of manufacturing and quality testing.
• Investigations are a major part of manufacturing and finished

product testing.
• Internal Investigations
– Any unexpected event
– Process test falling out of specification
– Environmental excursion does not meet quality specifications
– Finished product does not meet quality specifications

Investigations and Microbial Identification
USP <1113> Microbial Characterization,
Identification and Strain Typing
• Microorganisms, if detected in drug substances, water for

pharmaceutical use, the manufacturing environment,
intermediates, or finished drug products, typically undergo
characterization and identification.
• Microbial identification can be especially useful during an

investigation of product contamination, environmental
excursion, and for determining potential sources of the
contamination.

References and Resources
• United States Pharmacopeia (USP)
– General Chapters
• International Journal of Pharmaceutical Compounding (IJPC)
• Your Testing Laboratory
• Laura Gillikin, cGMP Validation
• Microbiology Network
• Andy Gunn, Kymanox
• CompoundingToday.com
• www.usp.org and www.fda.gov

Thank You!
Speaker Contact Information:
Thomas C. Kupiec, PhD

Phone: (405) 271-1144
Email: tkupiec@arlok.com

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Microbiology Testing:

USP Requirements for Sterile

Thomas C. Kupiec, PhD

© 2017 Quality Compounding Summit

Thomas Kupiec declare(s) no conflicts of

© 2017 Quality Compounding Summit

• Review of USP Microbiology General Chapters

• Biological Tests and Assays

• USP Nonsterile Drug Product Testing Requirements

• Investigations and Microbial Identification

© 2017 Quality Compounding Summit

2. Identify microbiological tests used in managing the production of

3. Interpret sterility test results and assess sterility test limitations

4. Identify areas of pharmacy operations that require environmental

© 2017 Quality Compounding Summit

© 2017 Quality Compounding Summit

• Scientific nonprofit organization that sets standards for

• USP’s drug standards are enforceable in the United

• The U.S. Federal Food, Drug, and Cosmetics Act

© 2017 Quality Compounding Summit

Monographs and General Chapters:

– A monograph consists of a series of tests, procedures

– General chapters less than 1000 are enforceable.

– General information chapters greater than 1000 are

© 2017 Quality Compounding Summit

General Tests and Assays – Biological Tests and Assays Chapter

© 2017 Quality Compounding Summit

© 2017 Quality Compounding Summit

• Challenge organisms are generally based on likely contaminants

• The sample scheme and acceptance criteria are based on its

© 2017 Quality Compounding Summit

• The ability of the procedure to detect challenge microorganisms in

• The growth-promoting capabilities of media used in this procedure

© 2017 Quality Compounding Summit

• The inoculated product is tested throughout a 28 day incubation

© 2017 Quality Compounding Summit

© 2017 Quality Compounding Summit

• Provides confirmation of the spore count on or in a biological

• Biological indicators are spore containing strips or ampules that are

• Viable spore count test is performed by subjecting the

© 2017 Quality Compounding Summit

• Verifies the resistance of the microorganisms contained within

• D-value, or resistance testing, is performed by subjecting the

© 2017 Quality Compounding Summit

© 2017 Quality Compounding Summit

• Used for bioburden determination in raw materials, during

• Bioburden monitoring is an important aspect of process control.

© 2017 Quality Compounding Summit

• The ability of the test to detect microorganisms in the presence of

• The growth-promoting capabilities of media used in this procedure

© 2017 Quality Compounding Summit

• Product is diluted to overcome antimicrobial properties and tested by

• At the completion of incubation, colonies are counted and results are

• USP <1111> provides guidance on bioburden limits

© 2017 Quality Compounding Summit

• The ability of the test to detect microorganisms in the presence of

• The growth-promoting capabilities as well as indicative properties

© 2017 Quality Compounding Summit

• The product is diluted in growth media and incubated to encourage growth

• Isolated organisms must be identified to verify the presence of the

• USP <1111> provides guidance on organisms to test based on drug’s route

© 2017 Quality Compounding Summit

Total Aerobic Total Combined