Microbiology Testing: USP Requirements for Sterile …acainfo.org/wp-content/uploads/2017/08/Session-7_Microbiology...USP Requirements for Sterile and Nonsterile Preparations ... At

© 2017 Quality Compounding Summit

Microbiology Testing:USP Requirements for Sterile and Nonsterile Preparations

Thomas C. Kupiec, PhDCEO / President

[email protected]

mailto:[email protected]


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 ObjectivesAt the conclusion of this program, the participatingpharmacist 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 ChapterAntimicrobial Effectiveness Testing <51>Biological Indicators-Resistance Performance Tests <55>Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests

<61>

Microbiological Examination of Nonsterile Products: Tests for Specified Organisms

<62>

Sterility Tests <71>

General Tests and Assays – Biological Tests and Assays ChapterBacterial Endotoxins Test <85>


General Information ChapterMicrobiological 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 General Chapters for Sterile and Nonsterile Products

Microbiological Testing Requirements


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 contaminantsto a drug product.

• The sample scheme and acceptance criteria are based on its physical attributes, formulation, and intended use.



• 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)



• 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.


For Category 1 Products - Parenterals, Nasal, and Opthalmic

Bacteria

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 from the 14 days' count at 28 days

Yeast and molds No increase from the initial calculated count at 7, 14, and 28 daysFor Category 2 Products - Topicals

BacteriaNLT 2.0 log reduction from the initial count at 14 days, and no increase from the 14 days' count at 28 days

Yeast and molds No increase from the initial calculated count at 14 and 28 daysFor Category 3 Products - Oral Products

BacteriaNLT 1.0 log reduction from the initial count at 14 days, and no increase from the 14 days' count at 28 days

Yeast and molds No increase from the initial calculated count at 14 and 28 daysFor Category 4 Products - Antacids

Bacteria, yeast, and molds No increase from the initial calculated count at 14 and 28 days


Table 1. Compendial Product Categories



USP 55Biological 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 55Biological 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.



Products: Microbial Enumeration Tests

• 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)



Products: Microbial Enumeration Tests• 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>Microbiological Examination of Nonsterile 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>Microbiological Examination of Nonsterile 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>

Route of Administration

Total AerobicMicrobial Count

(cfu/g orcfu/mL)

Total CombinedYeasts/Molds

Count (cfu/g orcfu/mL) Specified Microorganism(s)

Nonaqueous preparations for oral use

103 102 Absence of Escherichia coli (1 g or 1 mL)

Aqueous preparations for oral use

102 101 Absence of Escherichia 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 aureus (1 g or 1 mL)Absence of Pseudomonas aeruginosa (1 g or 1 mL)

Cutaneous use 102 101 Absence of Staphylococcus aureus (1 g or 1 mL)Absence of Pseudomonas aeruginosa (1 g or 1 mL)

Nasal use 102 101 Absence of Staphylococcus aureus (1 g or 1 mL)Absence of Pseudomonas aeruginosa (1 g or 1 mL)

Table 1. Acceptance Criteria for Microbiological Quality of Nonsterile Dosage Forms


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

Auricular use 102 101 Absence of Staphylococcus aureus (1 g or 1 mL)Absence of Pseudomonas aeruginosa (1 g or 1 mL)

Vaginal use 102 101 Absence of Pseudomonas aeruginosa (1 g or 1 mL)Absence of Staphylococcus aureus (1 g or 1 mL)Absence of Candida albicans (1 g or 1 mL)

Transdermal patches (limits for one patch including adhesive layer and backing)

102 101 Absence of Staphylococcus aureus (1 patch)Absence of Pseudomonas aeruginosa (1 patch)

Inhalation use (special requirements apply to liquid preparations for nebulization)

102 101 Absence of Staphylococcus aureus (1 g or 1 mL)Absence of Pseudomonas aeruginosa (1 g or 1 mL)Absence of bile-tolerant Gram-negative bacteria (1 g or 1 mL)


USP <61> and <62>

Total AerobicMicrobial Count(cfu/g or cfu/mL)

Total CombinedYeasts/Molds Count

(cfu/g or cfu/mL)Substances for pharmaceutical use

103 102

Table 2. Acceptance Criteria for Microbiological Quality of Nonsterile Substances for Pharmaceutical 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.



Table 2. Minimum Quantity to be Used for Each Medium



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 TestsInterpretation 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 IdentificationUSP <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


• 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.

Investigations and Microbial IdentificationUSP <1113> Microbial Characterization,

Identification and Strain Typing




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, PhDPhone: (405) 271-1144Email: [email protected]

mailto:[email protected]

Microbiology Testing: USP Requirements for Sterile …acainfo.org/wp-content/uploads/2017/08/Session-7_Microbiology...USP Requirements for Sterile and Nonsterile Preparations ... At

Documents