Microbiology testing is an indispensable process in the pharmaceutical manufacturing process. The microbial testing requirements for sterile and nonsterile preparations must adhere to the requirements outlined in the United States Pharmacopeia (USP). It is particularly important to understand the recommendations outlined in the USP Microbiology Test General Chapters (chapter number less than <999>) and USP Drug Standards are enforceable in the United States by the Food and Drug Administration (FDA).
United States Pharmacopeia (USP)
There are six USP General Chapters that are relevant to the microbiological testing requirements of sterile and non-sterile products and are listed below. These chapters contain some mandatory requirements, and these should be adhered to by the manufacturers.
General tests and assays (Microbiological tests)
- Chapter <51> Antimicrobial Effectiveness Testing
- Chapter <55> Biological Indicators-Resistance Performance Tests
- Chapter <61> Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests
- Chapter <62> Microbiological Examination of Nonsterile Products: Tests for Specified Organisms
- Chapter <71> Sterility Tests
General tests and assays (Biological tests and assays)
- Chapter <85> Bacterial Endotoxins Test
In addition, there are General Information Chapters that provide information on microbiological testing requirements. These general information chapters include (but not limited to)
- Chapter <1111> Microbiological Examination of Nonsterile Product: Acceptance criteria
- Chapter <1117> Microbiological Best Laboratory Practices
- Chapter <1229.3> Monitoring of Bioburden
Antimicrobial effectiveness testing
Many pharmaceutical products contain an antimicrobial or preservative to kill or inhibit unwanted microbial growth. An antimicrobial effectiveness testing (AET) is a microbial challenge methodology to assure that the added antimicrobial can produce the intended inhibitory effect against targeted microorganisms. This applies to injections, topicals, oral products and antacids packaged in multiple-dose containers.
The descriptions of AET methods are outlined in USP <51>. Sample schemes and acceptance criteria may differ between pharmaceutical products as the product’s physical attributes, formulation, and intended use are different. In addition, a suitable procedure to detect the challenge microorganisms (method suitability) and the growth-promoting capabilities of media (growth promotion) used for culture must be established before testing.
The test samples are typically inoculated by microorganisms that are likely contaminants to the products, and the inoculated test samples will be evaluated throughout a 28-day incubation period where the log-reduction in the microorganism’s population can be calculated.
Biological Indicators-Resistance Performance Tests
Biological indicators used in the sterilization process should be evaluated and their suitability for use established before they can be utilized unless there are existing data to support their use in these processes. One such evaluation is the resistance performance test that verifies the resistance of the microorganisms (contained in the biological indicator – typically a strip or ampule containing the spore) to the sterilization measures.
Resistance testing is performed by exposing the biological indicator to at least five different conditions that are consistent with its intended use, then incubating and counting the recovered colonies.
Microbial Enumeration Tests
For non-sterile pharmaceutical products, microbial contamination testing should be performed following the methodology described in the USP Chapter <61> Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests.
The ability to detect the likely microorganisms in the presence of the pharmaceutical products (method suitability) as well as the growth-promoting capabilities (growth-promotion) of the culture media used for this procedure must be established first before performing the enumeration test.
Microbial enumeration tests including Total Yeast and Mold Count (TYMC) and Total Aerobic Microbial Count (TAMC) analysis are typically employed for this purpose.
The products to be tested will be diluted with pH-balanced, isotonic diluents to overcome the effects of any antimicrobial, and the resultant samples are tested by membrane filtration or plate count method.
At the end of the incubation period, the colony-forming units (CFU) are counted, and the results are further extrapolated based on the dilution factor to arrive at the total aerobic microbial count and the total yeast and mold count.
For sterility testing, the absence of microbial in the product is tested to ensure the pharmaceutical product is truly free of microbial contamination. This applies to all substances, preparations, or articles that are required to be sterile according to the USP.
It is worth noting that a satisfactory result from sterility testing can only indicate that no microorganism has been found in the sample that is examined under the test condition, and the procedure itself is not designed to guarantee the product batch is sterile. Instead, sterility assurance is primarily achieved by the validation of the sterilization process or the aseptic procedures.
A positive microbiology test is considered when microbial growth is detected after 14 days of incubation at the appropriate temperature (7 days at 20 оC – 25 оC followed by 7 days of incubation at 30 оC – 35 оC).
There are two methods to perform sterility testing:
- Direct inoculation
- Membrane filtration can be subdivided into Open Membrane Filtration (OMG) or Close Membrane Filtration
When there is evidence of growth, the product is considered not sterile.
Bacterial endotoxin testing
Bacterial endotoxins are produced by gram-negative bacteria that disintegrate. At certain concentrations, these endotoxins could be fatal to the end users. USP Chapter <85> outlines the requirements for bacterial endotoxin tests on finished products to ensure the level meets the specific criteria.
Endotoxins are durable molecules that could persist in or on a product even after the gram-negative bacteria that produce the endotoxins have been destroyed. Hence, it is possible to detect the presence of endotoxin even if the product sample meets the requirement of the sterility test.
Bacterial endotoxin can be tested using the Limulus Amebocyte Lysate (LAL) Test. Over the past years, US FDA and USP have recognized the validity of different LAL tests for endotoxin testing. For example, the four FDA-approved methods are (1) the gel-clot, (2) the turbidimetric (spectrophotometric), (3) the colorimetric (Lowry protein), and (4) the chromogenic assay.