Why Is Microbial Testing Necessary?
“How do we determine that our substance or preparation complies with an established specification for microbial quality?” This is what many manufacturers or producers of pharmaceuticals or medical grade products ask themselves. When manufacturing a product for human use or consumption, there are many considerations involved, not least of which is proving that the product or sample is safe for use, and determining how long the product can stay safe without changing its original composition or nature. Much of this centers on ensuring that microbial contamination is within established limits and standards.
History of the Harmonized MLT
To be able to determine whether a substance, a product, or a preparation complies with an established specification for microbial quality, testing needs to be performed. Many standards have been created not only to guard the manner of production but ultimately protect consumers and avoid release of a product that is unsafe to use. Historically, the United States Pharmacopeia chapter <61> Microbial Limits Test, provided directives for the estimation of the numbers of viable aerobic microorganisms present in a manufactured nonsterile product. It contained instructions to perform testing for: Plate Count (TPC), which includes Aerobic Plate Count (APC) and Yeast/Mold Count (YMC), and Screening/ Detection for Specified Microorganisms. USP Chapter 61 was also equivalent to Chapter 35 “Microbial Limit Test (MLT)” of the Japanese Pharmacopoeia.
In order to provide for standards that are applicable not just in the United States, but around the world, and because certain chapters from the USP are nearly identical to sections of the European Pharmacopeia, a course of harmonization was decided upon. International harmonization of the chapters was initiated and made official by the USP on May 1, 2009. The previous version of the MLT, USP <61>, was separated into 2 chapters, USP chapter <61> Microbial Enumeration Tests (MET) which includes Total Aerobic Microbial count (TAMC) and Total combined Yeasts and Molds Count (TYMC); and USP chapter <62> Tests for Specified Microorganisms. These chapters are equivalent to European Pharmacopoeia (EP) chapters 2.6.12 Total Viable Aerobic Count (TAC) and 2.6.13 Tests for Specified Microorganism. Incidentally, these chapters are exactly identical to the British Pharmacopoeia (BP) chapter B1. Tests for Specified Microorganisms and B2. Total Viable Aerobic Count. Because of these equivalencies, the new USP Chapters are commonly referred to as the “Harmonized MLT” or the “Harmonized Microbial Limits Test.”
Microbial Testing Procedures for Manufactured Products
Microbial Limits Testing must be carried out under conditions designed to avoid accidental extrinsic microbial contamination of the product to be examined during the test. However, any product/preparation with antimicrobial component(s) needs to be neutralized to remove its antimicrobial activity before testing. The validity of the test results depends largely upon demonstrating that the test articles do not inhibit the multiplication of microorganisms that may be present under any test condition.
Classification of product plays an important role in the selection of the test method (Plate Count Method, Most-Probable-Number (MPN), and Membrane Filtration), type of organisms that need to be screened, and acceptance criteria to be imposed according to the microbial quality prescribed by the standards. Before any testing to occur, Growth Promotion, Suitability of the Counting Method (Validation), and Suitability Tests for Specified Microorganisms (Validation) should be performed first. The ability of the test to detect microorganisms in the presence of the product to be tested must be established, and suitability must be confirmed if a change in testing performance or a change in the product is introduced that may affect the outcome of the test.
Antimicrobial Preservatives and Testing Considerations
Products may contain antimicrobial preservatives added to prevent proliferation or limit microbial contamination that may occur subsequent to the manufacturing process. Preservatives may also be added to inhibit the growth of microorganisms during normal conditions of storage and use, such as when microbes might be introduced inadvertently from repeated withdrawing of individual doses of a product from a containment vessel. Without preservatives, continued use could results in spoilage of the product or render it hazardous to users. Always keep in mind, however, that “Antimicrobial preservatives must not be used as a substitute for Good Manufacturing Practice.”
In order to support a claim that the added preservative is adequate to provide protection from adverse effects that may arise from microbial contamination or proliferation of microorganisms during storage and use, the USP Antimicrobial Effectiveness Test (AET), also known as Efficacy of Antimicrobial Preservation (EAP) – European (EP) and British Pharmacopoeia (BP) and Preservatives-Effectiveness Tests (PET)-Japan Pharmacopoeia (JP) should be tested to. This test is not intended to be used for routine control purposes, but instead is focused on challenging the preparation in its final container with prescribed inoculums of suitable microorganisms.
Compendial articles for testing have been divided into four categories based on route of administration. Each category has its own acceptable criteria for tested microorganisms. Individual product samples are inoculated with high concentrations of specific organisms and incubated at room temperature for 28 days. The population of any surviving microorganisms will be determined at a specific time interval depending on which standard is to be followed (each standard has its own intervals). Determination of the surviving microbial population will be performed using the Plate Count Method and calculating the log reduction of each microbial strain. However, prior to performing the actual test, a Plate Count Method Validation must be completed for the specified item.
The first step to successfully test any product is to know the product itself, and be able to classify it according to its intended final use. The nature and frequency of testing vary according to the products, as some may require freedom from one or more species of selected indicator microorganisms. The significance of microorganisms in non-sterile pharmaceutical products should be evaluated in terms of the use and the nature of the product, and the potential hazard to the user as well. Certain categories of products should be tested routinely for total microbial count and for a specified indicator of microbial contaminants. Regardless, it is still essential to apply strict good manufacturing practices to assure the lowest possible load of microorganisms. For reliable results, testing should be performed by personnel with specialized training in Microbiology and in the interpretation of microbiological results and data.
Pacific BioLabs can provide you with USP <61> and <62> Microbial QC Testing Services, including microbial limits testing, antimicrobial effectiveness testing, and microbial enumeration.
USP Chapter <61>
USP Chapter <62>
USP FAQ on chapter 62: http://www.usp.org/USPNF/pharmacopeialHarmonization/genChapter62FAQ.html