The Harmonization of the Microbial Limits Test - Enumeration

The USP and the European Pharmacopoeia (EP, Pharm Eur) Microbial Limits Tests are in the final stages of harmonization. They were signed off to Stage 6A at the November, 2005 meeting of the Pharmacopeial Discussion Group (PDG) held in Chicago, IL USA (USP 2006a). However, the signed-off versions have yet to be published. This makes the description of the test a bit difficult, as the current tests will be disappearing, and the final, harmonized test is not yet public knowledge. However, we do know that the harmonized tests do not differ greatly from the drafts published in 2003 (USP 2003a, USP 2003b, USP 2003c), and so we will use those drafts as the description of the finalized test.
The Microbial Limits Tests are actually two chapters in the current USP: Current USP <61> Microbial Limits Tests (USP 2006b) and <1111> Microbiological Attributes of Nonsterile Pharmaceutical Products (USP 2006c). This will be modified in the harmonized version to mirror the European format:
USP
EP
<61> Microbiological Examination Of Nonsterile Products: Microbial Enumeration Tests
2.6.12 Microbiological Examination Of Nonsterile Products: Microbial Enumeration Tests
<62> Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms
2.6.13 Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms
<1111> Microbiological Quality of Nonsterile Pharmaceutical Products
5.1.4 Microbiological Quality of Nonsterile Pharmaceutical Products
Table 1: Harmonized Chapter Numbering Scheme
This review will only address the microbial enumeration portions of the harmonization effort – that which will become USP chapter <61> and Pharm. Eur. chapter 2.6.12.
The microbial enumeration test is a basic, simple design to count the number of CFU in a nonsterile product or raw material. The preferred method is to put the material into solution and then plate aliquots to determine the CFU/gram (or mL) of initial material. If the product cannot be put into solution, there are provisions to use the Most Probable Number method (MPN – see FDA BAM website). The method of plating can be either pour plate, spread plate or the filtration of material and then placing the membrane filter on the surface of an agar plate. The membrane filtration method should only be used when there are few expected colony forming units in the material to be tested as it is a good method to test a large volume of liquid, but can only count up to approximately 100 CFU/membrane.
The harmonized method provides a great deal more detail than any of the current pharmacopeial methods in terms of demonstration of method suitability (validation of the method) and in terms of media growth promotion.
The demonstration of method suitability should be performed using the challenge organisms listed (see Table 2 below) in accordance with the recommendations found in USP chapter <1227> (USP 2006d). Growth promotion is an area of some ambiguity in the compendial text. Although media growth promotion is not described in the tests, demonstration of media suitability is required, and the draft USP Chapter <1117> (USP 2004) provides assistance in designing the studies using 10-100 CFU of the challenge organisms.
A major concern of many QC workers is if the changes in the harmonized chapter will necessitate revalidation of existing assays to meet the requirements of the harmonized test. There are several considerations that might lead to revalidation – a required change in media, in volume of material required for testing, in general testing conditions. It is difficult to determine whether all product types would require revalidation, and so a summary table is provided (Table 2) describing the critical aspects of the current Microbial Limits Tests (Enumeration) and the draft harmonization text. The summaries provided in Table 2 are only meant as an aid, the decision as to whether or not revalidation is necessary rests with each individual facility for their particular products.