Pharmacopoeial harmony

As the world’s three major pharmacopeias are merged, what changes are in store for the microbiological examination of non-sterile pharmaceutical products? Maria Cristina Higgins gives us a handy breakdown of the changes you need to know about

HARMONISATION of methods for the microbiological examination of non-sterile pharmaceutical products in the world’s three major pharmacopoeias - the European Pharmacopoeia (Ph. Eur.), the Japanese Pharmacopoeia (JP) and the United States Pharmacopoeia (USP) - aims to reduce the overall cost of testing of pharmaceutical products worldwide and the time required for new products to become available to international markets. The revised chapters of the Ph. Eur. should have been implemented by the 1st January 2009. This article discusses some of the changes to the Ph. Eur. that have resulted from harmonisation.

Previously, the specifications and criteria for the microbial examination of non-sterile pharmaceutical products have differed between the world’s major pharmacopoeias.  Consequently, products intended for worldwide markets required extensive validation and testing in order to meet the requirements of each region’s pharmacopoeia. In order to reduce duplication, and the time taken for products to become available in different markets, the Pharmacopoeial Discussion Group (PDG) and the International Conference on the Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) have sought to harmonise the differing regional standards and specifications.

Like any product that is destined for human consumption or use, it is important to ensure that the microbial content of non-sterile pharmaceutical products falls within acceptable limits and does not present any risk to the consumer. Microbiological control is achieved through the implementation of current good manufacturing practice (cGMP) throughout the manufacturing process and is demonstrated by microbial examination of raw materials and finished products.

In order to satisfy international standards, the methods used for the microbiological examination of non-sterile pharmaceutical products should comply with the specifications outlined in the pharmacopoeias. These include microbial enumeration tests for total aerobic microbial counts (TAMC) and total yeast and mould counts (TYMC), in addition to tests for specified microorganisms, i.e. Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Salmonella, Candida albicans and Clostridium sporogenes. Similarly, the results obtained should meet the criteria specified within the pharmacopoeias.

The relevant chapters in the Ph. Eur. for the microbial examination of non-sterile products are  2.6.12 Microbiological examination of non-sterile products - microbial enumeration tests and 2.6.13 Microbiological examination of non-sterile products - tests for specified micro-organisms (equivalent to chapters 61 and 62 in the USP).  The major changes to these chapters are as follows1,2.

• Introduction (Ph. Eur. 2.6.12, Ph. Eur. 2.6.13)
Addition: “Alternative microbiological procedures, including automated methods, may be used provided that their equivalence to the Pharmacopoeia method has been demonstrated.”

• General procedures (Ph. Eur. 2.6.12, Ph. Eur. 2.6.13)
Addition: “If the product to be examined has antimicrobial activity, this is insofar as possible removed or neutralised. If inactivators are used for this purpose, their efficacy and their absence of toxicity for microorganisms must be demonstrated.”

“If surface-active substances are used for sample preparation, their absence of toxicity for microorganisms and their compatibility with inactivators used must be demonstrated.”

• Growth promotion test, suitability of the counting method and negative controls (Ph. Eur. 2.6.12); Growth-promoting and inhibitory properties of the media, suitability of the test and negative controls (Ph. Eur. 2.6.13)
Addition: “The ability of the test to detect micro-organisms in the presence of the product to be tested must be established. Suitability must be confirmed if a change in testing performance, or the product, which may affect the outcome of the test is introduced.”

• Preparation of test strains (Ph. Eur. 2.6.12, Ph. Eur. 2.6.13)
Addition: “Seed-lot culture maintenance techniques (seed-lot systems) are used so that the viable microorganisms used for inoculation are not more than 5 passages removed from the original master seed-lot.”

The preparation of microorganisms is described in more detail.

• Negative Control (Ph. Eur. 2.6.12, Ph. Eur. 2.6.13)
Addition: Recommendations on performing negative controls for testing conditions and testing of product. “A failed negative control requires an investigation.”

• Growth promotion of the media (Ph. Eur. 2.6.12); Growth promotion and inhibitory properties of the media (Ph. Eur. 2.6.13)
(For both Ph. Eur. 2.6.12 and Ph. Eur. 2.6.13)
Addition: “Test each batch of ready-prepared medium and each batch of medium, prepared either from dehydrated medium or from ingredients.”

Each test strain should be inoculated individually (no pool).

Add each strain at the time of mixing in the prescribed growth medium.

Addition: “For a freshly prepared inoculum, growth of the microorganisms comparable to that previously obtained with a previously tested and approved batch of medium occurs.”

(For Ph. Eur. 2.6.13)
The growth promotion section is expanded to include: which microorganism to use on which medium; and verification of nutritive, selective and indicative properties of the media.

Incubation times are also clarified:
o The shortest incubation period prescribed for growth promotion.
      The longest period of time specified in the test for inhibition.
o A period of time within the range specified in the test for indication.

• Suitability of the counting method in the presence of the product (Ph. Eur. 2.6.12)
The following is described:
o Preparation of the sample (this depends upon the physical characteristics of the product to be tested).
o Inoculation and dilution.
o Neutralisation/removal of antimicrobial activity.
o Recovery of micro-organisms in the presence of product.
o Results and interpretation.

In ‘Results and Interpretation’, there is reference to the maximum acceptable count being 2 times the criterion (rather than 5 times).

• Suitability of the test method (Ph. Eur. 2.6.13)
Addition: “If for a given product the antimicrobial activity with respect to a microorganism for which testing is prescribed cannot be neutralised, then it is to be assumed that the inhibited microorganism will not be present in the product.”

• Testing of products (Ph. Eur. 2.6.12)
The following is described:
o Amount of product used for the test.
o Examination of the product
- Membrane filtration.
- Plate-count methods.
- Most-probable-number method.
o Interpretation of the results
- Reference to total aerobic microbial count (TAMC) and total yeast and mould count (TYMC).

• Testing of products (Ph. Eur. 2.6.13)
The following is described for each test strain:
o Sample preparation and pre-incubation.
o Selection and subculture.
o Interpretation.

In addition, the test for bile-tolerant, Gram-negative bacteria contains a quantitative test and a test for absence. 

• Recommended solutions and culture media (Ph. Eur. 2.6.12, Ph. Eur. 2.6.13)
Note introductory statement: “The following solutions and culture media have been found to be satisfactory for the purposes for which they are prescribed in the test for microbial contamination in the Pharmacopoeia. Other media may be used provided that their suitability can be demonstrated.”

Detailed information is given.

In addition, the following changes to chapters 2.6.12 and 2.6.13 of the Ph. Eur. have just been published in Ph. Eur. supplement 6.53:
• A negative control should be performed when testing products and a failed negative control requires further investigation.
• in the section regarding Salmonella testing (indication properties) in chapter 2.6.13 (Tests for specified micro-organisms), the agar XLD (Xylose, Lysine, Deoxycholate) no longer requires to be inoculated with Escherichia coli, due to difficulties in growing this species on this medium.
• The description of the test for Clostridia has been reworded in order to improve consistency.
• The statement in the section regarding recommended solutions and culture media in chapter 2.6.13, “Other media may be used if they have similar growth promoting and inhibitory properties” has been reworded as it was considered misleading. The new wording is, “Other media may be used provided that their suitability can be demonstrated”.

Alternative methods to those described can be used if they are shown to be equivalent to the reference methods.  It is important, therefore, for alternative methods to be validated against the reference methods described in Ph. Eur. 2.6.12 and Ph. Eur. 2.6.13.

Considerable time can be saved by selecting microbiological products that have already been validated according to the recommended methods in the Ph. Eur, USP and JP, such as the Oxoid and Remel ranges of culture media for the microbiological examination of non-sterile pharmaceutical products4, which have been designed to help manufacturers comply with changing regulatory requirements5. The quality control of these media is performed quantitatively and qualitatively, using the recommended strains and incubation/temperature parameters outlined in relevant chapters of the Ph. Eur., USP and JP.

In addition, the complete range of specified ATCC microbial test strains recommended in the microbial examination of non-sterile products chapters of the Ph Eur, USP and JP are also available, ready-to-use, in vials that consistently deliver a specific range of colony forming units or in disposable bacteriological loops containing stabilised micro-organisms. These test strains are ideal for use in quality control procedures in the pharmaceutical industry, including performance testing, method validation, microbial examination, bioburden testing, bacteriostasis/fungistasis testing, and growth promotion testing.