Validation of microbiological methods

Microbiological methods are used in many applications in the pharmaceutical industry. Since it involves working with living organisms, various factors must be considered during an ongoing experiment. Many traditional methods have been established over the years and are listed universal methods in the pharmacopoeias. Considering the given requirements in the instruction their application doesn’t need a validation, only a verification is sufficient. Due to recent technological advancements, it is possible to use alternative methods developed by the laboratory or a company itself. To ensure the new method also demonstrably leads to correct results, a previous validation is must.

This means that test conditions and performance must be checked for suitability. A regulatory overview helps to understand the individual requirements and factors to be considered when validating microbiological methods.

 

What are the different types of microbiological methods?

In order to better understand the topic of validating microbiological methods, it is first necessary to define which types of microbiological methods are available. As a rule, one differentiates between three different variants, whereby these can also be combined:

a) Qualitative tests:

Qualitative microbiological testing is about checking for the presence of microorganisms in a sample. An example is sterility testing (e.g. according to USP <71> or Ph. Eur. 2.6.1). In general, the procedure is such that a specified portion of the sample is added either in broth as liquid medium or on a solid nutrient medium. The growth of potential germs can be detected after appropriate incubation based on turbidity of the liquid medium or by colony-forming units on the solid medium.

b) Quantitative tests:

Quantitative testing is not just about detecting microorganisms in a sample, but to find out in what quantity. An example is an enumeration test for living germs (according to USP <61> or Ph. Eur. 2.6.12) or flow cytometry.

c) Tests for identification:

In these tests, the presence of specific microorganisms is determined. Usually, biochemical test procedures are employed for this purpose. Different properties of the microorganisms such as morphological features (size, appearance or gram behavior) are investigated. Furthermore, physiological properties are considered and certain active enzymes in the bacterium are estimated to be determined. Additionally, for identification, DNA sequence or cell wall structure analysis can be performed.

 

What are the regulatory requirements and how should we proceed?

For the validation of microbiological methods, there are several regulatory requirements that must be considered, depending on the field of application:

USP (United States Pharmacopeia)

The American Pharmacopoeia contains several chapters relevant to microbiological methods. Particularly interesting are the chapters USP <1113>, <1223> and <1227>, the contents of which are summarized below:

USP <1113> (Microbial characterization, identification and strain typing):

This chapter describes the various ways in which certain types of microorganisms can be identified and the verification of such methods. The determination of a bacterial strain may become necessary if limits are exceeded or deviations occur in an aseptic filling process of a drug. To identify a bacterium, it is important to isolate it and then cultivate it under optimal environmental conditions. This is followed by the evaluation of appearance (e.g. color and shape of colony forming units) and gram or spore staining. Subsequently, a biochemical screening takes place, which includes tests for the detection of specific enzymes (oxidase, catalase, coagulase). When deemed necessary, further tests are possible:

• Phenotypic Methods: This includes e.g. the detection of certain fatty acids / fatty acid profiles or total cell composition by MALDI-MS. It is important that the bacteria grow as a monoculture under optimal conditions in sufficient quantity before analysis.
• Genotypic Methods: They are necessary for a much more specific typing of microorganisms (e.g. by PCR, 16S and 23S rRNA sequencing or hybridization techniques). They are technically complex and are usually carried out by contract laboratories.

 For the verification of the methods described above, the USP chapter also provides some suggestions. For example, reagents, reference organisms and instruments should be tested for suitability before use. Furthermore, for the functioning proof of a method up to 50 successful identifications may be required to be done. Parallel testing with a reference laboratory can be considered. Another variant is the comparison in relation to an already approved method or the successful identification of already known organism species. In general, a match of at least 90% should be used as acceptance criterion for verification. The most important verification parameters are trueness and reproducibility. In addition, sensitivity, specificity as well as positive and negative predictive values must be examined.

 

USP <1223> (validation of alternative microbiological methods):

The chapter <1223> of the USP is a very large chapter that sheds light on what companies need to consider if they want to use an alternative microbiological method. The chapter refers to both quantitative and qualitative tests. As mentioned earlier, microbiological tests depend on many factors. These include, among other things, the nutrient medium used, incubation conditions, physical state of the microorganisms and the sample material to be tested. All these factors have a potential impact on bacterial growth and can alter test results. If a company wants to use a new method in routine analysis, it should first define which internal requirements exist for the new method. This is listed in the USP as so-called "User Requirements". It should be determined in advance which equipment is necessary for the performance of the test and whether the alternative technology to be used is comparable to already established compendial methods. The possible sample volume to be tested should also be considered. If the equipment has been procured, it must be qualified before starting the validation. It must be checked whether the device fulfills the previously defined requirements. Details for such a process can be found e.g. in USP chapter <1058>.

For validation, the USP describes different validation parameters, which must be taken into account, depending on the test method. The following table gives a brief overview:

Validation parameter	Qualitative tests	Quantitative tests
Trueness	-	+
Precision	-	+
Specificity	+	+
LOD	+	+
LOQ	-	+
Linearity	-	+
Range	-	+
Robustness	+	+
Repeatability	+	+
Ruggedness	+	+
Equivalence	+	+
- Not required + required

One crucial parameter is “equivalence”. It must be shown that the alternative method is equivalent or better compared to the pre-existing compendial method. This can be done e.g. in case of a microbial enumeration method by showing no statistically significant difference in the results obtained by the two methods. An improvement may be, for example, a higher sensitivity in a quantitative test.

 

USP <1227> (Validation of microbial recovery after antimicrobial exposure to pharmaceutical products):

This chapter specifically addresses the validation of recovery methods for microorganism exposed to antimicrobial exposure. This is e.g. the case when the drug under study is an antibiotic and thus shows per se an antimicrobial effect. In doing so, the USP first describes which influencing factors exist and to what extent they should be regarded during validation. Great attention is paid to the neutralizing agent. There are three major options to eliminate the antimicrobial effect: chemical agents or enzymes, dilution and membrane filtration. In some cases, a combination of these variants might be necessary.

In the case of a validation, the following points must be addressed: Firstly, the neutralizing agent must demonstrably inhibit the antimicrobial effect of the product, secondly, the agent used must have no toxic effect on the microorganisms. To prove this, three different test groups must be prepared and analysed at least in triplicates:

• Product + neutralizing agent + microorganisms
• Neutralizing agent + microorganisms (without sample)
• Buffer + microorganisms (positive control)

To make the evaluation of the validation feasible, a certain number of colony-forming units on the solid agar must certainly grow. The chapter also provides useful information on determining this number.

 

European Pharmacopoeia (Ph. Eur.)

Section 5.1.6 of the current issue (9.2) of the European Pharmacopoeia provides a well-structured overview of how alternative microbiological methods are developed and validated. It is mentioned that although conventional methods are proven, they are sometimes very slow, and therefore alternatives to compendial methods are becoming increasingly important. Even if they are not used directly for the release of specific products, they are certainly useful for in-process controls (IPC) or as part of environmental monitoring.

The section details alternative methods and describes which principles underlie them and what the pros and cons are. Thereby, the pharmacopoeia refers to all three groups of microbiological tests. This listing makes it easier for companies to find a suitable method for their purpose.

The Pharmacopoeia also makes it clear what is required and which documents must be prepared for carrying out the validation. Like the USP, the Ph. Eur. first requires that internal requirements be specified in a URS (User Requirement Specification). This is followed by a risk-benefit analysis. This is important to work out the advantages the alternative method has over the pharmacopoeia method and whether new risks may arise. A detailed and well-thought-out risk analysis helps later to determine the validation parameters and to ensure that the requirements made in the URS are met.

In validation, the Ph. Eur. differentiates between primary validation and validation for a specific product (validation for the intended use). The primary validation is usually carried out by the manufacturer of the device to be used and serves to characterize the detection principle. The pharmaceutical company then must "only" carry out the validation of the method for its intended use. According to the usual procedure, the four phases of a qualification are necessary for this (DQ, IQ, OQ, PQ).

The actual method validation falls under the PQ category, in which the parameters listed below in the table, depending on the type of method, are to be checked:

Validation parameter	Qualitative tests	Quantitative tests	Identification tests
Trueness	+ (1)	+	+
Precision	-	+	-
Specificity	+	+	+
LOD	+	- (2)	-
LOQ	-	+	-
Linearity	-	+	-
Range	-	+	-
Robustness	+	+	+
Suitability testing	+	+	-
Equivalence	+	+	-
- Not required + required 1 could be used as an alternative to LOD 2 could be required in some cases

 

PDA TR 33 (Parenteral Drug Association Technical Report 33)

The report was first published in 2002 and revised last time in 2013. It deals with the implementation of alternative or rapid microbiological methods (RMM) and can be considered as being a supplement (and update) to the requirements made in the pharmacopoeias. The goal is to aid pharmaceutical companies to successfully validate new methods and meet regulatory requirements.

 

EU GMP Guidelines, Annex 15, Chapter 9 (ZLG Aide-mémoire 07123101)

Two aspects of the validation of microbial methods to be observed are also mentioned in Annex 15 of the EU GMP Guidelines, which are also taken up by the ZLG Aide-mémoire AiM 07123101:

• The validation of microbiological methods that are used a) to check the microbiological quality of products (à drug products or their active pharmaceutical substances and excipients) or b) for the microbiological monitoring of cleanroom surfaces should prove in each case that the recovery of microorganisms is not impaired by the product (a) or the cleaning agents and disinfectants (b).

The AiM also refers to Ph. Eur. chapter 5.1.4..

 

Conclusion

The validation of microbiological methods is a complex issue in which companies must consider many different factors in advance. Although a regulatory overview (Ph. Eur. 5.1.6 or USP <1113>, <1223> and <1227>) helps to familiarize with the requirements, it does not provide a standard solution. The respective requirements and test conditions must therefore be developed individually by the responsible employees in a team to establish a proven functioning method considering the advantages and risks. The technical report 33 of the PDA can be of good help for this.