The variety of microbiological tests makes it difficult, if not impossible, to prescribe a single, comprehensive method for
validating all types of tests. By their very nature, microbiological tests possess properties that make them different from
chemical tests. Consequently, the well-known procedures for validating chemical tests are not appropriate for many microbiological
tests. Yet, it is necessary to validate microbiological tests if they are to be useful for controlling the quality of drug
products and devices. Test-method validation provides assurance that a method is suitable for its intended use. Given this
definition, any rational company would want to be sure that its methods are validated.
Some tests, such as bioburden or viral titer tests, are quantitative in nature while other tests, such as those for the presence
of objectionable organisms, are qualitative. As with chemical tests, these differences necessitate different validation approaches.
The purpose of a test also may change the procedures for running and validating it. As an example, consider a drug that will
be orally administered. Normally, sterility is not a major issue, and the specification allows for a considerable number of
organisms. However, if the drug will be administered to immunocompromised cancer or AIDS patients, the bioburden level must
be reduced considerably, increasing the test sensitivity required in the validation study.
The nature of the test material itself changes how a test is run and the validation protocol. Consequently, testing for objectionable
organisms is different when testing a diuretic for hypertension or an antibiotic for treating pneumonia. Also, a procedure
that works perfectly well for checking the bioburden of granulated sugars may fail with sodium chloride. These differences
make full coverage of the topic impossible within the context of this primer. This article will present the general considerations
that apply to most microbiological tests. However, three excellent publications are available to analysts preparing validation
study protocols for microbiological methods (see Suggested Reading).
Note also that certain microbiological tests are already associated with well defined validation procedures. For example,
the endotoxin test and USP bacterial enumeration tests have clearly defined validation procedures. In addition, individual
countries may have specific requirements that modify or change standard procedures. If a test is associated with a compendial
or regulatory validation procedure, workers are advised to follow that procedure unless there are clear reasons for not doing
so. In such cases, the reasons should be documented and filed with the test procedure.
Media
The suitability of the medium used for cultivating organisms or cells obviously can have a major impact on the test results.
Some organisms are extremely fastidious and require a precisely defined medium with several complex nutrients, while others
grow in the presence of inorganic salt mixtures and simple carbon sources. It is commonly argued that delicate, fastidious
organisms cannot survive manufacturing processes and should not be of concern, but organisms as delicate and fastidious as
mycoplasmas can appear in final preparations of biologics.
In addition to the nutrient composition of the media, more general factors such as pH and ionic strength must be validated.
While it is commonly believed that media in the range of pH 6.0 – 8.0 are suitable for sterility and bioburden studies, individual
organisms may require a more restricted range. The same holds true for ionic strengths and osmolalities outside of the human
physiological range. Shifting the pH range from 6.0 – 7.0 to 7.0– 8.0 and raising the ionic strength to 300 mOsm may select
for a different set of organisms than those that would be present in the lower pH range at 150 mOsm.
Most validation schemes require the use of five or more "indicator organisms" to demonstrate the medium's ability to support
growth. In addition to aerobic bacteria, anaerobic organisms, yeasts, and molds are usually included. This is an important
step since a finding of "no growth detected" is meaningless if the medium was incapable of growing any organisms. This leads
to two important points.
