Validation of Microbiological Tests

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.

First, the indicator organisms are supposedly representative of the types of organisms that will be encountered during the testing, but this is not necessarily true. The indicator organisms are a subset of organisms that are known to grow on properly prepared media, but the organisms contaminating a manufacturing process may not belong to that subset. As a result the quality control laboratory may repeatedly face what appears to be a microbial contamination event despite monitoring cultures that show no growth. It is very important to know what organisms are normally present in the working environment and to include these environmental isolates in a validation program. There is little value in proving that a medium will support the growth of indicator organisms if the environment is full of organisms with very different cultivation requirements.

The second issue involves media handling. The qualification or validation study may require autoclaving the medium and then pouring culture plates as the autoclaved material cools. In laboratories with a low testing load, the excess material is often poured into large tubes or culture flasks to cool and solidify and then stored for future use, usually in a refrigerator. However, when future testing is done, the second heating of the medium may not be captured in the qualification or validation check and may not even be mentioned in the test procedure. If the agar is melted under gentle conditions and quickly poured, there may be no problem, but in some cases, technicians have placed the flasks in microwave ovens to heat the medium while taking a short break. With a powerful microwave oven it is easy to boil the medium for an unknown period of time. This can destroy nutrients or produce toxic or inhibitory substances. Consequently, in laboratories where this second heating is a common practice, this procedure must be captured in the validation and described exactly in the test procedures.

When preparing the validation protocol, the analyst should specify the recovery level expected for each of the indicator organisms. Generally, recovery of at least 80% of the inoculum or control is desirable. Recovery of less than 50% is usually unacceptable and should raise questions about the presence of inhibitory substances, especially when the testing is taking place in the presence of a raw material or product intermediate. It may be necessary to introduce — and validate the performance of — an agent that inactivates the inhibitor. It is important to set the specifications before the study is conducted and to hold to these specifications. If specifications are not pre-set and the test system cannot meet general acceptance specifications, it is very easy to set "acceptable" specifications that would otherwise have been unacceptable. The other problem is the "specification creep" that occurs when a recovery of 78% is found and the specification is 80%. A quality assurance or quality control worker who allows the 78% to pass will soon face the expectation that 75% should pass because it is "only slightly different from the other one." Over the course of a few years, an 80% specification can gradually turn into a 70%, then 65%, specification.

Environment

The incubation temperature can have a major effect on the ability of an organism to grow in a given medium. It is well known that yeasts and molds require a different incubation temperature than bacteria in a sterility test. Similarly, cells in tissue culture are often extremely sensitive to small changes in temperature, not only for their growth but also in their susceptibility to being infected or lysed by viruses. The analyst may need to develop temperature curves to justify the incubation temperatures used for the test. It is also important to verify the incubator's ability to maintain the set temperature within the specified range. If a four-degree temperature variation can cause a significant change in the test results, the incubator's ability to hold a ±1° C range at all internal locations is critical. This may not be covered in a validation study, but it should be included in the incubator's qualification studies.

In addition to the usual range from 20 – 40° C, it may be necessary to demonstrate the ability to grow organisms at extreme temperatures. If it is necessary to monitor the presence of microbes in a hot or cold room, it will be necessary to demonstrate an ability to cultivate thermophiles or psychrophiles in addition to organisms that grow under more normal conditions. While the significance of these extremophiles may be open to question, their presence and the possibility that they may leave residues such as endotoxins must be considered.

The atmosphere in which the test system is immersed can have a major effect. Anaerobic organisms cannot grow in the presence of oxygen, and tissue cultures may require the presence of 5% CO₂ to grow well. Certain facultative organisms will adjust their metabolic paths to cope with reduced levels of oxygen. This, in turn, can affect their growth rates. When media for general purposes, such as sterility tests, are being considered, it is normal to include one medium that provides anaerobic conditions. The detection of anaerobes is important as they include toxin-producing and other pathogenic bacteria.

Quantitative Issues

One of the problems with quantitative microbiological tests is that as microbe counts become smaller, straight-forward linear behavior is less common than that which follows the Poisson distribution. This is because random distribution is not even distribution. Most quantitative tests for microorganisms require the plating of dilute liquid samples, and it is normal to prepare samples to ensure the dispersion of microbes and a random distribution of bacteria or viruses. When concentrations are high, the lack of even distribution is not a problem; simple linear averaging methods can compensate for the uneven distribution. Problems arise with smaller numbers of microbes.

Consider an example where there are exactly 100,000 organisms per mL. If 0.1 mL is taken and mixed with 0.9 mL of a diluent, it is highly unlikely that the new suspension will contain exactly 10,000 organisms; it would not be surprising to have anywhere from 9,800 – 10,200 organisms. Back-calculating the result produces a range from 98,000 – 102,000 organisms in the original sample, and, if there were enough replicates, the results could be averaged to obtain a number indistinguishable from 100,000. This is the result that would be expected based on linear thinking.

However, if there were only 10 organisms per mL, it is quite possible that a 0.1 mL aliquot would not contain any organisms at all. In fact, in this situation about one third of the aliquots will not contain a single organism. This could lead to the conclusion, on averaging, that the sample only contained 6.7 organisms per mL, which is a significant deviation from the true value.

A transition occurred from a high density that produces a fairly smooth, homogeneous distribution of organisms to a low density that results in organisms that are distributed with significant distances between them. Under these conditions, the suspension behaves according to the Poisson distribution and assumptions related to a normal distribution no longer hold. The Poisson distribution is an exponential function. The problem is that parameters such as the standard deviations may be logarithmic in nature, and when attempts are made to make these numbers "real" by taking the antilogarithms, the results may actually have no "real" meaning. This can cause great difficulties when attempting to validate quantitative microbial test procedures.

When it is necessary to deal with the Poisson distribution, it is wise to consult a statistician who is versed in the use of this distribution. It appears that the transition to the Poisson distribution occurs when approximately 100 colonies or plaques are counted. This is unfortunate because at this level many analysts will declare a colony or plaque count to be "too numerous to count" (TNTC) to avoid the tedium of these measurements. Therefore, most colony or plaque counting procedures actually operate under the Poisson distribution and calculations based on the normal distribution will be incorrect.

Revalidation

The frequency of revalidation is a contentious question. There are many tests, such as the growth promotion test on culture media, that are essentially self-validating and are run frequently. It could be argued that if performance parameters (for example, percent recovery of indicator organisms) are monitored via control charting and no significant changes are seen, revalidation is unnecessary. However, control charting usually does not measure all the parameters included in validation studies. Consequently, it is wise to revalidate tests after any major change in constituents or procedures; in fact, revalidation may be needed to justify the changes. Changes in suppliers (especially of media components) and changes in the composition of test samples have resulted in major changes in microbiological tests. Finally, it is probably wise to revalidate procedures approximately every second year to protect against unseen or unreported changes. A media supplier may change its own suppliers or change its processing procedures without notifying customers. The supplier may have no idea of the impact these changes could have on the end use of their product. In addition, personnel changes in the laboratory and the maturing of analysts' techniques can have an effect.

Suggested Reading

Carroll MC. A multifaceted look at the microbial limits test. In: R Prince, editor.

Microbiology in Pharmaceutical Manufacturing.

Baltimore, MD: PDA; 2001. pp. 519–535.

PDA. Evaluation, validation and implementation of new microbiological testing methods: PDA Technical Report No. 33. PDA Journal of Pharmaceutical Science and Technology 2000; 54(Suppl. TR33).

Petitti DM. Practical considerations for the development, validation, and transfer of analytical test methods. In: R Prince, editor. Microbiology in Pharmaceutical Manufacturing. Baltimore, MD: PDA; 2001. pp. 723–746.

Steven S. Kuwahara, Ph.D., is the principal consultant and founder of GXP BioTechnology LLC, PMB 506, 1669-2 Hollenbeck Avenue, Sunnyvale, CA 94087-5042, 408.530.9338, stevekuwahara@yahoo.com.