| Validation of Microbiological Methods Roger Dabbah Tilntrsect Solitons, Rockvle, Maryland, U.S.A David A. Porter Vectech Pharmaceutical Consultants, nc., Farmington Hils, Michigan, U.S.A. INTRODUCTION Numerous microbiological methods in use today date back to the time of Louis Pasteur. These methods are still useful and effective but there is a substantial interest in the development and use of more modern techniques. In fact most pharmacopeial microbiological methods in USP, EP, and JP are closer to the methods of Pasteur’s time and. are the so-called classical methods. Advances in method- ologies and instrumentation should be incorporated in the pharmacopeial methods that are cited by the FDA for compliance purposes. Microbiological methods, ‘especially if intended for replacement of more conven~ tional approaches require validation. If the new methods that are desirable because of advantages speed, accuracy, specificity, and generation of more quantitative data are to replace the pharmacopeial methods, then they also have to be shown to be equivalent (equal or better) than the pharmacopeial methods. This situation exists in Europe, Japan and in the US.A., where regulatory agencies will accept alternative methods to the pharma- copeial methods for compliance purposes. ‘There are three types of microbiological methods that need to be validated, qualitative tests, quantitative tests, and identification tests. Each type of test that follows has a different pattern in their validation and ‘will be examined separately. ‘The special case of validation of equivalency between a pharmacopeial microbiological test and alternative test will de discussed in each of the sections, ‘Validation of any kind of assay requires that certain parameters be examined, including accuracy, precision, ‘specificity, limit of detection, limit of quantification, Linearity, range, ruggedness, and robustness. However, for microbiological assays and depending on the type of assay used, these parameters are not applicable. VALIDATION OF QUALITATIVE MICROBIOLOGICAL METHODS ‘The principal function of a qualitative microbiological method is to determine whether the sample under test contains any viable microorganism, An example of such a ‘Abbreviations used in this chapters ATCC, American type culture ‘collection; EP, European Pharmacopoeia; FDA, Food and Drog Admin- station; FP, Janese Pharmacopoc; NE, National Formulatory; USP, United State Pharmacopeis test is the sterility test. The validation parameters to be considered include, accuracy, precision, specificity limit ‘of detection, ruggedness, and robustness ‘Accuracy Assterility testis not used to provide assurance of sterility of an entire batch. The assurance of sterility of a batch of sterilized products is obtained through the validation of the sterilization cycle or process. Accuracy pertains to the ability of the test to detect the presence of viable microorganisms if present in the sample under test, ‘The issue ofa false positive and false negative {rom a sterility test can have serious repercussions. A false positive, if it cannot be ruled out decisively due to technical error, will likely result in discarded lots of products, costly in terms of labor and material. A false negative would declare a batch sterile while it is not and could result in harm to patients using the product and potential liability to the manufacturer. ‘The limitations of a sterility test are well known and will depend on the ability of media to permit the growth of surviving microorganisms, the ability of microorgan- isms to grow under temperatures and time of incubation used. If the microbiological method used for sterility testing does not require the growth of microorganisms, it might require the uptake of vital markers and sub- sequent ability to fluorescence For methods that require the growth of microorgan- isms to a level that can be detectable by turbidity examination, the validation of the method will include the testing of the capabilities of media used to support the growth of likely microorganisms. The growth promotion test used in the USP Sterility Test is an example of validation of media, The type of microorganisms used for USP growth promotion is arbitrary but is a compro- rise, since it includes aerobes, anaerobes, and fungi Often, environmental isolates can be included in a ‘growth promotion test, or microorganisms isolated from Positive sterility tests are also used. A growth promotion test will use very low concentration of microorganisms since one cannot expect a wholesale survival of micro- organisms following sterilization. Another issue is the possibility of the test sample itself being inhibitory to the growth of microorganisms, A validation will include qualification of the sterility test for each and every Product to be tested. This can be done using the same ‘microorganisms used for growth promotion of media that are inoculated at very low levels (less than 100 CFU) but666 LABORATORY METHODS AND QUALITY ASSURANCE in the presence of the product to be tested. Modification of the sterility test might be necessary if the sample is inhibitory to the challenge microorganisms. For methods that do not rely upon reproductive capability of viable microorganisms, the presence or absence of microorganisms will depend on the ability of the vital marker to be incorporated into microorganisms that are viable and not in microorganisms that are not viable, Using a low level of challenge microorganisms one ccan then validate the test using incorporation of a vital ‘marker and its ability to fuoresce under the conditions of the test. The special case of validation of an alternative microbiological test to a pharmacopeial introduces another complexity, since it involves the comparison of two tests and the determination of their equivalence. If one wants to establish equivalence of accuracy of two ‘microbiological methods used for qualitative determina- tion, the null hypothesis becomes one of inequality (the results ofthe two methods are significantly different) If the methods are not significantly different then the two ‘methods are equivalent. Precision Precision is the repeatability of atest, Ifa microbiological procedure is applied repeatedly to a specific lot of product or material, the results should be the same. The rate of false positive and false negative in samples inoculated with microorganisms at low levels using perhaps a serial dilution scheme would give an indication ‘of precision of the microbiological method for methods relying on growth of microorganisms. For methods not relying on growth, the precision can be determined using the quantitative data obtained for example by fluorescence measurement ‘The comparison of the precision of a pharmacopeial ‘method and an alternative method can be measured by subjecting the inoculated samples to the two procedures and determining their equivalency. Specificity For methods based upon growth of microorganisms, the specificity parameter can be measured as turbidity, development of CO2, and changes in media pH that can allbe shown to be due to microbiological growth and that must reach a threshold in order to be detectable For methods not based upon growth of microorgan- isms, the presence of interfering components in the test sample must be ruled out, For example, for a method that indicates viability via the fluorescence of a viable marker it should be established that no other component in the sample, medium, or diluents or reagents used can produce fluorescence. ‘The specificity parameter for an alternative method to a pharmacopeial method is not comparable per se, since specificity for each method is dependent on the mechanisms and concepts used for each, Limit of Detection ‘The limit of detection is the lowest number of micro: organisms that must be present in the test system to elicit 1 positive response that is a signal that can be discerned. from the underlying noise. For methods relying on growth of microorganisms, ‘single microorganism given enough time to grow by incubation at an appropriate temperature, the limit of detection should be one. This does not take into account slowly growing microorganisms or injured microorgan: isms that take more than 1d days for recovery and growth. For methods that do not rely on growth of micro- organisms for detection, the limit of detection is more ‘complex. One has to first establish that a signal picked up by the instrumentation is actually the result of microbial activity. Setting a threshold of detection too low will provide “blips” that would indicate potential microbial activity when it might be due to residual signals from ‘other components. The threshold of detection should be set following a risk assessment determination on the significance of the signal of certain quantitative value. In situations when a microbiological method is to bbe shown equivalent to a pharmacopeial method, and oth methods rely upon growth of microorganisms, a method using serial dilution for both methods should be ‘conducted with appropriate microorganisms to determine the limit of detection. When the alternative method to a ppharmacopeial method does not rely on growth of micro- organisms, the equivalence of the methods in terms of limit of detection should follow a risk assessment of the results obtained to determine the threshold of detection that must be above the noise level. Ruggedness A method is rugged when it will resist producing divergent results, when it is performed by different microbiologists, in different laboratories, on different days, using different instruments or lots of reagents. Establish ruggedness is straightforward using the same lot of materials tested under different conditions as. cited above. The ruggedness of an alternative method to a pharmacopeial method is done separately and the results are compared and if they do not significantly differ then the methods are equivalent. Robustness The robustness of a microbiological method is an indi cation of the resistance of the method to small and deliberate differences introduced in the method itself Growth-based methods might be tested using different lots of a given medium or slightly different pH might be used, For methods not relying on growth of microorgan- isms, use of different instruments or variations in reagent lots or temperature and /or ime conditions could be done to determine the robustness of the method. When an alternative test to a pharmacopeial method is validated for robustness, results of the tests should be compared and relative robustness between the methods established. VALIDATION OF QUANTITATIVE MICROBIOLOGICAL METHODS The principal function of a quantitative microbiological ‘method is to determine how many viable microorganismsa sample contains. An example of a quantitative microbiological test is found in USP chapter <61> Microbial Limits. In the case of these methods parameters of vali dation include, accuracy, precision, specificity, limit of quantification, linearity, range, ruggedness, and robustness. ‘Accuracy Accuracy refers to the closeness of the value determined to the true number of microorganisms present in a sample at the time of testing. Contrary to physicochemical tests where samples are homogeneous, the microbial load is not distributed homogenously in a sample, thus the accuracy will depend on sampling. Other factors involved include the ability of the media used to support the growth and detection of microorganisms present in a sample. Results of quantitative microbiolo- gical tests are at best estimates and no amount of sophisticated statistical procedures applied would change the accuracy. If a need for accuracy is necessary, perhaps because a regulatory agency requires it, then a “spiking” experiment with known challenge microorgan- isms could give a better measure of accuracy. The spiking. experiment could use a mixture of well-defined micro organisms rather than several experiments with individual challenge microorganisms ‘Methods that rely on growth of microorganisms for quantification use, for example, CFU as a quantitative ‘measurement. However, the assumption that one colony is the product of one microorganism is arbitrary and generally not correct. Another level of complexity is encountered when a microbiological specification is expressed as a limit, such as less than 100 CFU, with a sample acceptable if the quantitative value is equal or Tess than 100 CFU, but not acceptable if itis greater than 100 CFU, This approach does not take into consideration the inherent large variability of microbiological quan- titative tests. This has been recognized in the harmonization work among USP, JP, and EP where it has been proposed that a specification of 100 CFU will be acceptable even if the result of the test gives 200 CFU; specification of 1000 CFU will be acceptable ifthe result of the test is 2000 CFU; and so forth. For tests that do not rely on growth of microorgan- isms for quantification the issue is more complex. A pharmacopeial test counts CFU while an alternative test could count each and every microorganism present, ‘The use of differential specifications depending on the microbiological method type used could be logical, although few individuals have tackled this issue. The relationship between metabolic activity and the number of microorganisms present will have to be quantified by the development of standard curves relating the number of microorganisms with the signal given by the test. Since the bioburden of a product is not homogeneous in terms of distribution of type of microorganisms and varies with seasons and suppliers, the standard curve approach will have to be carefully developed, perhaps using a mixture of most likely microorganisms present. Introducing accuracy measurements for these tests is not appropriate, from a theoretical, experimental, or practical point of view. St: VAUDATION OF WICROBIOLOGICAL METHODS 667 Ifa test is to be validated against a pharmacopeial test as being equivalent, the comparison will be between “apples” and “oranges” casting doubt on the meaningful- ness of requiring that an alternative testbe equivalent toa pharmacopeial test. Precision The precision of an analytical method is the degree of agreement among the individual results when the method is applied repeatedly to multiple samplings of a homogeneous material, The first issue one has to deal with is that the microbiological content of a sample is not generally distributed homogeneously within a product. Away to approach the issue of precision is to indicate that the precision of a quantitative microbiological method is a function of the precision of a number of steps involved in the procedure. These steps include but are not limited to sampling, pipetting, temperature of the ager if plate counting is used, temperature of incubation and length of incubation. These apply to methods based on ‘growth of microorganisms. For methods that do not rely fon growth and are based on instrumental procedures, calibration of the instrument and temperature of the reagents used are factors that have to be taken into ‘consideration in the determination of precision. Replicate of the testing will give a general estimate of the precision of a method through statistical analysis of results using more likely standard deviations. One has to remember however that as the number of microorganisms become smaller, the error as a percentage of the mean increases. ‘A microbiological quantitative method might have a very good precision but it might not be very accurate. Specificity ‘The measurement of the quantity of microorganisms in a sample must be specific. This requires, for methods involving growth of microorganisms that a differentiation ‘must be made between CFUs and debris. In general ths is not too difficult, and in case of doubt use of magnification ‘could resolve this issue. In the case of methods that do not rely on growth of microorganisms, the discriminatory power of the instrumentation will come into play. The instrumental noise level should be established as well as the specificity of the method for a variety of products since each product will contain different components that might give a signal similar to a microorganism, When a microbiological quantitative method is designed for the determination of specific category of microorganisms—for example yeasts and molds—the specificity of the method for enumeration of these specified microorganisms should be validated Limit of Quantification Most quantitative microbiological tests are used as limit test. Regardless of the type of method used, the method should be able to quantify microorganisms above and below that limit. If the specification for a product is 100CFU and the method cannot detect less than 00 CFU, the method should not be used. If one needs to demonstrate the equivalence of an alternative method to a pharmacopeial method, their limit of quantification should be comparable.668 LABORATORY METHODS AND QUALITY ASSURANCE Linearity Linearity for an analytical procedure is determined using. a least five different concentrations of analyte. For a ‘microbiological test a serial dilution of the sample to achieve a five different concentration of microorganisms could be used. For growth-based tests the linearity factor will not be very useful and will only test the precision of pipetting and the maintenance of the conditions of the test throughout the procedure. For non-growth-based tests linearity becomes important depending on the statistical analysis done on an automatic mode by the instrument. The linearity parameter becomes important if one needs to develop a standard curve that correlates a growth-based test to an instrumental test. These standard curves are used for example, in antimicrobial effective- ness testing (sce chap. <51> in the current USP-NF) to determine the size ofthe inoculum without having to use a growth-based test Range The precision of a microbiological quantitative test is a function of the number of microorganisms in the sample. To obtain an acceptable precision the range (upper and ower limit) should be defined and confined to a narrow ‘window. In plate counts it is customary to use “countable plates” which are in the range of 10 to 300 CFU. Dilution of the sample should be manipulated to obtain countable plates in order to get a decent precision, Ruggedness Ruggedness will be determined using different micro- biologists, at different locations, on different days, using. different instrumentation, reagent lots, ete, but using the same sample The variability of a microbiological test in the estimation of bioburden is very wide and we expect that the determination of ruggedness will be rather difficult if not impossible to establish, Robustness It is an indicator of how a quantitative microbiological test will perform if small changes in the parameters of the test are introduced under routine usage. An alternative method to a pharmacopeial method should have equivalent ruggedness and robustness. VALIDATION OF MICROBIAL IDENTIFICATION METHODS Microbial identification tests are tests that would determine the presence or absence of specific microorgan- isms in a sample. The validation of these tests is similar to the validation of qualitative microbiological test such as a sterility test that checks samples for presence or absence of microorganisms. Parameters of validation to be considered include accuracy, precision, ruggedness, and robustness. ‘Accuracy The presence or absence of microorganisms feature of these tests depends more on sampling than on the accuracy of the method used. Once a procedure detects ‘2 presumptive specified microorganism, the accuracy of the test will depend on the accuracy of the identification scheme that will be used to confirm the identification. The classical identification methods depend on the ‘metabolism of specified microorganisms in the media provided, their phenotypic characteristics, and special- ized biochemical tests. Other identification methods depend on the use of specialized instrumentation that compares the biochemical reactions of the isolate on specialized media with a reference library of microorgan- isms. Calibration of the biochemical method will depend ‘on the reactions of a specific strain of ATCC microorgan: isms as a comparator. The issue of concern is that isolated. microorganisms from samples do not always behave as. the ATCC strain of reference, and the results cannot be predictable. Some instrumental methods will use as a comparator the percentage of similarity between the isolate and the reference strain, In the special case of equivalency between a method and a pharmacopeial procedure, inoculation of samples with ATCC strains of interest will provide a relative measurement of accuracy when compared to the pharmacopeial method. Precision Precision of an identification method can be measured or estimated by replicating the scheme repeatedly using the same sample. Since, in general, alternative methods to a compendial method will sometimes use the same general procedure but will use instruments for some portions of the testing procedure, the precision of each step can be determined for the pharmacopeial method and the alternative method and the results compared. However, it is best to modify the pharmacopeial method then run the same sample, inoculated and non-inoculated by specified microorganisms by both methods, from enrich: ‘ment to isolation to identification Ruggedness ‘The determination of ruggedness for microbial identifi cation schemes is really a function of the training of the personnel that do the testing and the calibration of the instrumentation if itis used. The stability and repeat- ability of the identification schemes will be a function of the drifting of the electronics of the instruments and the accuracy of the databases used for final identification, and the accuracy and idiosyncrasies of the software used. Robustness The robustness of an identification scheme, especially when instrumentation is used, is determined by the manufacturers of these systems. The manufacturers should provide the users with the parameter ranges that might be encountered during routine use. ‘THE PHARMACOPEIAL PERSPECTIVE FOR VALIDATION OF USP MICROBIOLOGICAL METHODS ‘The USP microbiological tests, and for that matter those from the JP and EP, are considered to be validated methods. Someone who uses the pharmacopeial methods will not have to re-validate those procedures.However, each raw material, excipient, drug substance, drug product will have to be qualified, since they have possibly the components that are inhibitory to the measurement of microorganisms. The USP provides for modification of the pharmacopeial tests to adjust for material characteristics and activities. These modifi cations do not have to be qualified each time that a sample is tested. However, re-qualification should be done when there are significant changes in the manufac- ture of raw materials, changes in suppliers, or changes in components. Information on validation of pharmacopeial ‘methods is offered in the USP as general information chapters that are not enforceable by the regulatory agencies. The current USP includes chapter <1225> Validation of Compendial Methods that covers validation of tests from the analytical chemistry perspective and does not reflect the special case and characteristics of microbiological tests. This chapter, however, contains information on the general concepts of validation of test methods and should be consulted. ‘The USP Expert Committee on Analytical Micro- biology has developed and proposed another information chapter <1223> Validation of Alternative Microbiolo- gical Methods that has received considerable public comments that will require a total rewrite of that proposed chapter, ‘The USP microbiological tests in <51> Antimicro- bial Effectiveness Testing, <71>Sterility Tests, and <61> “Microbial Limits Tests require thatthe recovery methods for microorganisms be qualified for each material tested. Procedures for qualification are included in these chapters. These procedures are simple and refer to visual exami- nation to assess the level of recovery or the inhibitory properties of the sample itself. Requests from interested parties to develop an information chapter to quantitatively determine the recovery of microorganisms resulted in the development and establishment of chapter <1227> Vali- dation of Microbial Recovery from Pharmacopeial Articles. This chapter deals mainly with validation of neutralization procedures to be used in preparatory testing as well as in bacteriostasis and fungistasis| determination. A list of common neutralizers is provided along with methods of neutralizations using chemical inhibition, dilution, or membrane filtration. Italso discusses the erzors associated with recovery of microorganisms, and procedures for assessing the recovery of microorganisms, CONCLUSIONS The validation of microbiological methods is more complex than the validation of physicochemical methods. This is in part due to the inherent variability of microbiological methods compounded by the lack of homogeneous distribution of microorganisms within samples to be tested, Pharmacopeial microbiological methods are vali- dated per se, and need only to be qualified for each material being tested. Qualification procedures are included in the compendial methods, ‘Advances in technologies represented by rapid microbiological methods have introduced another level of complexity to validation of microbiological tests. For compliance purposes, regulatory agencies in the US.A., St: VAUDATION OF MICROBIOLOGICAL METHODS 669 Japan, and Europe accept the use of alternative methods provided that the alternative method is shown to be equal ‘orbetter than the pharmacopeial method. This also applies to microbiological tests. ‘The classification of microbiological tests as quali- tative, quantitative, or identification was done since validation of methods depends on its classification. 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