Introduction
Rapid microbiology methods have long been essential tools of the clinical and food industry microbiology laboratories. Swift diagnosis of infectious diseases by clinical labs and the need for prompt test results from perishable food items have been strong incentives for the use of rapid methods. The pharmaceutical industry, however, has not been as quick to embrace rapid microbiology despite the potential advantages. Faster microbiology test results would provide better control over the manufacturing process. More rapid microbiology assays would also allow for earlier release of product. One of the explanations offered by the pharmaceutical industry for not using rapid microbiology methods is the uncertainty over regulatory acceptance. The process of evaluating, validating and implementing rapid microbiology test methods can be an expensive and time consuming task. Industry has been reluctant to expend precious resources when regulatory approval of the new method may be in doubt.
This article will provide an overview of microbiology testing in the pharmaceutical industry and will look at where rapid microbiological testing methods could fit into the manufacturing process. The issues involved with validation of rapid microbiology methods will be considered with regard to CDER review expectations. Finally, current FDA initiatives that could facilitate the use of rapid microbiology will be described.
Microbiological Test Methods
Microbiological tests for pharmaceuticals fall into several categories. One category involves release tests for drug products such as microbial limits testing of non-sterile drug products or sterility testing of parenterals and ophthalmics. Environmental monitoring tests include testing air, water, surfaces and personnel for viable microorganisms. Bulk drug product or components can also be tested for bioburden. Finally, some drug products are required to demonstrate antimicrobial effectiveness as part of the pharmaceutical development process. All these tests traditionally involve growth of microorganisms. Many of these tests involve compendial methods such as USP Chapters <51> (Antimicrobial Effectiveness Testing) [1], <61> (Microbial Limits Tests) [2] and <71> (Sterility Tests) [3]. The use of compendial test methods provides several advantages. Compendial test methods require minimal validation work on the part of the applicant before acceptance by regulatory authorities. The current compendial tests are also simple to execute and the results are easy to understand (colonies on a plate or turbidity in a liquid culture) even for a non-microbiologist. However, these growth-based tests have some drawbacks. They are slow, requiring several days to two weeks or more incubation. Growth of the microorganisms is limited by the medium and the incubation conditions used for the assay. Even though they have performed more or less adequately, the existing traditional microbiological methods are far from perfect.
There are many examples of rapid microbiology methods either in use, or in development, that could be of use for pharmaceutical manufacturing. The test methods can be grouped into the following three categories according to their uses: qualitative, quantitative and identification. Qualitative tests provide a yes/no answer to the question of microbial contamination. Asterility test is probably the most common qualitative test. Quantitative rapid methods provide a numerical value for the microbial contents of a sample and include conventional microbial limits tests. Quantitative test methods can also replace sterility tests, as long as they are sensitive enough to detect a single organism. Identification tests provide a name (or at least a description) of a microorganism. Various types of rapid identification tests have been available for many years, largely due to advances in clinical microbiology. However, test methods designed for clinical isolates are not always effective at identifying organisms found in a manufacturing environment. This is because the phenotypic databases used to support the clinical ID systems are drawn primarily from clinical isolates and may not contain adequate representation from other sources of microorganisms.
Validation
A rigorous scientific validation is a critical part of obtaining regulatory approval for a rapid microbiology method. There are two aspects of a new test method that should be addressed. Does the method work (in general), and is the method appropriate for the specific sample(s) to be tested? The general evaluation of a test method can be performed by the prospective user or literature references can be used to support the method. It is important to note that the user should not have to repeat the general method evaluation if the method is already in widespread use for a similar purpose. The suitability of the test method for the proposed samples should be established by using the samples with the method to determine inhibition or enhancement.
A new method should be at least equivalent (not inferior) to the existing method. Since the existing methods are predominantly the growth-based compendial methods, this comparison should not be a large hurdle for a new method. In order to determine equivalence between the old and new method, a number of criteria should be examined [4]. The essential elements for validation can vary somewhat depending on the type of test being studied (i.e., quantitative, qualitative or identification). The validation criteria are best examined experimentally using samples spiked with known organisms. Although parallel testing using the old and new methods has been suggested in determining equivalence, this is often not necessary or advisable. Parallel testing of samples that contain few microorganisms (or none) can be particularly problematic due to sample variation. Multiple samples taken from the same batch may give different results. The following sections will cover the various elements involved in validation of a rapid microbiology method.
Accuracy – A test for accuracy is a determination of the closeness of the results of a new test method with the expected results. Since the expected results are usually based on the existing method, a test of accuracy is essentially a comparison between the old and new methods. The results for a new quantitative method should not be significantly lower than the old method, although it is acceptable for the new method to give higher counts. Higher counts using the new method can be an indication of increased accuracy. Assessing the accuracy of an identification method can be more complex since the new and old method may give different results.
Specificity – A measure of the range of organisms a test method can detect. Using the term specificity can be somewhat misleading, since the wider the range the test has, the better the test. A new method should not have a narrower range than the existing method. The range of organisms used to test specificity should be representative of the types of organisms a test is likely to encounter. Using organisms isolated from the product/production facility would be very useful for assessing specificity. It should be recognized that an experimental protocol cannot be expected (nor is it necessary) to test every potential isolate.
It should be noted that some rapid microbiology methods can detect a significantly wider range of organisms than the traditional methods they would replace. This is particularly true for rapid methods that do not rely on microbial growth. This could potentially lead to results above the traditional alert and action levels. In order to allow for the difference between the new and old methods, the acceptance limit can be increased. The reason(s) for increasing the limit should be clearly explained and justified scientifically.
Precision – A measure of the repeatability/reproducibility of the test method. Anew method should be at least as precise as the old method. Precision is particularly important for identification methods since trending isolates can be difficult if the same organism is assigned different identities each time it is isolated.
Limit of Detection/Limit of Quantification – The lowest number of organisms that can be detected or counted. Limit of detection is particularly important for qualitative tests such as sterility. A sterility test should be able to detect a single viable organism. However, experimentally testing this capability is difficult due to problems associated with preparing a challenge sample that contains only one viable organism. Using samples with low numbers of organisms (e.g., 1-10 CFU) should be sufficient to assess limit of detection.
Range – The upper and lower limits of quantification. Range is an important characteristic for a potential user of a test method but is not necessarily important for determining equivalence. A test with limited range may require testing of multiple dilutions to be sure that the microorganisms in the sample are within the useful range of the method. Since the traditional method of enumeration (plate count) already has a fairly limited range (30-300 CFU/plate [2]), it should not be difficult for a rapid method to demonstrate equivalence to the existing method.
Linearity – The results of a quantitative method should be linear over the useful range of the method.
Ruggedness/Robustness – These characteristics reflect the ability of a test method to tolerate slight deviations in test parameters and still provide accurate results. The parameters varied often include reagents, operators and test equipment. Ruggedness and robustness are probably best assessed by the test vendor who will have ready access to multiple lots of reagents and multiple pieces of equipment.
FDA Initiatives
As was stated at the beginning of this article, one of the reasons cited by industry for not using rapid methods has been uncertainty over FDA acceptance. FDA has started several initiatives that should facilitate industry adoption of rapid microbiology methods. The first of these programs is the Process Analytical Technology initiative (PAT) [5]. Traditionally, pharmaceutical manufacturers use batch processes with end-product testing to confirm product quality and thus verify the suitability of the process. The PAT initiative was started specifically to encourage industry to use new innovative technologies to analyze and control the manufacturing process. One of the goals of PAT is continuous real-time quality assurance. Even though the current rapid microbiology methods may be primarily end-product tests, they can still offer increased control and understanding of the process by providing faster results. The PAT initiative will encourage use of rapid microbiology by providing specific regulatory handling for PAT submissions. Users of PAT are encouraged to meet with the agency to discuss their proposed uses of new technologies. Applications containing rapid microbiology will be reviewed by CDER reviewers trained in the technologies, and inspections will be carried out by FDA personnel who are trained in rapid microbiology. It is hoped that the PAT initiative will encourage industry to take advantage of rapid microbiology.
Another FDA program that should have an impact on rapid microbiology is the Pharmaceutical cGMPs for the 21st Century initiative. There are several aspects of this initiative that should prove helpful for introduction of rapid microbiology. First, there will be a dedicated and specially trained pharmaceutical inspectorate. These investigators will be provided advanced training specific to inspecting pharmaceutical manufacturing facilities. In addition to the pharmaceutical inspectorate, there will be product specialists available to go on inspections. These product specialists will include microbiologists with experience in rapid microbiology. These product specialists will provide further expertise as part of pharmaceutical inspections and ensure that rapid microbiology methods are evaluated in an appropriate scientific manner. Finally, a formal dispute resolution process will be set up to resolve disagreements that arise during pharmaceutical inspections [6]. The dispute resolution process could be helpful in settling differences of opinion involving introduction of rapid microbiology methods. The various aspects of the Pharmaceutical cGMPs for the 21st Century initiative should provide assurance to industry that their efforts to utilize rapid microbiology methods will be scientifically assessed.
Regulatory Submissions
The appropriate pathway for rapid microbiology submissions to FDA is best determined through direct dialogue with the agency. As mentioned above, the PAT initiative recommends discussion with FDA regarding all aspects of implementation for new process analytical methods. Until rapid microbiology methods become more widespread in the pharmaceutical industry, it may be simplest to implement new rapid methods on a post-approval basis. It may also be useful to employ a comparability protocol to implement rapid microbiology methods for multiple products and/or facilities [7]. A comparability protocol for an analytical method is a detailed plan that specifies the validation experiments and acceptance criteria. The approval of a detailed validation protocol should greatly reduce requests for additional information to support the subsequent changes. An approved comparability protocol can also designate a reduced reporting category for changes covered by the comparability protocol.
Summary
The use of rapid microbiology methods by the pharmaceutical industry should offer many advantages. Receiving microbiology test results sooner will provide for better control and understanding of the manufacturing process via faster feedback. Appropriate validation of rapid microbiology methods is necessary to ensure that the test is suitable for its intended purpose. However, it should be noted that the existing traditional microbiological test methods leave a lot of room for improvement. Therefore, it is not necessary to demonstrate that a new rapid method is flawless, only that it is not inferior to the current method, and will thereby provide equivalent assurance of microbial quality. Current FDA initiatives described in this article should help assure industry of the agency’s understanding of the potential importance of rapid microbiology methods. These initiatives should also convince industry that FDA will assess rapid methods scientifically and not place undue regulatory burdens on firms interested in using these methods. There are many exciting potential uses for rapid microbiology methods in the pharmaceutical manufacturing process, and industry should not feel that FDA will be a hindrance to the appropriate use of these methods.
References
1. Chapter <51>, Antimicrobial Effectiveness Testing, USP 26, USPC, Inc., Rockville, MD, p. 2002, (2003).
2. Chapter <61>, Microbial Limits Tests, USP 26, USPC, Inc., Rockville, MD, p. 2006 (2003).
3. Chapter <71>, Sterility Tests, USP 26, USPC, Inc., Rockville, MD, p. 2011 (2003)
4. Technical Report No. 33, Evaluation, Validation and Implementation of New Microbiological Testing Methods, PDA Journal of Pharmaceutical Science and Technology, Supplement TR33, vol. 54, no 3 (2000).
5. Draft Guidance for Industry, PAT – A Framework for Innovative Pharmaceutical Manufacturing and Quality Assurance, FDA (2003).
6. Draft Guidance for Industry, Formal Dispute Resolution: Scientific and Technical Issues Related to Pharmaceutical CGMP, FDA (2003).
7. Draft Guidance for Industry, Comparability Protocols – Chemistry, Manufacturing, and Controls Information, FDA (2003).
