RAPID METHODS SUPPLEMENT


visual detection and they can detect microorganisms earlier in the growth phase. Other types of RMM do not need a growth phase at all. These non-growth based methods are therefore faster than the growth-based methods, in some cases even providing real- time results. Examples of the non-growth based systems include cytometers (either flow or solid phase) which may require a staining step or utilise auto-fluorescence. The first phase of selecting a RMM is to


determine your user requirements. For example, what microbiological test samples require faster results than are achievable with traditional methods? Is a qualitative or quantitative test required and what Limits of Detection or Quantification (LOD or LOQ) are needed? The nature of the sample is also important as some RMM are only suitable for filterable samples, while other RMM can only use direct inoculation. The maximum acceptable time to results is also important. Selecting a RMM that cannot provide results in a timely fashion (as determined by the user) is not going to be satisfactory. However, it is also important not to rule out a RMM that is suitable for your intended use just because it is not the fastest method available. A thorough understanding of your user requirements and the parameters that are critical for your purposes is crucial when selecting a RMM. Once an RMM has been selected, then it must be validated for its intended use1. There are


several documents that provide guidance on how to validate a RMM. These documents include PDA Technical Report 33 – Evaluation, Validation and Implementation of New Microbiological Testing Methods2


, EP Chapter


5.1.6 Alternate Methods for Control of Microbiological Quality3


, and USP Chapter


<1223> Validation of an Alternate Microbiology Method4


. Each of these documents describes


the basics of validating a RMM including each of the validation parameters (e.g., accuracy, precision, specificity, etc) and discusses acceptance criteria that might be appropriate for each parameter. While it is important for each validation parameter to be addressed, it may not be necessary for the user to do all of the work themselves. For some validation parameters, it is much easier for the RMM vendor to perform the validation experiments. For example, ruggedness and robustness are best addressed by the vendor who has ready access to multiple reagent batches and equipment needed to


04


European Pharmaceutical Review Volume 16 | Issue 5 | 2011


“The most common techniques to address LOD have


involved serial dilutions of microbial suspensions”


standards. Therefore, a combination approach is typically utilised. The initial part of this plan is to start with a panel of microorganisms taken from the compendial method suitability tests (e.g. EP 2.6.12 or USP <71>). This panel for a sterility test might consist of S. aureus, P. aeruginosa, B. subtilis, C. sporogenes, C. albicans and A. niger. The panel would thus contain a representative of each of the major groups of microorganisms (gram-negative, gram-positive,


perform these studies. In addition, a vendor can provide other data to support validation. A RMM vendor should have extensive data demonstrating the effectiveness of their method. The vendor can make this data available to a potential user either by providing it directly to the user or by submitting the data to the FDA in a Drug Master File (DMF)5,6


. The


data in the drug master file can be accessed by FDA reviewers who are assessing the validation studies for a RMM. The user would still have to


spore former, anaerobe, yeast and mould). However, additional organisms should also be considered for inclusion in a test panel. Many firms will use microorganisms isolated from their environmental monitoring program on the theory that these isolates represent potential contaminants. It is also useful to include microorganisms that would pose a challenge to the specific RMM being validated. For example, with a growth-based RMM the use of slow growing microorganisms is a good challenge to the method. Propionobacterium acnes has been used by a number of firms validating growth- based RMM because it is a slow grower and has proved to be a challenge even for the compendial test methods. Another validation parameter that has been


perform their own studies to cover validation parameters not completely addressed by the vendor studies. Some of the data that the user would need to supply would include product specific data. One of the validation parameters for a RMM


that stimulates a great deal of discussion is specificity. Ideally, a RMM would be able to detect/count all microorganisms. Considering the broad range of microorganisms that exist, it is clearly unrealistic to expect a method to detect all microorganisms, not to mention the difficulty in validating a method to those


somewhat contentious has been the limit of detection (LOD) for a rapid sterility test. In theory, a sterility test should be able to detect a single viable microorganism that is present in the test sample. Therefore, it stands to reason that the validation of a rapid sterility test should be able to demonstrate that the proposed test method has a limit of detection at (or very near) one microbial cell. However, there are technical difficulties associated with accurately spiking a validation sample with a single viable microbial cell. Potential RMM users have employed several approaches to deal with this issue. The most common techniques to address LOD have involved serial dilutions of microbial suspensions. The dilutions start with a known population and are then continued until less than one CFU is present in each test sample. The dilutions are tested using the RMM. In some cases, the samples are tested in parallel using the compendial test method and the results of the RMM are compared to the compendial results to determine if they are equivalent7,8


.


In other examples, the RMM results are used to determine a most probable limit of detection, which can then be assessed to determine suitability9


. Although the use of RMM in the pharma -


ceutical industry has slowly increased over the last few years, it is still more the exception than the rule. Consequently, there are still many questions that potential RMM users have regarding how to validate a RMM and what regulators expect in validation studies. There has been communication between potential RMM users and regulatory authorities while the firms are considering RMMs and planning validation studies. Firms have been able to tell regulators


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