This article is excerpted from a book chapter on microbial methods to be included in Specification of Drug Substances and Products: Development and Validation of Analytical Methods, to be published by Elsevier.
Compendial microbiological methods have been in existence for many years, with only minor changes being incorporated. Such methods include United States Pharmacopeia (USP) Chapter <61> "Microbiological Examination of Non-sterile Products: Microbial Enumeration Tests"1, USP Chapter <62> "Microbiological Examination of Non-sterile Products: Tests for Specified Microorganisms"2, and USP Chapter <71> "Sterility Tests."3 Recently, more rapid technologies have emerged and, in some cases, have received regulatory approval as alternatives to the traditional compendial tests.
Rapid microbiological methods
Alternative technologies are available for the rapid detection of microorganisms. In 2000, the Parenteral Drug Association (PDA) published the first guidance document on how to validate and implement alternative rapid microbiological methods.4 The USP and European Pharmacopeia (EP) have also published guidances on alternative methods.5,6
EP 5.1.6 placed rapid microbiological methods (RMMs) into three categories: growth-based methods, direct measurement, and cell component analysis. Growth-based methods detect a signal after a short incubation period in liquid or on solid media; examples include detection of CO2 production by colorimetric methods or a change in head space pressure and detection of adenosine triphosphate (ATP) by bioluminescence. Direct measurement methods can detect cell viability without requiring growth of the microorganism. One example of a direct measurement method combines fluorescent labeling and laser scanning cytometry to enumerate organisms. The sample containing microorganisms is filtered onto a membrane and treated with a combination of stains to fluorescently label viable organisms without the need for growth. The membrane is scanned by a laser, fluorescent light is detected, and a membrane scan map is produced which captures the position of each fluorescent event, which is then verified by visual examination using an epifluorescent microscope. The third type of RMM is cell component analysis or indirect measurement; expression of certain cell components correlates to microbial presence. One example is amplification of DNA or RNA by polymerase chain reaction (PCR). RMMs can be qualitative (presence/absence) or quantitative (enumeration), destructive or nondestructive, and can be applied to filterable or non-filterable products.
In 2006, the FDA's Center for Drug Evaluation and Research (CDER) published a paper on the use of alternative microbiological methods.7 The authors stated, "New microbiology methods can offer advantages of speed and precision for solving microbiological problems associated with materials or environmental influences. Neither corporate economics nor regulatory attitudes should be a barrier to the use of new testing technologies or different measurement parameters."
Rapid sterility test methods
Sterility testing has also undergone an evolution. It was first introduced in the 1930s for testing of liquid products (USP XI) as a seven-day test using one medium at 37oC targeted for human pathogens. By the early 1940s, an incubation temperature of 22 to 25oC was added specifically for yeasts and mold with a 15-day incubation period. By the mid-1940s, a sabouraud-based medium was used for ten days instead of 15 days and fluid thioglycollate medium (FTM) for seven days. In the mid-1960s, the incubation conditions for FTM changed to 30 to 32oC for seven days. Several changes were incorporated into the test in 1970, including different incubation times for aseptically filled products (14 days) versus terminally sterilized products (seven days), incubation temperature ranges were increased to 30 to 35oC for FTM and 20 to 25oC for soybean casein digest medium (SCDM), and the incubation period was used to differentiate the membrane filtration test (seven days) from the direct inoculation test (14 days). Over the course of several years, efforts led to incubation times being harmonized to 14 days in 2004, and by 2009 (USP 32) the remaining portions of the sterility test were harmonized with only a few exceptions.8
Use of a rapid method as an alternative to the traditional sterility method requiring 14 days has several advantages. A shorter incubation minimizes the time needed for recovery of microbial contaminants, enabling more rapid implementation of corrective actions that would prevent cross contamination to other product batches and can reduce product release time.
The FDA's Center for Biologics Evaluation and Research (CBER) has evaluated three growth-based rapid sterility methods: two qualitative methods utilizing CO2 monitoring technologies and one quantitative method incorporating ATP bioluminescence technology. A total of 14 different microbial strains (ATCC and environmental isolates) representing bacteria (Gram negative, Gram positive, aerobic, anaerobic, and spore forming), yeast, and fungi were used. The sensitivity of the rapid microbiological methods was compared to the compendial membrane filtration (MF) and direct inoculation (DI) methods with regard to observation of growth at various low levels of inoculations. Results showed that the ATP bioluminescence technology was the most sensitive of the methods, the CO2 monitoring technologies were more sensitive than the compendial methods, and the compendial membrane filtration method was more sensitive than the direct inoculation method.9
In 2010, a leading pharmaceutical company implemented a rapid sterility method consisting of a five-day incubation as compared to the traditional 14-day incubation. The ATP bioluminescence technology system was selected because it is growth based, uses membrane filtration which is similar to the compendial method, and can detect one colony-forming unit (cfu) following incubation.10
The system uses ATP bioluminescence to detect and quantitate micro-colonies. The first step is to filter a sample through the system's filter unit and place the membrane onto a solid media cassette. The media cassette is incubated to allow for the formation of micro-colonies and the detection of ATP. The filter is removed from the media cassette and sprayed with an ATP releasing agent that makes the cell wall of the microorganism permeable to ATP. A bioluminescent enzyme reagent is then applied, which reacts with the ATP to produce light (photons). The membrane is moved to the detection tower where image processing takes place. The photons are converted into electrons and multiplied in the photomultiplier tube (PMT). The location of the photons correlates with the location of the micro-colonies. The image forms on a charge coupled device (CCD) camera, a computer algorithm then processes the data and enumerates the micro-colonies in colony forming units (CFUs), and a 2D and 3D image map is generated.11
Taking into consideration the compendial guidelines (USP Chapter <1223>, Ph. Eur 5.1.6), the pharmaceutical company validated the rapid method and was able to demonstrate that it delivered robust, reliable results. Equivalent performance to the compendial sterility test method in terms of robustness, ruggedness, repeatability, limit of detection, specificity, accuracy, and precision was reported. In 2010, the company achieved regulatory approval by the FDA, EMA, and MHRA to use the alternative method in lieu of the compendial method.12
Rapid bioburden methods
Quantitative rapid methods can be used as alternatives to the traditional bioburden test. The ATP bioluminescence technology system combines two proven technologies: membrane filtration and fluorescent staining. Membrane filtration is the standard method for microbial bioburden testing due to the capacity to remove any inhibitory agents and the ability to process larger volumes. After filtration and a short incubation time (approximately one-third shorter than traditional incubation times), reagent is applied to the membrane and any viable and culturable microorganisms retained on the filter are stained with a fluorescent marker. The active microbial metabolism of the microorganism causes an enzymatic cleavage of the non-fluorescent substrate and, once cleaved inside the cell, the substrate liberates free fluorochrome into the microorganism cytoplasm. As fluorochrome accumulates inside the cells, the signal is naturally amplified. The cells are then exposed to the excitation wavelength in the system's reader to visually counted.13
Validation of alternative methods
USP Chapter <1223> states, "Validation studies of alternate microbiological methods should take a large degree of variability into account. When conducting microbiological testing by conventional plate count, for example, one frequently encounters a range of results that is broader (%RSD 15 to 35) than ranges in commonly used chemical assays (%RSD 1 to 3). Many conventional microbiological methods are subject to sampling error, dilution error, plating error, incubation error, and operator error."5 The USP goes on to state that the characteristics such as accuracy, precision, specificity, detection limit, quantification limit, linearity, range, ruggedness, and robustness are applicable to analytical methods and less appropriate for alternate microbiological method validation. Yet, the general present regulatory expectation is to apply these analytical performance characteristics to alternative rapid microbiological method validation. Additionally, USP includes these validation parameters in Chapter <1223>.
It is more than appropriate for vendors of new alternative technologies to apply these analytical performance characteristics during validation. The data generated from the validation testing should be analyzed using statistical tools to show that the method meets the applicable requirements. However, once the technology is validated, the end user should not have to repeat the in-depth validation that was conducted by the vendor. Rather, the end user should focus on whether or not the alternate method will yield results equivalent to, or better than, the results generated by the conventional method when testing their product.
In 2011, FDA CDER published A Regulators View of Rapid Microbiology Methods14 and stated, "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."
The author goes on to say that the end user would still have to perform their own studies not addressed by the vendor, which include product specific data.
A RMM may incorporate portions of the compendial test up to a certain point. For example, a sample may be processed using conventional membrane filtration and the membrane placed on a recovery medium and incubated. However, at that point the presence of viable cells may then be demonstrated by use of some alternative rapid technology. Hence, validation would be required on the recovery portion of the method rather than on the entire test.
When evaluating the range of the method, the vendor needs to ensure that the upper end of the range is challenged. New technologies that enumerate micro-colonies verses macro-colonies can count a higher population. Traditional pour plate or membrane filtration methods are limited in the numbers of macro-colonies counted, with 300 cfu being the maximum number. New technologies can count much higher cfu in some cases.
1. United States Pharmacopeia <61>, 35th revision. United States Pharmacopeial Convention, Rockville, MD, December 2012.
2. United States Pharmacopeia <62>, 35th revision. United States Pharmacopeial Convention, Rockville, MD, December 2012.
3. United States Pharmacopeia <71>, 35th revision. United States Pharmacopeial Convention, Rockville, MD, December 2012.
4. PDA Technical Report No. 33, Evaluation, Validation and Implementation of New Microbiological Testing Methods. PDA Journal of Pharmaceutical Science and Technology. 2000. Volume 53(3) Supplement TR33.
5. United States Pharmacopeia <1223>, 35th revision. United States Pharmacopeial Convention, Rockville, MD, December 2012.
6. European Pharmacopeia 5.1.6, PhEur 7.5. Council of Europe, 2012.
7. Hussong, D. and Mello, R., Alternative Microbiology Methods and Pharmaceutical Quality Control. American Pharmaceutical Review. 2006. 9(1): p. 62-69.
8. Cundell, A., The History of the Development, Appplications and Limitations of the USP Sterility Test. Rapid Sterility Testing. 2011. 7: p. 127-169.
9. Parveen, S., Kaur, S., Wilson David, S.A., Kenny, J.L., McCormick, W.M, Gupta, R.K., Evaluation of Growth Based Rapid Microbiological Methods for Sterility Testing of Vaccines and Other Biological Products. Vaccine. 2011. 29: p. 8012-8013.
10. Gray, J.C., Staerk, A., Berchtold, M., Mercier, M., Neuhaus, G., Wirth, A., Introduction of a Rapid Microbiological Method as an Alternative to the Pharmacopoeial Method for the Sterility Test. American Pharmaceutical Review. October 2010.
11. Millipore Corp., Milliflex® Rapid System Operator's Manual. 5/2006. Publication No. PF09390 Rev. B.
12. Gordon, O., Gray, J.C., Anders, H.J., Staerk, A., Schaefli, O., Neuhaus, G., Overview of Rapid Microbiological Method Evaluated, Validated and Implemented for Microbiological Quality Control. European Pharmaceutical Review. 2011. 16(2): p. 9-13.
13. Millipore Corp., Milliflex® Quantum Rapid Detection atp User Guide. 2/2010. Publication No. PF11940 Rev A.
14. Riley, B., A Regulators View of Rapid Microbiology Methods. European Pharmaceutical Review. 2011. 16(5): p. 3-5.