Over the past 80 years, few changes have been made to compendial microbiological methods, which 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
Although the compendial methods are the standard, rapid technologies have recently emerged and, in some cases, have received regulatory approval as alternatives to traditional microbiological methods.
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 similar guidances.5,6
Rapid microbiological methods (RMMs) are grouped into three categories (EP 5.1.6): 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. Direct measurement methods can detect cell viability without requiring growth of the micro-organism. One example of a direct measurement method combines fluorescent labelling and laser scanning cytometry to enumerate organisms. 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.
The FDA’s Center for Drug Evaluation and Research (CDER) published a paper in 2006 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… 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
The evolution of sterility testing began in the 1930s when it was introduced for liquid products (USP XI) as a 7-day test using one medium at 37°C targeted for human pathogens. Incubation temperatures, periods and conditions changed significantly over the preceding decades, but by 2004, incubation times were harmonised to 14 days, and by 2009 (USP 32) the remaining portions of the sterility test were harmonised with only a few exceptions.8
Rapid methods have several advantages over the traditional sterility method. Shorter incubation minimises the time needed for recovery of microbial contaminants, enabling quicker implementation of corrective actions that would prevent cross-contamination to other product batches and can reduce product release time.
Shorter incubation minimises the time needed for recovery of microbial contaminants, enabling quicker implementation of corrective actions
The FDA Center for Biologics Evaluation and Research (CBER) has evaluated three growth-based rapid sterility methods: two qualitative methods using CO2 monitoring technologies and one quantitative method incorporating ATP bioluminescence technology (Milliflex Rapid Detection System). 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 with 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 Milliflex system 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 5-day incubation instead of the traditional 14-day incubation. The Milliflex 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. Step one is to filter a sample through the system’s filter unit and place the membrane onto a solid media cassette, which 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 micro-organism 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 CFUs, and a 2D and 3D image map is generated.11
The pharmaceutical company validated the rapid method, taking into consideration the compendial guidelines (USP Chapter <1223>, Ph. Eur 5.1.6), and demonstrated that it delivered equivalent performance to the compendial sterility test method in terms of robustness, ruggedness, repeatability, limit of detection, specificity, accuracy and precision. 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
Quantitative tests
Quantitative RMMs can also be used as alternatives to the traditional bioburden test. The MilliPROBE real-time detection system for Mycoplasma uses industry standard membrane filtration combined with nucleic acid technologies to provide qualitative results in four hours (compared with 28 days for traditional detection methods). After separation, the reagent containing Background Reduction Technology (BRT) probes is added to the Mycoplasma-retentive membrane. Once the Mycoplasma is lysed, rRNA target is released and BRT probes are activated to bind the rRNA target. The stabilised rRNA is purified by automated Target Capture and the lysate is transferred to a tube containing the Target Capture Reagent.
Target rRNA is captured on the magnetic beads and cell debris, proteins, and non-specific DNA and RNA are washed away from the target. Only BRT-modified target rRNA is amplified and detected in real time by the hybridisation of fluorescently labelled probes called molecular torches. The molecular torch probes are non-fluorescent unless bound to an amplified target. As the rRNA is amplified, a fluorescent signal is generated, measured and recorded.
The Milliflex Quantum growth-based 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 (around one third shorter than traditional incubation times), reagent is applied to the membrane and any viable and culturable micro-organisms retained on the filter are stained with a fluorescent marker. The active microbial metabolism of the micro-organism causes an enzymatic cleavage of the non-fluorescent substrate and, once cleaved inside the cell, the substrate liberates free fluorochrome into the micro-organism cytoplasm. As fluoro-chrome accumulates inside the cells, the signal is naturally amplified. The cells are then exposed to the excitation wavelength in the system’s reader to be visually counted.13
Methods validation
As stated in USP Chapter <1223>: ‘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 also states that 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 current regulatory expectation is to apply these analytical performance characteristics to alternative rapid microbiological method validation. Additionally, the USP includes these validation parameters in Chapter <1223>.
Once the technology is validated, the end user should not have to repeat the in-depth validation that was conducted by the vendor
It is acceptable for vendors of new alternative technologies to apply these ‘analytical’ performance characteristics during validation. The data generated from validation testing should be analysed 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. Instead, the end user should focus on whether or not the alternative method will yield results equivalent to, or better than, the results generated by the conventional method when testing their product.
The FDA CDER published A Regulators View of Rapid Microbiology Methods in 201114 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 also states that the end user would still have to perform their own studies not addressed by the vendor, which include product specific data.
RMMs can 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. Therefore, validation would be required on the recovery portion of the method rather than on the entire test. To properly evaluate the range of the method, the vendor needs to ensure that the upper end of the range is challenged.
In summary, rapid methods for microbiological, sterility and bioburden testing may provide alternatives to compendial methods. Traditional pour plate or membrane filtration methods are limited in the numbers of macro-colonies counted; new technologies can count much higher CFU in some cases. Additionally, new technologies that enumerate micro-colonies verses macro-colonies can count a higher population, and rapid microbiological technologies can shorten incubation time, while significantly decreasing time to result. However, variability must be accounted for and traditional analytical performance characteristics should be considered in method validation.
This article is an excerpt from a chapter on microbial methods to be included in the book ‘Specification of Drug Substances and Products: Development and Validation of Analytical Methods’ to be published by Elsevier.
References
1. USP<61>, 35th revision. United States Pharmacopeial Convention, Rockville, MD, December 2012.
2. USP <62>, 35th revision. Ibid.
3. USP <71>, 35th revision. Ibid.
4. PDA Technical Report No. 33, PDA Journal of Pharmaceutical Science and Technology. 2000. Vol 53(3) Supplement TR33.
5. USP <1223>, 35th revision. United States Pharmacopeial Convention, Rockville, MD, December 2012.
6. European Pharmacopeia 5.1.6, Ph. Eur. 7.5. Council of Europe, 2012.
7. Hussong, D. and Mello, R., American Pharmaceutical Review. 2006. 9(1): p62-69.
8. Cundell, A., The History of the Development, Applications and Limitations of the USP Sterility Test. Rapid Sterility Testing. 2011. 7: p127-169. ISBN Number: 1933722568
9. Parveen, S., et al, Vaccine. 2011. 29: p8012-8013.
10. Gray, J.C. et al, American Pharmaceutical Review. October 2010.
11. Millipore Corp, Milliflex Rapid System Operator’s Manual. 5/2006. Publication No. PF09390 Rev. B.
12. Gordon, O., et al, European Pharmaceutical Review. 2011. 16(2): p9-13.
13. Millipore Corp, Milliflex Quantum Rapid Detection System User Guide. 2/2010. Publication No. PF11940 Rev A.
14. Riley, B., European Pharmaceutical Review. 2011. 16(5): p3-5.
