The Encyclopedia of Rapid Microbiological Methods is a culmination of many years of research, development and implementation of new technologies by a number of industry sectors, including pharmaceuticals, medical device, cosmetic and personal care, health and clinical, food and beverage, and municipal water, as well as government agencies and their subsidiaries, including bio-defense laboratories, first responders and homeland security. Furthermore, support for novel ways in which to conduct microbiological assays is becoming the norm for both regulatory agencies and pharmacopoeias, as demonstrated in recent initiatives and guidance documents provided by the FDA, EMEA, USP and Ph. Eur.
The encyclopedia attempts to pull together the opinions of these organizations, suppliers of new microbiology platforms, and the laboratories and endusers of the technologies that will be discussed within its pages.
Volume 1 provides an overview of microbiological methods and opportunities for industry, regulatory and pharmacopoeial perspectives, and validation strategies. Topics include the history of microbiological methods, risk-based approaches to pharmaceutical microbiology, the realities and misconceptions of implementing rapid methods in the manufacturing environment, the use of rapid methods in bio-defense and the food industry, PAT, comparability protocols, 21 CFR Part 11 and practical guidance on RMM validation and implementation.
Volumes 2 and 3 explore specific rapid microbiological methods, technologies and associated instrumentation, from both a supplier and an end-user viewpoint. Volume 2 concentrates on growth-based and viability-based rapid microbiological technologies, including flow and solid phase cytometry, ATP bioluminescence, impedance microbiology, and a variety of microbial identification platforms relying on physiological responses.
Volume 3 concentrates on artifact-based and nucleic acid-based technologies, the detection of Mycoplasma, and the use of microarrays, biochips and biosensors. Some of the platforms that are discussed include fatty acid analysis, MALDI and SELDI-TOF mass spectrometry, portable endotoxin testing, 16S rRNA typing, DNA sequencing, PCR, advances in Micro-Electro-Mechanical Systems (MEMS) including Lab-On-A-Chip systems, and a novel instantaneous and real-time optical detection technique for airborne microorganisms.
The detection of food pathogens is crucial for food safety; detection methods must be fast, sensitive, and accurate. Yet, almost all techniques used today to identify specific pathogens in foods take at least 48 hours, and some take as long as a week. Further confounding the challenge is the need to address "zero tolerance," a standard that mandates that no viable pathogens are allowed in certain foods. To meet zero-tolerance levels, detection methods need to be sensitive down to a single pathogen in a prescribed sample. Current methods require several days to achieve this standard, because they rely on culturing the pathogen to increase its numbers to detectable levels.
Regulatory and Compendial Aspects of Developing "Rapid" Microbiological Analytical Methologies for Detecting, Speciating and Enumerating Microorganisms in Samples.
The risk for patients through spoiled or otherwise adulterated pharmaceuticals has been acknowledged for many centuries and led to the establishment of Good Manufacturing Practice (GMP) and pharmacopoeial guidelines. Besides chemical purity, pharmaceuticals also have to
meet microbiological standards, the latter primarily depending on the administration route. Drug products which are injected directly into blood vessels or tissues or that are applied directly into eyes and ears represent a greater infection risk than products which are administered orally or
onto intact healthy skin. While parenteral drug products are required to be free from any viable microorganism (USP <71>, Ph. Eur. 2.6.1), oral and topical products are not required to be sterile, but are subject to strict guidelines limiting the number and types of acceptable microorganisms (USP <61> and <62>, Ph. Eur. 2.6.12 and 2.6.13).
It is the responsibility of the pharmaceutical industry that these microbiological standards are maintained until secondary packaging of the drug product. Knowledge of the microbiological quality of the used excipients and active ingredients, microbiological monitoring of the environment in which the pharmaceuticals are produced, as well as release-testing of the final drug product contribute to maximising patient safety. Testing for microbiological quality requirements relies on traditional methods based on visual detection of a large enough population of microorganisms, either as a colony on solid nutrient medium or as turbidity in liquid nutrient medium. The duration until microbial growth can be detected visually is dictated by the generation time of the microorganisms present; whilst fast-growing microorganisms like E. coli can be seen within hours, visual detection of slow-growing microorganisms can take days or even weeks. Therefore, microbiological quality control often represents the bottleneck for release of drug products after manufacturing. In addition, the late detection of a microbiological quality issue complicates subsequent investigations for the root cause of the contamination. Accordingly, there is high interest throughout the pharmaceutical industry to
replace traditional test methods by faster alternative methods. The encouragement by several health authorities to implement such alternative microbiological test methods, as well as official validation guidance documents for the pharmaceutical industry (USP <1223>, Ph. Eur 5.1.6,1)
heralded a start to the transition to the use of alternative, faster test methods. In this article, several Rapid Microbiological Methods which were evaluated or validated by Novartis will be presented.
The methods used in most microbiological test laboratories originated in the laboratories of Koch, Lister, and Pasteur. While numerous changes have occurred in the chemistry laboratory, there have been limited improvements in methods used for microbiological testing.
In the past decade, many researchers have focused on the study and implementation of improved methods for isolation, early detection, characterization, and enumeration of microorganisms and their products. This translates into better methods, automated and miniaturized methods, methods that require less time or those that are less costly. All of these changes are collectively grouped into the category known as rapid microbiological methods (RMM). In some compendia, these are also called alternative microbiological methods. Although these methods are called rapid microbiological test methods, many of them have their roots in other sciences, e.g., chemistry, molecular biology, biochemistry, immunology, immunochemistry, molecular electronics, and computer-aided imaging.
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.
Rapid Microbiological Methods in the Pharmaceutical Industry. M.C. Easter (2003), Hygiena International, Ltd., Hertsfordshire, UK
In recent years there has been increased interest in the possibility of rapid microbiological methods offering enhanced potential error detection capabilities. However, these methods raise a number of questions, such as how to validate new methods, will they be accepted by the pharmacopoeias, and, most importantly, how will the regulators respond? Rapid Microbiological Methods in the Pharmaceutical Industry answers these questions and more.
The Advent of Rapid Microbiological Methods: Background, Applications, and Validation. Ball, P. R., L. Arbizzani, et al. (2007). White Paper
Microbial contamination poses enormous risks to consumers of pharmaceuticals. There is also the associated financial liabilities and potential for damage to the pharmaceutical manufacturer’s reputation. To guard against these risks, pharmaceutical manufacturers have typically collected hundreds of samples per week, incubated them on agar plates for 7 to 14 days, and then counted the colonies to judge for the presence or absence of bacteria. This approach is very time-consuming and runs the risk that by the time a problem is discovered, a large amount of money may have been invested in manufacturing the product. An even worse scenario is that the product may have already been shipped, creating a potential risk to consumers of the drug. These challenges help to explain the increasing interest by pharmaceutical manufacturers in rapid microbiological methods (RMMs) that provide the ability to detect microbial contamination in a fraction of the time of traditional methods. RMM instruments have been on the market for a number of years, but recent developments, such as performance improvements and cost reductions in the technology, have made them more attractive than in the past. The U.S. Food and Drug Administration (FDA) has also helped to spur the introduction of RMMs through its Process Analytical Technology (PAT) initiative which encourages real-time process monitoring. The result is that a recent survey shows that more than 70% of biopharmaceutical manufacturers either are currently using or plan to introduce RMM technology within three years.
Good manufacturing practices (GMPs) are a prerequisite for commercial production in the pharmaceu-tical industry. They are a basic set of requirements to ensure patient safety. For different reasons, GMP conditions may be lost, including, for example, as part of a planned shutdown for maintenance or con-struction or due to an unplanned disruptive event. This article shares a case study in which rapid mi-crobiological methods (RMMs) were used to evaluate risk and expedite recovery of GMP conditions after the devastation of Hurricane María in Puerto Rico.
The Growth Direct™ System that automates the incubation and reading of membrane filtration micro-bial counts on soybean-casein digest, Sabouraud dextrose, and R2A agar differs only from the tradi-tional method in that micro-colonies on the membrane are counted using an advanced imaging system up to 50% earlier in the incubation. Based on the recommendations in USP _1223_ Validation of New Microbiological Testing Methods, the system may be implemented in a microbiology laboratory after simple method verification and not a full method validation.
A real-time polymerase chain reaction (RT-PCR) assay was developed to detect Burkholderia cepacia in pharmaceutical products contaminated with low levels of bacteria. Different pharmaceutical suspen-sions were artificially contaminated with B. cepacia, Escherichia coli, Staphylococcus aureus, and Bacillus megaterium. After a 24 h incubation in trypticase soy broth with Tween 20, samples were streaked on mannitol salt, phenyl ethyl alcohol, eosin methylene blue, MacConkey, and pseudomonas isolation agar. Microbial DNA was extracted from each sample by using a Tris-EDTA, proteinase K, Tween 20 buffer. Regular PCR targeting the 1.5 kilobases 16S rRNA eubacterial gene and cloning showed the predominant DNA in the extracted mix belonged to E. coli. Selective media isolation of bacterial contamination showed B. cepacia only detected on pseudomonas isolation while eosin meth-ylene blue and MacConkey detected only E. coli. RT-PCR using primers PSL1 and PSR1 amplified a 209 bp 16S rRNA fragment using a Roche LightCycler 96® system with SYBR green I, a common double-stranded binding dye. The cycle at which fluorescence from amplification exceeds the back-ground fluorescence was referred to as quantification cycle.