RMM Scientific Tutorial Pages:
Nucleic Acid Amplification
Micro-Electrical-Mechanical Systems (MEMS)
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When the science of microbiology was in its early stages of development, scientists used liquid media for the cultivation of microorganisms. For those who were in need of a method to segregate individual types of organisms, the use of liquid media proved to be a significant disadvantage. This was the case for Dr. Robert Koch, who, in 1881, was determined to find an alternative method for his experiments. His laboratory first used aseptically cut slices of potato as a solid culture medium, and later turned to liquid culture supplemented with gelatin, which was subsequently poured into glass plates and allowed to solidify. This technique permitted the scientists to obtain pure cultures of the bacteria that were found to be growing in the form of discreet colonies on the surface of the plates.
Unfortunately, on hot summer days, the gelatin medium would liquefy, rendering it useless for its intended application. Furthermore, this phenomenon was accelerated when certain types of bacteria growing on the surface would produce enzymes capable of digesting the gelatin medium. One of the scientists, Dr. Walther Hesse, was frustrated by these events, and he turned to his wife and laboratory assistant, Angelina for help. Walther recalled that his wife's jellies and puddings remained solid, even in the heat of the day, and when he inquired as to what her secret was, she provided an unusual but simple answer. Angelina was previously made aware of a cooking ingredient called Agar-Agar, which had been used as a gelling agent by Asian chefs for many centuries, and as a result, used the material in many of her recipes. Dr. and Mrs. Hesse discussed the possibility of using Agar-Agar as the basis for a stable, solid microbiological medium, and subsequent experiments showed that this worked magnificently.
This simple kitchen ingredient revolutionized the science of microbiology as it made what had been a difficult task of separating and culturing microorganisms on solid surfaces a routine procedure. Interestingly, more than 125 years later, all microbiology laboratories, in every industry sector, continue to use agar as the most important and widely accepted material for growing microorganisms today. Angelina would be proud...but should we be proud as well?
Although the growth of microbial cells on agar surfaces provides the laboratory with critical information about the amount and the type of organisms that may be present in a sample under evaluation, the time to result is usually longer than what is desired. Days and even weeks may elapse before microbial colonies are visually detected, and in most cases, confluent growth prevents individual organisms from being isolated, necessitating sub-culture onto additional agar media, delaying the time to result even further. Additionally, many laboratories are discovering that microorganisms, when stressed due to nutrient deprivation, or following exposure to sub-lethal concentrations of antimicrobial agents, such as preservatives, disinfectants, heat or decontaminating gases, may not replicate when cultured on artificial media, because the environment is not truly optimal for the resuscitation and subsequent proliferation of organisms that may be present. For these reasons, the modern microbiological laboratory should look toward developing innovative approaches to the detection, quantification and identification of microorganisms. From a quality risk management perspective, this direction is critical for the pharmaceutical and biopharmaceutical industries.
Quality risk management (QRM) is an important part of science-based decision making which is essential for the quality management of pharmaceutical manufacturing. The ICH Q9 guideline defines QRM as a systematic process for the assessment, control, communication and review of risk to the quality of drug product across the product lifecycle. Similarly, the FDA's cGMPs for the 21st Century: A Risk-Based Approach, states that using a scientific framework to find ways of mitigating risk while facilitating continuous improvement and innovation in pharmaceutical manufacturing is a key public health objective, and that a new risk-based pharmaceutical quality assessment system will encourage the development of new technologies, such as process analytical technology (PAT), to facilitate continuous manufacturing improvements via implementation of an effective quality system.
Effective monitoring of our manufacturing processes can help to ensure that a state of control is maintained (providing assurance of the continued capability of processes and controls to meet product quality), areas for continual improvement are identified (helping to understand and reduce process variability), process and product understanding is enhanced, and manufacturing agility and efficiencies are realized (by reducing waste and wasteful activities, reduce lead time and increase manufacturing capacity). From a microbiology perspective, we can apply QRM principles in order to design a process to prevent contamination, investigate ways to correct a contamination event, and assess the potential impact of failing results on the patient. Fortunately, recent advances in alternative microbiological monitoring platforms, such as rapid microbiological methods (RMM), provide the analytical tools necessary to accomplish these tasks.
During the past 20 years, the field of alternative and rapid microbiological methods (RMMs) has been gaining momentum as an area of research and application across a number of technology sectors. In fact, much of the development of new systems for the detection and identification of microorganisms has been driven by consumer and patient needs within the food, beverage, environmental and clinical or health care industries. Recent advances in rapid technologies have also encouraged the pharmaceutical and biopharmaceutical industries to validate and implement RMMs in place of their traditional microbiology methods within QC/QA labs and on the manufacturing floor.
Many rapid microbiological method technologies provide more sensitive, accurate, precise, and reproducible test results when compared with conventional, growth-based methods. Furthermore, they may be fully automated, offer increased sample throughput, operate in a continuous data-collecting mode, provide significantly reduced time-to-result (e.g., from days or weeks to hours or minutes), and for some RMM platforms, obtain results in real-time. These methods have also been shown to detect slow-growers and/or viable but non-cultural microorganisms as compared with standard methods used today. Most importantly, a firm that implements a RMM in support of sterile or non-sterile manufacturing processes may realize significant operational efficiencies during the monitoring and controlling of critical process parameters, reducing or eliminating process variability, and reducing the risk to patients. Additional benefits may include the elimination of off-line assays and a reduction in laboratory overhead and headcount, lower inventories (raw material, in-process material, and finished product), a reduction in warehousing space, and a decrease in repeat testing, deviations, out-of-specification investigations, reprocessing or lot rejection.
Rapid methods are now being used for a wide range of applications. As an example, a number of companies within the pharmaceutical industry have implemented RMMs as alternative methods to conventional microbiological tests for the following assays: finished product, in-process and raw material bioburden analyses, sterility testing, environmental monitoring, pharmaceutical grade water testing, endotoxin analysis, microbial identification and the detection of Mycoplasma.
Before purchasing a RMM, there are a number of technical, quality, business and regulatory due diligence activities that should be considered. One of the most important is to understand what technology platforms are available in order to match the most appropriate RMM with its intended application. For example, technical or method considerations may include:
Current rapid method technologies can detect the presence of diverse types of microorganisms or a specific microbial species (qualitative RMMs), enumerate the number of microorganisms present in a sample (quantitative RMMs), and/or can identify microbial cultures to the genus, species and sub-species levels (identification RMMs). The manner in which microorganisms are detected, quantified or identified will be dependent on the specific technology and instrumentation employed.
For example, Growth-based technologies rely on the measurement of biochemical or physiological parameters that reflect the growth of microorganisms. These types of systems require the organisms in a sample to proliferate, either on a solid or liquid medium, in order to be detected and/or quantified.
Viability-based systems use viability stains and laser excitation for the detection and quantification of microorganisms without the need for cellular growth. Flow cytometry and solid-phase cytometry technologies are examples.
Cellular Component-based RMMs rely on the detection and analysis of specific portions of the microbial cell, including ATP, endotoxin, proteins and surface macromolecules.
Optical spectroscopy methods utilize light scattering and other optical techniques to detect, enumerate and identify microorganisms. Real-time analysis with Mie scattering and Raman spectroscopy are now possible.
Nucleic acid amplification-based technologies employ a variety of scientific principles, including PCR-DNA amplification, RNA-based reverse-transcriptase amplification, 16S rRNA typing, gene sequencing and other novel techniques.
Micro-Electrical-Mechanical Systems (MEMS) utilize microarrays, biosensors, Lab-On-A-Chip or micro-fluidic systems, and nanotechnology, all which provide miniaturized technology platforms as compared with conventional, bench-top instrumentation.
To learn more about the science behind each of these RMM technologies, please click on one of the blue links found above.
Our RMM Product Matrix provides detailed comparison tables of more than 60 different RMM product configurations, with specific information about applications, throughput, time to result, sample size, sensitivity, ID libraries, cost per test, capital cost, and methods of analysis.