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Volume 2010 Supplement, Issue 1
Pharmaceutical Technology talked to drugmakers, equipment vendors, and service providers to learn more about the advantages and disadvantages of rapid microbial-detection methods.
Traditional methods of microbial detection tend to be labor-intensive and take more than a day to yield results. Rapid methods for microbial detection can be sensitive, precise, and quick. Yet the pharmaceutical and biopharmaceutical industries have been slow to embrace these techniques. To understand rapid microbial-detection methods better, Pharmaceutical Technology discussed the benefits, application, and challenges they entail with Orla M. Cloak, commercial development manager of rapid testing solutions at Lonza (Basel); Lori Daane, vice-president of Celsis Rapid Detection (Chicago); Maitry Ganatra, program manager at Pall BioPharmaceuticals (Port Washington, NY); Wayne Miller, field marketing manager, and Martha S. Rook, marketing and regulatory manager at Millipore (Billerica, MA); Geert Verdonk, senior research scientist for quality at MSD (Oss, The Netherlands); and John A. Williams, leader of the rapid microbiological methods research and development program at Baxter (Deerfield, IL).
Definition of rapid microbial methods
PharmTech: What are rapid microbial methods?
Ganatra: Rapid microbial methods (RMMs), also known as alternative microbiological methods, are the technologies that allow the user to get microbiology test results faster compared with traditional culture-plate methods.
Miller: "Faster" means in a matter of hours, as opposed to days or weeks in some cases. The various techniques do not share a common principle.
Cloak: In general, rapid methods can be grouped into three distinctive categories in accordance with their application. These categories include qualitative, quantitative, and identification methods. Qualitative rapid methods provide a presence or absence result that indicates microbial contamination in a sample. Quantitative methods provide a numerical result that indicates the total number of microbes present in the sample. Identification methods provide us with a species or genus name for the microbial contaminant in a sample.
Various rapid methods are available and in use in the industrial microbiological market today. They tend to be based on various technology platforms. The more common technologies include nucleic-acid-based detection, which uses DNA or RNA targets; antibody-based detection; biochemical; enzymatic detection such as adenosine triphosphate (ATP) methods; impedance methods; and flow-cytometry-based methods.
Advantages and disadvantages
PharmTech: What are the benefits of RMMs?
Daane: From a manufacturing perspective, a faster time to result enables companies to release raw materials quickly, transfer in-process work to the next stage, and bring finished products to market, which shortens the production cycle, reduces inventory requirements, and frees up working capital. Realizing these benefits requires a rapid method that can match the requirements of the manufacturing line in terms of testing throughput.
Significant savings from rapid methods also come from the ability to identify, contain, and recover from a contamination event quickly. The financial, supply-chain, and brand benefits of being able to recall affected products from distribution centers before they reach customers are obvious.
In addition, some rapid methods help laboratories reduce worker subjectivity. Automated systems that provide test results in a clear, pass–fail approach take the guesswork out of plating and waiting.
Cloak: Rapid methods' advantages include ease of use, high-throughput capabilities, minimal training requirements, compliance with process-analytical-technology (PAT) initiatives, high specificity and sensitivity, ability to interface with laboratory information management systems, and data-trending ability. These characteristics could potentially contribute to better control, quality, and efficiency in the manufacturing and product-release processes.
PharmTech: What are the disadvantages of RMMs?
Miller: One disadvantage is that not one RMM has been able to replace traditional methods in total. I believe that is coming, but it is a slow process.
Ganatra: The rapid methods require upfront capital investments, and the cost per test is high compared with that of culture tests. Rapid methods are technically more complex than culture methods.
Comparison of rapid and traditional methods
PharmTech: How do rapid methods differ from traditional microbial-detection methods?
Ganatra: All the traditional methods require the growth of microorganisms in media. Scientists need to examine cultures visually to check for microorganisms. Because of our limited vision, humans can see those microbes only when growth reaches a high number of colony-forming units. Rapid methods typically use markers that can be detected by an instrument. Many instruments detect those markers even at a low number of colony-forming units. This ability significantly reduces the time to detection.
Cloak: Traditional methods can be slow, labor-intensive, and subjective. Rapid methods usually target an alternative analyte such as a DNA or RNA sequence unique to the organism. They tend to be easier to use and provide a much faster time to result. This differential is similar across the board, regardless of whether the target is bacterial, fungal, or viral.
Williams: Rapid methods may be more sensitive and objective in detecting bacteria and fungi compared with the traditional methods. For viruses, special detection methods are used because viruses are unable to proliferate on the substrate used for bacterial or fungal growth.
PharmTech: Do rapid methods require special equipment?
Rook: Rapid methods do require special equipment, which ranges from handheld to capital equipment.
Cloak: The type, quantity, and cost of instrumentation required varies, depending upon the rapid method in question. The manufacturers of rapid methods generally provide all the components necessary to the run the test.
Daane: Some equipment such as luminometers are compact instruments that fit on the laboratory bench and are designed for industrial microbiology. Other systems require a dedicated room or the purchase of multiple modules for volume testing, which takes up valuable bench space.
PharmTech: Is there a quantitative or qualitative difference between the results that rapid and traditional methods achieve?
Rook: Qualitative or quantitative differences can exist, based on each particular technique. This is where validation of the rapid method can get quite complex. Comparing traditional results, in colony-forming units, to a new method that detects bacteria in terms of relative light units can be challenging.
Ganatra: In traditional methods, detection is highly dependent on growth conditions that do not allow all the microbes to proliferate. Rapid methods target metabolic markers, so they can also detect viable but nonculturable microorganisms. So, rapid methods generally give higher counts than traditional methods.
Cloak: Qualitative or quantitative result differences depend on the specific methods in question and also the application of those methods. In most cases, the rapid method performance is benchmarked against performance requirements of traditional methods. However, some rapid methods can provide more sensitive and specific results in a shorter timeframe than the more conventional approach.
Daane: Quantitative tests are useful when contamination is present, but are useless otherwise since there's nothing to count. That's why a qualitative, absence–presence screening makes sense. It provides rapid results, including confirmation of negative results within 24 hours, that a company can act on to save time and money. Rapid quantitative tests such as flow-cytometry methods are currently limited to filterable liquids only. Other product types require sample preparation such as dilution, enrichment, and incubation that limits the results to that of a more expensive qualitative test.
PharmTech: Are certain methods best for bacteria or for yeast?
Williams: Each rapid microbial-detection method has its own strengths, which depend on the type of testing being performed and the product being tested. One rapid method may be ideally suited for a particular product or test, but may not be applicable to other areas.
Rapid methods that are useful for detecting bacteria usually are also valuable for detection of yeast and mold as well. But specific methods are required for virus detection because viruses grow differently. Bacterial or eukaryotic cells are used as a growth substrate for viruses.
PharmTech: Do the detection limits of rapid methods differ?
Cloak: The various rapid microbial-detection methods in use have different strengths and limits of detection. It is very difficult to comment on which methods are better because it depends upon the customer's application and use of that method. Some rapid microbiological methods will fit better with an individual company's process or work more effectively with a specific sample or product type. When choosing a rapid method, the user typically determines what performance criteria are important for their manufacturing or laboratory process. Some of the attributes of a rapid microbiological method that a user will assess would be performance criteria, including specificity, accuracy, limits of detection, limits of quantification, linearity, ruggedness, and robustness. In addition, the user will take into account the cost to implement, validate, and also run the method on a routine basis. The return on investment of the rapid method has become an important factor in method evaluation in these challenging economic times. In addition, regulatory acceptance of the method is a critical factor that the user must consider.
Miller: One must consider tradeoffs when evaluating these strengths and weaknesses. For instance, an end user may require that any positive results identify the organisms. In this case, they would require the test method to be nondestructive. The tradeoff may be a longer time to result, but the method would meet the requirement of being nondestructive. Different methods are better for bacteria, yeasts, or viruses. For instance, rapid ATP detection works best with yeasts because they have high levels of ATP, thus increasing the method's limit of detection and sensitivity. Methods' detection limits are slightly different, although most are similar to the traditional method today.
Daane: Detection levels vary depending on the test method. Most systems have some interference from background noise. Sensitive assays have a way to increase the signal-to-noise ratio, thus enabling the assay to detect one-cell preenrichment after a 24-hour incubation. For return on investment, it makes sense to select a system that can screen the broadest range of your products for bacteria, yeast, and mould. Different systems present different challenges. A rapid system that detects carbon dioxide will have difficulty with anaerobic bacteria or buffered products. A system that looks for color changes will not be able to test some pigmented products. Systems that require filtering will have difficulty with solid or viscous samples. A flexible system will be able to test the widest range of products.
Williams: The detection limit varies with different rapid methods. In general, enumerative and DNA-based methods have a lower limit of detection. Growth-based methods offer a greater sensitivity, but take more time and are less accurate. Nongrowth-based rapid methods may have greater accuracy than growth-based rapid methods. Organisms present in the sample may be viable but nonculturable because of injury or media conditions, and therefore they cannot be detected by growth-based rapid methods.
Adoption in the pharmaceutical industry
PharmTech: RMMs were first developed in the 1960s, but it wasn't until the 1970s and 1980s that manufacturers began to invest in them. RMMs were first commercialized in the early 1990s, and Wyeth Pharmaceuticals (Madison, NJ) was an early adopter. What are the pharmaceutical and biopharmaceutical industries' current attitudes toward the methods?
Cloak: The pharmaceutical and biopharmaceutical industries have been reluctant to adopt rapid microbial methods despite the possible advantages. Historically, one of the main obstacles to adoption has been the uncertainty about regulatory acceptance. Why invest in an alternative method when regulatory approval is uncertain? Other factors that come into play include the perceived cost associated with validation and implementation and the cultural change from compendial methods.
Ganatra: Overall, the industries show increased awareness and interest in adopting new methods. Of course, many companies remain skeptical and expect to have one big magic box to replace all microbiology testing. This expectation is gradually changing, and more companies accept the idea that microbiology laboratories, like chemistry laboratories, will need to adapt to more than one type of technology for a specific test. In addition, many companies are looking at rapid methods as a way to improve their bottom line by lean manufacturing and obtaining quicker product release to market. Industry is looking at serious return-on-investment calculations to justify new rapid methods.
PharmTech: Do regulatory authorities accept the validity of rapid methods?
Ganatra: Yes. GlaxoSmithKline (London), Genzyme (Cambridge, MA), and Alcon (Hünenberg, Switzerland) have all received regulatory approval for rapid microbial methods.
Daane: Regulatory agencies around the world are increasingly familiar with rapid methods as submissions and approvals become more commonplace. Global regulatory agencies are committed to accepting process changes or improvements if the proposed change is equivalent to or better than the existing process. The European, Japanese, and US Pharmacopeias all clearly state that an alternative method may be used. For products regulated by the US Food and Drug Administration, the comparability protocol helps streamline the submission and approval process. Submitted for review and approval before initiating the validation process, the comparability protocol simply outlines the studies that will be performed and how the study results will be interpreted. The goal is to show that the proposed change produces results that are equivalent to or better than the existing method.
Williams: Regulatory and industry guidance now exists on the implementation of rapid methods. As long as a rapid method is scientifically evaluated and validated to demonstrate equivalence to the compendial method, the regulatory pathway, at least in the United States, is well defined.
PharmTech: To what extent has industry adopted RMMs?
Verdonk: The larger pharmaceutical companies all have rapid microbial-method programs running. For smaller companies, it is difficult.
Rook: The pharmaceutical and biopharmaceutical industries have been slow to adopt rapid methods because no one method generally meets all of the industry needs with regards to detection limit, enumerative capability, and low cost. Regulatory authorities understand the value of rapid methods but require a demonstration that the rapid method can meet or exceed the performance of the current growth-based methods. While there have been some individual well publicized regulatory approvals and adoption of rapid methods, they are still used primarily in screening and specialized applications.
Williams: The transition to rapid methods has been slow because of the need to evaluate new technologies thoroughly and minimize the risks associated with changing existing methods. Some manufacturers have implemented rapid methods for routine use successfully, and the number appears to be growing yearly.
PharmTech: What new rapid microbial-detection methods have emerged recently?
Daane: The newest rapid methods use DNA and ribosomal RNA to improve time-to-result and specificity. These molecular-based methods appear in both high-end instruments that can strain-type organisms at the genetic level and in easy-to-use assays that detect objectionable organisms quickly. They are used currently to supplement traditional or other rapid methods.
For example, molecular strain typing by sequencing or enzyme digest patterns can be useful to see whether the organism is the same strain as previously observed contaminants. This test requires a pure culture of the microbial contaminant to get useful information. For actionable information, the ability to detect an objectionable organism in 2 hours in a pure or mixed culture is a next-step option following a positive result from a rapid screening.
Cloak: Improvements in the automation of molecular methods have contributed significantly to reductions in operator variability and method complexity, thus facilitating improved accuracy in the application of these technology platforms in the laboratory. Exciting advances in microarray technology for organism identification have made these methods more affordable for routine use. Other technologies such as Raman spectroscopy, microfluidics, and flow-cytometry-based methods continue to be miniaturized and simplified, making them more applicable for the users' requirements. Platforms like the aforementioned will continue to provide more accurate, sensitive, and effective tools that can address the industry's current testing needs.
Miller: The two newest methods are nucleic-acid-amplification methods such as polymerase chain reaction, and fluorescence-based detection methods. Both have been used in research environments for quite some time, but only recently have they been applied to industrial rapid microbiology. For nucleic-acid-amplification methods, instead of detecting the microbial cell itself, the instrument amplifies and detects the nucleic acid in the cell. For fluorescence detection, either a cell's natural autofluorescence or fluorescence resulting from the absorption or activation of a fluorescent dye is used to detect a bacterial cell. Often both of these methods require a short growth based pre-enrichment to allow an acceptable limit of detection.
Williams: Newly emerging microbial-detection techniques include solid-phase cytometry and flow cytometry. These methods are nongrowth-based and have the potential to be more sensitive in comparison with previous rapid methods. An increase in sensitivity seems to be a direction of rapid development despite the disadvantages of these systems.