Detection limits

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.

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.

Innovation

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.