Improved testing technology aids QC Microbiology Laboratories

Advances help establish robust manufacturing environments

By RICHARD PRINCE

The role of the typical Quality Control (QC) Microbiology Laboratory is to test water, raw materials, in-process materials and/or finished products in support of product-release activities. To successfully accomplish this controlled testing, a variety of capable and calibrated laboratory equipment is needed.

Sterility testing

During the 1970s and 1980s, sterility testing was typically performed in open laminar flow hoods which provided controlled Class 100 conditions during the testing process. (Laminar flow is now referred to as unidirectional flow and the international term M3.5 replaces Class 100.) However, by the 1990s, the isolator concept had been developed as a replacement for aseptic filling manufacturing and was being used by selected pharmaceutical firms as a primary means to perform sterility testing.

Whereas the laminar flow cabinet represents an open system, the isolator literally represents a closed system, thus permitting the testing of presumed sterile articles in a pre-sterilized and enclosed work environment. Companies that have made the switch to these isolator systems report that false positive sterility results have virtually disappeared. Clearly, more and more QC laboratories that perform large volumes of sterility testing will continue to move in the direction of installing these superior, albeit more expensive, barrier systems.

Water testing

Testing of high purity water systems used to manufacture either sterile drugs or drugs that may be susceptible to microbial growth is another crucial function of QC laboratories. Water testing is composed of both analytical analyses such as conductivity, pH and Total Organic Carbon as well as microbial analyses such as Total Plate Counts and endotoxin levels. While analytical tests are relatively easy to perform and results can be garnered in minutes, it has always been a different story for microbiological analyses. For example, Total Plate Count typically requires several days to perform and is dependent on multiplication rates.

Organisms are notoriously temperamental to deal with and require special nutritive media and designated culture conditions to allow individual cells to multiply and be expressed as discernible macroscopic colonies on an agar plate. Since companies use various types of culture media and culture conditions to determine microbial counts in this poorly standardized area, inter-laboratory data is not easy to evaluate in the attempt to troubleshoot a problem or in developing a preferred approach by consensus.

In addition, the recovery of organisms that may be stressed from residence in a nutrient-poor habitat may be marginal. Several new techniques, however, have been developed, particularly since the 1980s, for microbial counting including indirect methods such as impedance and ATP bioluminescence assays. Both of these procedures measure intracellular metabolism by means of photon pulses and then correlate these signals in quantifiable terms.

Another technique relies on the fluorescent labeling and laser scanning of intact cells. This approach can differentiate between viable and non-viable cells since only the viable cells are able to enzymatically cleave and retain the fluorochrome from the non-fluorescent substrate. The fluorescent-labeled cells enter a sample flow channel, separate into a linear fluid stream, and emit a signal that is subsequently detected and counted by optical detectors.

The benefits of this technique are obvious since it can bypass time-dependent proliferation thereby determining both viable and non-viable cell levels quickly and with sufficient sensitivity. From a microbiological perspective, such an assay offers hope for real-time process control of water systems and filling operations. However, since there are three types of bacterial cells (viable, and thus can multiply; resting, and thus can metabolize; and dead, and thus can metabolically shut down), the system must be validated to distinguish among the three.

Microbial speciation

Since microorganisms possess unique metabolic properties, it is possible to identify organisms on the basis of assimilative and dissimilative biochemical reactions of intermediate metabolism. However, microbial speciation is another area that is fraught with difficulty.

Historically, identification schemes have evolved through several phases. The first-generation approach (c. 1910 to 1960) consisted of agar plates and broth tubes designed to give visible biochemical reactions, while the second-generation approach (c. 1960 to 1970) consisted of paper strips designed to give single reactions. The next approach consisted of miniaturized lyophilized cupules, and this was followed by fatty acid analysis by means of gas chromatography. The current approach now consists of automated (computerized) identification systems that have the capacity to optically read the fingerprints of microbes as they react with biochemical reagents, thus permitting identifications in a matter of hours. The next advance in microbial speciation may come from the better use of either nucleic acid probes that hybridize with complementary base sequences within the cytoplasm or fluorescent-labeled antibody probes that attach to antigens on the surface of the targeted cell.

One quirky aspect of the science of microbiology is the practice of a steady stream of taxonomists who, without due regard to medical, environmental or regulatory predicates, change the names of bacteria from time to time. Scientists and reg ulators are therefore urged to stay abreast of these taxonomic changes since, international nomenclature aside, the organisms do not know their own names!

Richard Prince Ph.D., is president of Richard Prince Associates, Inc. (Short Hills, NJ), a pharmaceutical-based consultancy. Prince has been providing contract testing and consulting services to the pharmaceutical and allied industries for 12 years. He has a bachelor of arts degree in microbiology from Rutgers University and a doctorate in zoology and physiology from Rutgers. He can be reached at (973) 564-8565 or E-mail: [email protected].