Alternative Microbiological Methods in the Pharmaceutical Industry: The Need for a New Microbiology Curriculum

Claudio D. Denoya, Ph.D. , Stephen T. Colgan, Ph.D. , and Gary C. du Moulin, Ph.D.
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Pfizer Global R&D, Genzyme Corporation

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Abstract The degree of training in techniques and instrumentation required for many of the laboratory functions in the pharmaceutical industry has evolved at a rapid pace during the last couple of decades. In order to keep up with rapidly changing concepts and technologies and to keep themselves current in a constantly developing environment, chemists and biologists have had to keep adding training seminars and courses to their curriculum. Genomic and proteome microarrays, Matrix-Assisted Laser Desorption/Ionization and Liquid Chromatography Mass Spectrometry (MALDI-MS and LCMS, respectively), Fluorescence Resonance Energy Transfer (FRET), gel and capillary electrophoresis, High Performance Liquid Chromatography (HPLC) equipment of all types and configurations, microcapillary flow cytometers, and many more, are some of the technologies that have dramatically transformed the blueprint and functions of the chemistry and biology labs throughout the biotech/pharma industry. The same cannot be said for the Quality Control (QC) microbiological testing laboratories. Most of the techniques developed more than 100 years ago, are still more or less unchanged today. This picture, however, is about to change. A new era of equipment and methodologies, termed Alternative Microbiological Methods (AMM) and Rapid Microbiological Methods (RMM), have been developed from cutting edge concepts in molecular microbiology, genetics, biology, and computer science, and are slowly making their way into the pharmaceutical microbiology laboratories. The challenges, and the pros and cons of introducing these new technologies to supplant wellproven conventional microbiological testing procedures, is one of the main subjects of this review. Introduction A late nineteenth century microbiologist walking into any of todays pharmaceutical microbiology laboratories would feel at ease, seeing that the testing techniques and instrumentation, developed more than 100 years ago, are still more or less unchanged today. The obvious need for a technological update combined with the declining productivity of pharmaceutical research and development in the last decade have forced industrial microbiologists to re-evaluate their practices and develop more efficient technologies as a means to assess the microbiological characteristics of our products. As a result, alternative microbiological methods of analysis are gaining traction with industry and acceptance with regulators. General acceptance of alternative microbiological methods (AMM), which also include the new rapid microbiological methods (RMM) is approximately 10 years behind process analytical technology (PAT). PAT now is broadly viewed to include chemical, physical, microbiological,

mathematical and risk analysis. PAT applications developed to monitor chemical reactions usually employ probes placed in-line or at-line and report results in real time (instantaneously). On-line analyses improve worker safety (samples are never isolated), business efficiency (immediate results), leading to nimble process control. PAT has additional advantages when reaction monitoring is needed for unstable intermediates, or when reaction temperatures are extreme (cryogenic or elevated). AMM generally are much quicker than the traditional methods, but cannot provide data as quickly as most PAT methods. AMM also may be automatable, easier, exhibit better accuracy, and have higher throughput. Classical microbial tests require extended periods of incubation to allow the growth of microorganisms. The cultivation of microorganisms present in pharmaceutical ingredients, intermediates, products and manufacturing environments usually require a long recovery phase before initiating a frank growth where cells are actively dividing. In addition, most microorganisms isolated from pharmaceutical samples are initially stressed, and the time for the microbial detection and enumeration typically ranges from two to more than 14 days of incubation. The technologies available to augment detection, quantitation, isolation and identification of microorganisms have gained momentum in recent years. These advances have been particularly notable in areas outside the realm of the pharmaceutical microbiologist emerging from disciplines such as clinical diagnostics, food microbiology, and bio-defense. Since AMM have been embraced to a much greater extent outside of the pharmaceutical industry [1], scientific or technological hurdles are not the main barriers. Regulatory and economic concerns have been cited as main reasons for the delayed adoption of AMM in the pharmaceutical industry. The initial cost to purchase these instruments can be quite high and validation is expensive and time consuming. The pharmaceutical industry generally is riskadverse when regulatory approval is at stake. Inclusion of an AMM as part of an initial NDA has the risk of delaying preliminary approval. AMM, PAT, and Regulatory Status AMM generally are molecular-based techniques combined with new detection platforms that usually require fewer or even single microbial cells for detection. Results are, in most cases, more accurate than those obtained in previous methodologies, and, very importantly, are faster to obtain. Applications of these emerging technologies have been slow to be evaluated in pharmaceutical science, but where there has been an evaluation there have been some successes. With continued validation, these technologies can provide better sensitivity and improved time to detection, in-line or at-line testing, automation and high test throughput potential, high labor efficiency and precise characterization of contaminating species. Feasibility studies have identified that these technologies can reduce high overhead and cycle times for products in quarantine. Moreover, these technologies have found value in the testing of components and monitoring of utilities, in process testing, finished product testing and support or

validation testing [2]. With the publication of chapters related to the validation of alternative microbiological methods in the USP and the European Pharmacopoeia [3-4] there will be undoubtedly an acceleration of the acceptance of these technologies by regulatory agencies. To facilitate the adoption of PAT and AMMs, the FDA has issued a guidance document on PAT and considers PAT to be a system for designing, analyzing and controlling manufacturing through timely measures (i.e. during processing) of critical quality and performance attributes of raw and in-process materials with the goal of ensuring final product quality [5]. The FDA is also championing the principle of Quality by Design (QbD) wherein critical sources of process variation are identified and controlled and where processes are continually monitored and updated to allow for consistent quality over time [6]. AMM and PAT allow processes to be monitored and adjusted before isolation of the final products thus increasing the probability of delivering a quality product. Since PAT applications may more likely be submitted as a postapproval change, the FDA also has provided guidance on submitting PAT, AMM, or applications as comparability protocols [7]. Approved comparability protocols may have the advantages of reduced regulatory risk or a reduced reporting category for implementation. Technological Status To begin to review these technologies it is important to note that there are three general areas of measurement pertaining to the pharmaceutical microbiological sciences [8]. Measurements can be qualitative, that is, determining if contaminants are either present or absent in a test sample. Measurements can be quantitative, that is to precisely determine the number of organisms present in a test sample. Finally, there are measurements derived from technologies designed to specifically identify the microorganism associated with contamination. The range of technologies that are available is broad and those interested in developing a technology to suit a particular application or need should apply an analytical approach in the evaluation of a proposed technology. Fung considers ten attributes that should be included in any review of proposed technology [9]. These are, (1) accuracy for the intended purpose, (2) speed in productivity, (3) cost, (4) acceptability by the scientific community and (5) regulatory agencies, (6) simplicity of operation, including training requirements, and reagents, (7) the reputation of the vendor, (8) technical services provided by the vendor, and finally, (9) utility and (10) space requirements. Detection platforms for alternative microbiological methods have been generally classified as growth based, viability based, artifact based and nucleic acid based. Growth Based Technologies Growth-based technologies utilize either biochemical or physiological measures that can reflect the growth of microorganisms. Test samples are transferred to traditional or enhanced media formulations that will encourage microbial proliferation. The primary advantage of these systems

is that microorganisms can be recovered for failure investigations and other microbial characterization methodologies. Recently, a system that detects CO2 produced by the growth of the microbial contaminant has been successfully validated for use as an alternative method of sterility testing [10]. In this system, organisms are detected by pH sensitive sensors incorporated into the media bottle. These sensors will change color in proportion to the amount of CO2 produced by the growth of a microbial contaminant. The color change of the sensor is interrogated by a light emitting diode whose signal is then translated into reflectance units. Sensitivity of the system is 1 CFU/ml. This system has been successfully employed to monitor sterility in cell therapy products that exhibit short shelf lives [10]. Other growth based systems use ATP-bioluminescence or electrical impedance as detection platforms. Viability Based Technologies Viability based technologies do not require cells to grow. These technologies are based on detecting the presence of individual living contaminants through vital dyes, stains, or cell surface markers [11]. Detection occurs for cells labeled by a specific fluorochrome metabolic substrate collected on a membrane. The fluorescein substrate is cleaved by the activity of an esterase present within the cell cytoplasm. Retention of the substrate is dependent on the integrity of the cell membrane. An intact plasma membrane indicates the presence of a microbial contaminant and can be detected by laser scanning cytometry. Artifact Based Technologies This technology platform analyzes cellular components or molecular probes that are designed specifically for a particular microbial species. For example, individual species can be characterized by unique patterns of fatty acid composition after samples of whole cells have been saponified to induce the formation of methyl esters [12]. Detection of fatty acid methyl esters (FAME) is determined by gas chromatography. Fatty acid profiles of individual species are compared to a database consisting of thousands of profiles. Other technologies utilize time of flight mass spectrometry. Nucleic Acid Based Technologies Partly based on the enormous increase in the development of detection technologies that support biodefense, nucleic acid based technologies are fast entering the world of the pharmaceutical microbiologist. Detection is based upon polymerase chain reaction (PCR) DNA amplification, 16s or 23s rRNA typing, and gene sequencing. Some technologies identify microorganisms by sequencing a portion of the chromosome of an unknown organism and comparing the sequence of 16s rRNA to a data base [13]. DNA fragments of approximately 500 base pairs can be amplified using PCR and subsequently, sequenced. This technology is capable of identifying fungi, mycoplasma, bacteria including slow growers and non-fermenters, as well as yeast. An entirely new generation of technologies is now being evaluated for application in pharmaceutical science. These promising technologies include biochips, micro arrays, biosensors, and

instantaneous detection systems. Using microfluidics, miniaturization, and nanotechnology advances, sample preparation, analysis, and detection can take place on a Lab-On-a-Chip [14]. The Need for a New Microbiology Curriculum for the Pharmaceutical Microbiologists The technologies discussed in the preceding section have enormous potential. However, in order for their capabilities to be fully realized the fundamental acceptance needs to be gained by the pharmaceutical microbiologist and the senior management who must support a sometimes arduous process of validation. Paradigms of validation are now available to facilitate comparability evaluation and regulatory acceptance and there is widespread consensus by the U.S. and international pharmacopeias who are providing the guidance to evaluate and validate new methods. These technologies can result in economic benefits to laboratory operations, enhance sensitivity, and improve time to detection while providing the pharmaceutical microbiologist with the tools to ensure the safety and quality of pharmaceutical products. However, to facilitate the adoption of AMM, the microbiology curricula offered by the academic institutions will need to change. It is evident that the great majority of microbiologists graduating over the past decades received a very traditional foundation in the area of microbiological testing. More recently, there were plenty of undergraduate and graduate microbiology courses dealing with the current concepts in molecular biology, genetics, molecular microbiology, immunology, virology, etc. Unfortunately, there was no systematic linkage between these advanced fields, and the microbiological concepts related to the actual microbiology tests required for quality control of pharmaceutical samples. In addition, there appears to be a complete absence of courses dealing with the specific needs and roles that a Quality Control microbiologist plays in the pharmaceutical industry. We have recently conducted an Internet search to assess the number and content of microbiology courses offered from a broad range of colleges. The search revealed that there is an abundance of courses covering the application of microbiology in medicine, agriculture, food and biotechnology industries. There is also a number of courses covering the study of fundamental microbial life processes, emerging infectious diseases, veterinary medicine and dentistry related to microbiological issues, as well as courses that cover the complexity of microbial life represented by prokaryotes, eukaryotes, and viruses. The understanding of fundamental principles of microbial growth, physiology, genetics, biochemistry, and ecology, general microbiology, biology, biochemistry, mathematical sciences, and physics, immunobiology, MCDB (molecular, cellular, and developmental biology), neuroscience, toxicology, and water resources as related to microbiology are also part of most curricula. However, our Internet analysis indicates that, for most of the colleges, there is a lack of coursework describing the specific microbiological skills and techniques applicable to the

pharmaceutical industry as their relationship to the current regulatory requirements for the manufacturing of drug products (see list below). It is also apparent in this search that there is a general claim by many institutions that those majoring in microbiology will find career opportunities in a wide variety of areas, including: hospital and clinical laboratories; federal, state, and local government agencies; research and development; dairy and food processing; as well as the pharmaceutical and fermentation industries. In spite of these claims, we could not find courses directly related to a professional preparation for a career in pharmaceutical microbiology. Additionally, there appeared to be an absence of courses connecting the latest advances in science and technology in microbiological methods to the area of pharmaceutical Quality Control (QC). The latter probably played a significant role delaying the acceptance and implementation of AMMs in the pharmaceutical microbiology labs. Based on all the above-mentioned points, we would like to put special emphasis on the urgent need to create a new microbiology curriculum. Such a curriculum should include the development of courses specifically focused in pharmaceutical QC microbiology. The new curricula should include many of the already offered general microbiology courses noted above, plus some or all of the following areas: Product development in the pharmaceutical industry, from discovery to manufacturing and commercialization. Microbiological tests applied to each of the pharmaceutical steps present in the development of a new product. Regulatory environment in the pharmaceutical industry General understanding of Good Manufacturing Practices (GMP) guidance, and regulatory compliance in general Compendial requirements in microbiological testing (USA, European, Japan). Full understanding of method development, validation, and comparability studies. Comparative view between traditional microbiological assays and the alternative and rapid microbiological methods. An integrated view of the microbiology discipline, including the fundamental concepts and how they apply to traditional and new methodologies. An integrated view of recent scientific and technological developments taking place in microbiology, cell biology, tissue culture, bioprocess, chemistry, nanotechnology, biochemistry and other related disciplines and how they apply to the pharmaceutical R&D world. Conclusions

Once the potential of AMM is fully recognized and implemented across the pharmaceutical industry, well prepared QC microbiologists will be required to perform an important role combining traditional and novel technological approaches (see Table I).

To assure success in this enterprise, the professional organizations and the academic institutions will need to act now to promote the development of curricula appropriate to meet this challenge. Concomitantly, a new curriculum focusing on the pharmaceutical field will also need a faculty better prepared to provide a strong foundation both in the traditional and the new microbiological techniques, including the latest concepts in pharmaceutical manufacturing, regulatory guidelines, and a comparative overview of the pharmacopoeia from the US, Europe, and Japan. References 1. May, M. Building a better biosensor. The Scientist. 100:36-38, 2004. 2. Sutton S.V.W. Opportunities for the Pharmaceutical Industry (Chapter 6) in Encyclopedia of Rapid Microbiological Methods, ed. Miller M., Parenteral Drug Association, Bethesda, MD, pp273-290, 2005. 3. U.S. Pharmacopoeia <1223> Validation of Alternative Microbiological Methods. USP in Pharmacopoeial Forum, 31:1475- 1486, 2005. 4. European Pharmacopoeia , 5.1.6 Alternative Methods for Control of Microbiological Quality. in PharmEuropa, Suppl. 5.5, 4131-4142. 5. Guidance for Industry PAT A Framework for Innovative Pharmaceutical Development, Manufacturing, and Quality Assurance. U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER), Center for Veterinary Medicine (CVM), Office of Regulatory Affairs (ORA), Pharmaceutical CGMPs, September 2004.

6. Nasr, Moheb M., Risk-Based CMC Review and Quality Assessment: What is Quality by Design (QbD). 2006 FDA/Industry Conference. School of Pharmacy Temple University, March 29, 2006. 7. FDA Guidance for Industry, Comparability Protocols Chemistry, Manufacturing, and Controls Information, issued February 2003. 8. Korcznski, M.S. The Importance of Emerging rapid Methods technology to Regulators and Industry (Chapter 3) in Encyclopedia of Rapid Microbiological Methods, ed. Miller M., Parenteral Drug Association, Bethesda, MD, pp273-290, 2005. 9. Fung D.Y.C. Rapid Methods and Automation in Microbiology Comprehensive Rev. in Food Science and Food Safety 1:3-22, 2002. 10. du Moulin, GC, Kielpinski G, Prinzi S, Duguid J, and Price A. Detection of microbial contaminants for cell therapy products: Validation of an automated microbial detection system in Encyclopedia of Rapid Microbiological Methods, ed. Miller M., Parenteral Drug Association, Bethesda, MD, pp273-290, 2005. 11. Newby P. Obstacles and Opportunities to the Introduction of Pharmaceutical rapid Microbiological Methods (Chapter 4) in Encyclopedia of Rapid Microbiological Methods, ed. Miller M., Parenteral Drug Association, Bethesda, MD, pp273-290, 2005. 12. Olsen WP, Groves MJ Klegerman ME (1990) Identifying bacterial contaminants in a pharmaceutical manufacturing facility by gas chromatographic fatty acid analysis. Pharm Technol 14:32-36. 13 ].Eldering JA, Felten C, Veilleux CA, Potts BJ (2004) Development of a PCR method for mycoplasma testing of Chinese hamster ovary cell cultures used in the manufacture of recombinant therapeutic proteins. Biologicals 32:183-193. 14. Miller MJ. Foreword in Encyclopedia of Rapid Microbiological Methods ed. Miller, M., Parenteral Drug Association, Bethesda MD ppxix-xxiv, 2005.

Claudio Denoya, Ph.D., is a Research Fellow at the Microbiology section of the Parenteral Development Center of Emphasis, Pfizer Global R&D, Groton, Connecticut. Dr. Denoya joined Pfizer in 1988 and he has worked on the engineering of Streptomyces, E. coli, and mammalian recombinant cell lines for the production of antibiotics, and Biologics. During the last 2 years, he implemented a Biologics & Microbiological Testing group. Prior to Pfizer, he worked in Bacillus subtilis molecular genetics at the Public Health Research Institute in New York, and before that, he worked in animal viruses and vaccine production, and formed and led a genetic engineering group at the first South American biotech company, BioSidus. He has more than 60 patents,

book chapters, and peer-reviewed journal articles in the areas of biochemistry, enzymology, microbiology, cell and molecular biology, genetics, and pharmaceutical sciences. Dr. Denoya received his B.S., M.S., and Ph.D. degrees from the University of Buenos Aires. Stephen T. Colgan, Ph.D., is an Associate Research Fellow in Regulatory Chemistry Manufacturing and Controls, Pfizer Global R&D, Groton, Connecticut. Dr. Colgan joined Pfizer in 1987 and he has worked in Analytical R&D, Chemical R&D, and the Pfizers Supply Chain. He has approximately 30 book chapters and peer-reviewed journal articles in the areas of physical chemistry, analytical chemistry, organic chemistry, microbiology, and pharmaceutical sciences. Current areas of interest include Process Analytical Chemistry, Rapid Microbiological Methods, Chromatography, Flow Injection Analysis and Supply Chain Management. Dr. Colgan received a B.S. in Chemistry from the State University of New York, College at Cortland, a M.S. in Forensic Chemistry from Northeastern University, and Ph.D. in Analytical Chemistry Northeastern University. Gary C. du Moulin, Ph.D., M.P.H. is Vice President of Quality Systems for Genzyme Biosurgery. Dr. du Moulin joined Genzyme in 1995 after working for six years developing quality systems for cellular therapies for the treatment of renal cell carcinoma. Prior to his industrial experience, he spent 15 years in the Department of Anesthesia at Harvard Medical School (Beth Israel Hospital) in Boston. He has more than 130 publications in the areas of microbiology, epidemiology, and the regulation and quality control of living cells as a therapeutic modality. Dr. du Moulin received his B.S. in 1969 from the Military College of Vermont-Norwich University, an M.S. degree from Northeastern University, and M.P.H. and Ph. D. degrees from Boston University. Dr. du Moulin currently serves on the Cell and Gene Therapy Expert Committee of the United States Pharmacopoeia and is the past Chairman of the Editorial Board of the Regulatory Affairs Professionals Society Magazine, RAPS Focus. He recently retired from the U.S. Army Reserve at the rank of colonel after 30 years of military service. To correspond with the authors, please contact the editor at: jk@russpub.com