Improvements in technology have continued, resulting in the appearance of new or modified detection methods for microorganisms that may not have been covered within the contents of the book. All of these technologies and methods have been developed and evaluated for application within the food and clinical industries. None has been validated or adopted for industrial pharmaceutical applications.
MicroFoss system (Foss Electric, Denmark) and Biosys (Remel, USA) are detection systems based on classical cultural methods such as color change and redox potential. The results that are generated give a time to detection that is similar to that of impedance. They are able to enumerate microorganisms and correlates well with plate counts. They also claim to detect specific groups of microbes such as coliform bacteria and yeasts, as well as specific organisms such as Salmonella and
Listeria.
Detection methods using microscopy can yield very rapid results but they usually require extensive sample preparation and a skilled operator, and, as such, these methods are usually labor intensive, have low sample throughput, and poor sensitivity. However, developments in confocal microscopy may extend the scope of this oldest of detection methods. Confocal microscopy can be applied to samples without extensive preparation processes, thus avoiding artifacts, and observations can be made in three dimensions without physically sectioning the specimen. It can be used to study the location, attachment, viability, and survival of microbes (see Takeuchi and Frank 2001). This technology is in its infancy and is currently used for research application in the food industry.
The Cellfacts system (Microbial Systems Ltd, UK) uses the well-established Coulter Counter principles of particle detection to detect microbes in clinical and food samples. The method generates information about both the number and size of particles that is directly related to their metabolic state. The system provides a novel and effective quality control methods for industrial fermentation processes to optimize yield and performance. Incorporating specific metabolic markers is also claimed to provide a method for the detection of specific organisms such as Salmonella. The system has not been evaluated for pharmaceutical applications.
Immuno-magnetic particles have been used for novel applications or in conjunction with other technologies to produce new detection methods. Origin, produced by IGEN International Inc. (USA and UK), has coupled very small ferro-particles with a ruthidium ion to provide a novel end detection system for pathogens such as Salmonella. Matrix Technology (UK) has used immuno-magnetic beads to separate and concentrate pathogens during conventional enrichment procedures. This selective capture process reduces the time and labor required for sample preparation and enrichment, and automatically increases the number of available target organisms, reduces the number of interfering organisms, and thus increases the probability of detection of specific organisms, e.g., Salmonella. The technology has been validated for food samples but not for pharmaceutical applications.
Alternative detection systems using the specificity of bacteriophages have also been developed. Alaska Diagnostics (UK) use phage-specific lysis coupled to enhanced luminescence by the detection of adenylate kinase to detect pathogens. The technology is being commercialized for food applications. BioTec Ltd. (UK) uses phage lysis in a more conventional plague- type agar plate assay to detect broad groups or indicator organisms and possibly even specific pathogens. Some research has
been targeted at objectionable organisms in the healthcare sector but the technology has not been validated or commercialized. BIND (BioControl [formerly IDEXX], USA) uses genetically engineered phage carrying the ice nucleation genes to produce a novel end detection system for pathogens such as Salmonella. The technology has been validated and approved for food applications only.
Automation in DNA analysis has been established (Cockerill and Smith 2002). PCR is set to revolutionize the clinical market by reducing the time and cost for the detection of Group A Streptococci in throat swabs. DNA-based detection systems are becoming established for the identification of microbes as well as the detection of specific organisms. Very rapid identification has also been shown possible by the use of powerful physico-chemical techniques such as MALDI-TOF Mass Spectroscopy. Whole untreated bacterial cells of USP objectional organisms can be differentiated and identified using cluster analysis performed on the relative intensity and mass of 8 to 20 major peaks from their spectral fingerprints.
ACCEPTABILITY
Significant advancement has been made in establishing the criteria for evaluating and validating alternative microbiological methods. An all-party industry Task Force led to the publication of the PDA Technical Report 33 (Bauer et al. 2000), and more recently the USP published as a draft a similar guidance document for the General Information Chapter <1223> (Anon 2002).
The acid test for the acceptance of any alternative method is its recognition and acceptance by regulator agencies as part of Market Authorization for licensed pharmaceutical products. The first and only method to receive this accolade to date is ATP Bioluminescence for nonsterile products. Both the validation process and the regulatory review process were simple and easy with both user and supplier working together to generate the necessary support information. Interestingly, this is a presence and absence test that is replacing a Microbial Limits Test for products with little or no expected bioburden. This is a clear example where the new alternative method is superior to the conventional method that is itself no longer fit for the purpose.
ATTITUDE
Experience has shown that there are no obstacles to the implementation of alternative microbiological methods. Everyone wants better methods that deliver improvements in detection speed, capability, and reliability to ensure the safety of pharmaceutical products. However, improvements, not just simpler, faster, more sensitive methods targeted at end product screening, need to come from several sources. We need a more holistic pragmatic approach that includes a needs assessment based on actual hazard and risk and the practicalities of sampling and testing statistics. That is, we need a modern day system based quality assurance along the whole manufacturing process.
The rate of implementation has been slow due to number of factors including the inherent inertia in the system and a fear of what the regulators may say. Regulators themselves are not immune to change, and many recognize the need for change and want to see the use of best available technology. However, inspectors have to enforce a wide range of standards, and many have only limited microbiological knowledge and experience. It is therefore unreasonable to expect them to be experts in everything. Many industrial microbiologists carry great responsibility but are rarely invested with the appropriate authority. Consequently, they fail to recognize their own strengths and are often forced to blindly follow the established prescriptive procedure, conveniently avoiding consideration of the real practical problems of industrial microbiology. Hence, both industrial microbiologists and regulators adopt the path of least resistance by following the pharmacopoeial guidelines that are intended as a common reference point and include methods based on historical custom and practice. Accordingly, the status quo is the continued use of outdated methods.
In August 2002 the FDA announced its intent to merge science-based risk management with an integrated quality systems approach to focus the agency’s cGMP requirements more squarely on potential risks to public health. This initiative will integrate the most current quality systems and risk management approaches and will encourage the adoption of modern and innovative manufacturing technology. It includes such innovations as regulatory process changes to encourage manufacturing innovations, emphasize a risk-based approach to quality control, and enhance key aspects of FDA inspections.
There are several systems for the assessment and management of risk that cover complex manufacturing processes. The identification of the real (and not perceived) hazards is an essential component and foundation of all risk management systems. Accordingly the outcome of any new risk management strategies and procedures must include a rational reassessment of the real microbiological hazards, including appropriate, practical analytical methods to monitor microbiological quality control and ensure safety.
Clearly there is no shortage of ideas and technologies for improved or alternative methods, only the will to make the change for the better. Opinion leaders and influencers in the industry are preparing the ground for change and improved methodology. The desired revolution will occur only if we all have the courage and determination to work together to challenge the status quo in a nonadversarial environment to make it happen. Failure to do so will result in another 100 years
blindly following and complaining about out-dated methods that are no longer fit for the purpose in the ultraclean manufacturing environment of the twenty-first century.
You do not get what you deserve—you get what you negotiate!
Martin C.Easter Anthony M.Cundell January 2003 REFERENCES
Anon. 2002. Validation of alternative microbiological methods. Pharmacopeial Forum 28(1): 154–160.
Bauer, B., M.Claerbout, W.Casey, A.Cundell, M.Easter, E.Fitzgerald, C.Gravens, D.Hussong, M. Korcynzski, R.Lerchen, F.Marsik, A.Meszaros, J.Moldenhauer, M.Sethi, S.Sutton, M.Tricarico, A.Turton, C.Vojt, K.Wills, J.Wuannlund. 2000. Evaluation, Validation
and Implementation of New Microbiological Testing Methods. PDA Technical Report 33, Vol. 54, no. 3.
Cockerill, F.R., and T.F.Smith. 2002. Rapid-cycle, real-time PCR: a revolution for clinical microbiology. ASM News 68(2):77–83.
Takeuchi, K., and J.F.Frank. 2001. Confocal microscopy and microbial viability detection in food research. Journal of Food Protection 64 (12): 2088–2102.
Contributors
Martin C.Easter, Book Editor
Dr. Martin Easter is General Manager at Hygenia International Ltd., which provides rapid and convenient testing devices for microbiology methods. He was educated in the United Kingdom, where he received his first degree in applied biology (specializing in microbiology and bio-chemistry) and his higher degree in microbial biochemistry. During his formative years, Dr. Easter developed an interest in alternative and rapid methods. As Director of Scientific and Regulatory Affairs at Celsis Ltd., he led the validation and approval team implementing ATP bioluminescence methods in the pharmaceutical and healthcare industries. Now recognized as an expert in alternative methods and systems for microbial safety and QA, he has developed a diagnostic media for use as a rapid test for Salmonella, using impedance, which has achieved AOAC Final Action Approval status. Dr. Easter was instrumental in initiating the PDA Task Force on rapid and alternative methods and has published widely.
Anthony M.Cundell
Dr. Tony Cundell currently directs a small corporate microbiological development and statistics group serving all the domestic manufacturing sites of Wyeth Pharmaceuticals. He has graduate degrees in biochemistry and microbiology from the Victoria University of Wellington and Lincoln University in New Zealand. After his post-doctoral studies in environmental microbiology at the University of Rhode Island and Harvard University, he spent 25 years working in Quality Control in the medical device, biologics, and pharmaceutical industries. Dr. Cundell was a member of the PhRMA Task Force on Environmental Monitoring in Non-Sterile Production Areas and chaired the PDA Task Force on the Evaluation, Validation, and Implementation of New Microbiological Testing Methods that resulted in the development of PDA Technical Report #33 published in July 2000. Subjects of recent publications are environmental monitoring, stream sterilization, and validation of rapid microbial enumeration methods. Most recently he was appointed to the 2000–2005 USP Microbiology Committee of Experts.
Jeffrey M.Farber
Dr. Jeffrey M.Farber is the Director of the Bureau of Microbial Hazards, Food Program, Health Products and Food Branch section of Health Canada. He has a PhD from McGill University and holds an Adjunct Professor position with the University of Ottawa in the Department of Biochemistry, Microbiology and Immunology. Dr. Farber’s lectures in these departments focus on pathogenic foodborne microorganisms. He accepts students in his laboratory for both undergraduate and graduate research projects. His research interests focus on listeria monocytogenes and include modified atmosphere packaging of foods, fresh-cut produce, molecular typing of foodborne pathogens, risk assessment, DNA microarray (biochip) technology for detection and molecular characterization of foodborne pathogens, and assessment of novel technologies for detection of foodborne pathogens. Dr. Farber is the Editor of the International Journal of Food Microbiology, serves on the Editorial Board of the Journal of Food Protection, is co-chairman of the Canadian Listeriosis Reference Service, and is a member of the ICMSF.
Edward A.Fitzgerald
Dr. Edward Fitzgerald has 30 years’ experience in the quality control testing of biological products and their related regulatory aspects. He has worked for the Center for Biologics Evaluation and Research (CBER) and the Food and Drug Administration and its predecessor organizations since 1967. During that time, Dr. Fitzgerald was appointed Deputy Director (1974) and Director (1990) of the Division of Product Quality Control, CBER, FDA. Following his retirement in 1997, he formed Fitzgerald Consulting to provide regulatory and technical advice for biopharmaceutical firms regulated by FDA.
Dr. Fitzgerald received his BS degree in biology from Georgetown University and his PhD in microbiology from Catholic University. He has been a member of the U.S. Pharmacopeia Committee of Revision, now called the USP Council of Experts (COE), since 1980 and was recently elected to his fifth term (2000–2005). He is currently assigned to the Analytical Microbiology subcommittee of the COE. Dr. Fitzgerald is also a member of the International Association for Biologicals, the American Society for Microbiology, and the PDA.
Klaus Haberer
Dr. Klaus Haberer is consultant and managing director at Compliance Advice and Services in Microbiology, GmbH in Cologne, Germany. He studied biology and biochemistry at the Universities of Tübingen and Cologne, where he earned a PhD in physiological chemistry. He did postdoctoral studies in microbiology at the University of Rochester, USA, and at the University of Ulm, Germany. Prior to his association with GmbH, Dr. Haberer held major positions in Quality Control at Hofmann-LaRoche-AG and Hoechst Marion Roussel AG. He is associated with the Working Group Microbiology of the German Pharmacopoeia Commission, and the European Pharmacopoeia Group 1 CM, where he has been an Associate Expert since 1995. He is a founding member of the European chapter of the PDA and a member of ISO TC 198 WG9 Aseptic Processing, where he is German Delegate and Convenor of the working group; FIP Working-party microbiology; and the editorial board of the European Journal of Parenteral Sciences.
H.Donald Hochstein
Dr. H.Donald Hochstein obtained a Doctor of Public Health degree in 1970. He worked at the NIH as a microbiologist from 1958–1972, when the FDA replaced the NIH. Dr. Hochstein remained with the FDA’s Bureau of Biologics, where he was responsible for all aspects of LAL, until his retirement in 1997. He has over 50 scientific publications.
Robert Johnson
Dr. Robert Johnson is the Global Quality Assurance Director for Primary Operations within GlaxoSmithKline. His role also includes establishing microbiological strategies for the GlaxoSmithKline operations. He holds a BS and a PhD in biology and a PhD in microbiology from Portsmouth Polytechnic.
Dr. Johnson has worked in the pharmaceutical industry for over 20 years in both the Quality and manufacturing areas. Initially employed within the R&D microbiological laboratories of Cyanamid, UK, before moving into pharmaceutical microbiology QC laboratories, he has worked in sterile and nonsterile processes as well as medical devices. Dr. Johnson was technical manager in production before taking on the role of QA manager for the Davies and Geck medical devices division of Cyanamid.
Larry J.Kricka
Dr. Larry J.Kricka is Professor in the Department of Pathology and Laboratory Medicine at the University of Pennsylvania and Director of the General Chemistry Laboratory at the University of Pennsylvania Medical Center. He received his BA and PhD degrees in chemistry from York University, England, and was Reader in Clinical Chemistry at the University of Birmingham, England, prior to taking up his appointment at the University of Pennsylvania. Dr. Kricka is a Fellow of the Royal College of Pathologists, the Royal Society of Chemistry, and the National Academy of Clinical Biochemistry, and past- president of the American Association for Clinical Chemistry.
Amy Meszaros
Amy Meszaros currently serves as Site Director of Ann Arbor Operations for STATPROBE, Inc., a leading privately held Clinical Research Organization. Ms. Meszaros and her staff guide biotechnology and pharmaceutical companies through the CONTRIBUTORS 171
clinical trial process in order to secure FDA product approvals. Prior to joining STSTPROBE, she managed product development activities for Difco Laboratories, serving the clinical diagnostic and industrial microbiology markets. Ms. Meszaros was a member of the PTA Task Force that wrote the Technical Report #33 on the Evaluation, Validation, and Implementation of New Microbiological Methods.
Marc W.Mittelman
Dr. Mittelman is a principal with Mittelman & Associates, an independent consulting firm specializing in microbiological contamination control. He holds a PhD from the University of Tennessee. He was previously an Associate Professor at the University of Toronto. Dr. Mittelman’s career experience includes positions in the pharmaceutical industry, a consulting engineering practice, and an independent testing laboratory. Over the past 20 years, his research and consulting specialization has been bacterial biofilm interactions with engineered materials in medicine and industries.
Fiona C.Mortimer
Dr. Fiona Mortimer studied for a pharmacy degree at Kings College, London, and qualified as a pharmacist. Subsequently she carried out research in rapid methods, resulting in a PhD with the thesis titled The Application of Rapid Methods for the
Preservative Efficacy Testing of Pharmaceuticals. A number of methods were assessed including impedance, in vivo bioluminescence and flow cytometry. After the PhD Dr. Mortimer continued with research in rapid methods while a Maplethorpe Postdoctoral Teaching Fellow within the Pharmacy Department at Kings College, London.
Paul J.Newby
Dr. Paul J.Newby is currently Team Manager in the Biological Quality Group, GlaxoSmithKline, Bernard Castle, UK, responsible for microbiological method validation and implementation of new technology. He studied microbiology at the University of Dundee, Scotland, obtaining a PhD in microbial physiology. Dr. Newby has worked in the field of pharmaceutical microbiology for more than 13 years. He was a research fellow investigating the introduction of rapid microbiological techniques into the food sector. With GSK, he has worked both in research and development and in manufacture. He has published and presented extensively in the area of rapid microbiological methods.
Thomas J.Novitsky
Dr. Thomas J.Novitsky is President/CEO of Associates of Cape Cod, Inc., a leading supplier of Limulus amebocyte lysate (LAL). He obtained a PhD in microbiology in 1973. Dr. Novitsky has published over 75 articles and has been awarded 13 patents related to LAL and endotoxin. His current research interest is to develop a synthetic replacement for LAL.
Richard Owen
Dr. Richard Owen is a member of the PA Consulting Group, London, UK. He holds a BS in biological science from Birmingham University and a PhD in molecular microbiology from Leicester University. Dr. Owen has been affiliated with DKO Diagnostic Ltd., Ely, UK, and Celsis plc, Cambridge, UK.
Anthony Sharpe
Dr. Tony Sharpe currently manages Filtaflex Ltd., which manufactures specialized HGMF and sampling apparatus. He began his career as a physical chemist with Unilever Ltd. in the UK, graduated to food microbiology, and joined the Health Protection Branch of Health Canada in Ottawa in 1973. He has been a member of the ICMSF, the AOAC Microbiology, Canadian Advisory Committee on ISO, and various Health Canada and Agriculture Canada Committees. Editor and contributing editor on various scientific journals and books, Dr. Sharpe has written 2 books, 20 book chapters, and 90 scientific papers and has given over 100 conference presentations and several courses in Spanish on rapid methods. His main