Rapid Microbiological Methods: Where Are They Now?

• Genzyme, a Sanofi Company

Introduction

Microbiological testing is slowly evolving as traditional methods with microorganism detection requiring days or weeks yield to technologies collectively known as Rapid Microbiological Methods (RMM), which may detect the presence of a single organism within hours. Probably the first RMM, the venerable Gram stain, set the stage over a century ago with the rapid application of a few chemicals to a sample allowing microscopic detection of 105 colony forming units (CFU). More recently, the Pharmaceutical Inspection Convention and Pharmaceutical Inspection Co-operation Scheme (PIC/S) has acknowledged that compendial sterility testing “suffers from significant statistical limitations and this contributes to the low probability of detecting anything less than gross contamination” [1]. Where microbiology laboratories still employ the Gram stain despite its limitations, there continues to be a reluctance to embrace the newer technologies of detection. The advent of cell-based therapeutics has accelerated interest in RMM implementation. In 2004, the United States Food and Drug Administration (FDA) approved an RMM for lot release of Genzyme’s cell therapy product, Carticel^®, after extensive validation [2]. Globalization of pharmaceutical quality systems, which emphasizes risk assessment and reduction using process analytical technology (PAT), has provided an impetus for accelerated acceptance of these technologies. Standards-setting agencies have convened groups of expert microbiologists to modify validation paradigms for compatibility with the unique requirements of RMM. As a result, RMM manufacturers have taken note and begun to incorporate cGMP compliance elements into their instrumentation facilitating regulatory agency acceptance. As these technologies gain wider acceptance in the pharmaceutical and biotechnology industries, confidence in RMM should improve. These technologies can identify microbiological risks faster than traditional Pasteurian methods, monitor process critical control points in real time, and contribute to continual improvement of manufacturing processes, which enhances the safety profile of the products with the possibility of decreasing the cost of quality.

Advantages, Limitations, Encouragement and Barriers

Advantages of RMM

Miller’s magnum opus, Encyclopedia of Rapid Microbiological Methods, documents the advantages of RMM by describing the available technologies and detection platforms [3]. In addition to reducing risk and improving patient safety, RMM can reduce product release cycle time, cost, and labor via automation, high throughput, real-time data analysis, and time-sensitive outputs. They also facilitate investigations by generating earlier results. Choosing an RMM compatible with the product’s critical quality attributes, cGMP/GLP regulations, and validation requirements is crucial. Considerations include 21CFR §11 compliance, ability to identify microorganisms for failure investigations, robust detection algorithms, degree of accuracy and specificity, minimal operator variability, ease of use, low false positive rate, and effectiveness with a range of sample configurations. RMM can improve manufacturing consistency by allowing faster implementation of corrective actions and fostering opportunities to improve the safety of the process. RMM technologies provide an opportunity to improve the quality of the science of microbiology, especially in assessing the significance of viable but non-culturable (VBNC) or stressed microorganisms [4]. Because the experience of pharmaceutical microbiologists and their exposure to traditional microbiology has waned, new technologies can partially compensate for the educational gap [5].

Limitations of Conventional Microbiological Testing

Conventional microbiological methods have well-known inherent limitations. These include small test sample volumes, prolonged incubation periods, incompatibilities with membrane filtration, and ambiguity associated with using turbidity as a detection endpoint. Since aseptic processing has become the foundation of most biotechnology drug manufacturing, increased microbiological risks are inherent in this form of manufacturing. Real-time control and early warning of contamination risks is essential before committing raw materials and other critical product components to the process, validating manufacturing and filling environments, or distributing finished products. This is particularly true for cell and gene therapy or tissue-engineered products with short shelf lives. Risk-based assessment tools, such as Hazard Analysis and Critical Control Points (HACCP) and Failure Mode and Effects Analysis (FMEA), can quantify the risks and potential for compromised microbial integrity in manufacturing processes. Taking advantage of these risk management tools is problematic when using conventional microbiological techniques. Cundell summarized standard incubation times for in-process, finished product, and supporting studies as one impediment [6].

Regulatory Encouragement

Regulators have never discouraged the pharmaceutical industry from exploring RMM as an alternative to traditional methodologies. In fact, providing increased assurance that a product meets microbiological specifications is inherent in 21CFR §610.9, which describes “equivalent methods and processes.” The applicant can present evidence “…demonstrating that the modification will provide assurances…equal to or greater than the method…specified” [7]. PAT and “cGMPs for the 21st Century” present a set of scientific principles and tools supporting innovation and continual improvement. Moreover, publications by scientists at regulatory agencies about their experience with RMM instrumentation provide further evidence of support. The French regulatory agency, AFSSAPS, the German Health agencies at the Paul Ehrlich Institute, and FDA have all published evaluations of a number of detection platforms [8, 9, 10]. Additionally, the United States Pharmacopeia (USP) and the European Pharmacopeia (EP) have recently published relevant chapters:

• USP<1223>“Validation of Alternative Microbiological Methods” [11]
• Ph. EUR. 5.1.6. “Alternative Methods for Control of Microbiological Quality” [12]
• Ph. EUR. 2.6.27 “Microbiological Control of Cellular Products” [13]

FDA recently published Guidance for Industry defining validation criteria for growth-based RMM [14]. Finally, the Parenteral Drug Association is in the process of revising Technical Report #33, “Evaluation, Validation, and Implementation of New Microbiological Testing Methods” to better align with USP, EP, PAT initiatives, and Quality-by-Design (QbD) principles [15].

Barriers to Change

With all this regulatory encouragement, the pharmaceutical industry has still been slow to adopt RMM. The barriers to change have centered on the perceived risk that applicants cannot easily broach the regulatory process. This argument has been getting consistently weaker. At a recent meeting of USP’s Science and Standards Symposium, Dr. Rajesh K. Gupta, Deputy Director in the Division of Biological Standards and Quality Control at FDA’s Center for Biologics Evaluation and Research (CBER), summarized the reluctance of industry to develop new methods. In his remarks, he observed that management may not readily support changes to existing applications because the methods submitted and approved continue to work, so there is no incentive to implement new assays. There is fear of the unknown, including the fear that methods with lower detection limits may uncover microbiological concerns that less robust methods cannot detect. From a business perspective, the cost of validation may be too high or the return on investment too long. Finally, the lack of microbiological expertise can prevent development and execution of appropriate comparability protocols to establish equivalency of the RMM with the official method [5].

Success Factor: Technology Choice

Innovations in RMM have provided the ability to move microbial detection and identification operations from routine multi-day activities to in-line or in-process activities. Visualization of microbial growth either in a broth medium or on agar plates usually requires a number of days of incubation. New RMM have shortened this time to a few days and in the case of surface monitoring to a few minutes. Choosing the appropriate technology for the application is critical to success.

Microbial Detection – Qualitative

Impedance

There are four commercial instruments that use principles of impedance or conductivity measurement to detect bacteria. The relationship between capacitance at the electrode surface and conductance from ionic changes in the media from byproducts produced during bacterial growth allows calculation of impedance. Increases in capacitance and conductance result in decreased impedance indicative of bacterial growth. Each instrument type uses variable design principles that measure conductance based upon frequency and electrode quantity and type.

CO₂ Detection

Growth-based technologies use either biochemical or physiological measures to reflect microorganism growth. Traditional or enhanced media formulations encourage microbial proliferation in test samples. A major advantage of these systems is the ability to recover microorganisms for failure investigations or identification after analysis. These systems use an internal colorimetric CO₂ sensor incorporated into each media bottle during manufacture. The sensor, separated from the media by a semi-permeable membrane, is impermeable to most ions and other media components but is freely permeable to CO₂. Carbon dioxide produced by microbial metabolism diffuses across the membrane, dissolves in water in the sensor, and generates hydrogen ions which result in a color change detected by a colorimetric detector. Light emitted by the detector reflects off the sensor onto a photometer. The resulting voltage signal is proportional to the intensity of the reflected light and to the concentration of CO₂ in the bottle.

Headspace Pressure

Headspace pressure platforms detect growth as a result of consumption or production of gases in the headspace of sealed media bottles causing conformational changes in the geometry of the septum. High resolution laser scanning is the detection platform that can detect these changes. When corrected for barometric pressure, an algorithm analyzes the rate of change in pressure to indicate the presence of a positive culture. Developers claim that this technology does not rely solely on production of CO₂, but will respond to any gas produced or consumed by microorganisms (CO₂, H₂, N₂, or O₂). This methodology has been particularly successful in the detection of fastidious organisms such as Neisseria.

Nucleic Acid-based Technologies

Qualitative nucleic acid-based detection technologies commonly use polymerase chain reaction (PCR). After extraction and purification of the genomic DNA, broad-range (universal) primers amplify highly conserved specific regions of the bacterial, mycoplasmal, or fungal genomes. Conventional PCR uses end-point detection on an agarose gel stained with ethidium bromide.

Flow Cytometry

Flow cytometry, a well-established detection platform and investigative tool first applied to studies of eukaryotic cells, can also be adapted to detect the presence of viable bacteria in samples. The ability to stain microorganisms with dyes such as propidium iodide, which is impermeable to cells with intact membranes, and thiozole orange, which is permeable to all cells, allows a differentiation of viable and nonviable bacterial cells in fluid media. Routine point-of-use screening of blood components for microbial contaminants, such as platelets, is one application of flow cytometry that could readily be adapted to the needs of the pharmaceutical microbiology laboratory [16].

Endotoxin

Automated Limulus Amebocyte Lysate (LAL) testing can provide results within minutes regarding the presence of bacterial endotoxin in raw materials, buffers, or in-process intermediates right in the warehouse or on the manufacturing floor. One such automated system uses the kinetic chromogenic method with endotoxin reagents contained in a plastic cartridge analyzed using a specialized reader to kinetically monitor the chromophore produced during the reaction.

ATP Bioluminescence

Industry has incorporated rapid detection of microbial contamination using ATP bioluminescence for many applications. One such application involves surface swabs dispersed into a liquid matrix for filtration. Addition of a substrate to the membrane surface yields fluorescence following exposure to microbial ATP. Adenosine triphosphate (ATP) is the main chemical energy source of all living cells. Detection systems based on bioluminescence exploit the chemical release of ATP from microorganisms. ATP reacts with luciferase and a photon counting imaging tube detects photons released by this reaction. A computer monitor then represents the photons detected. There are both qualitative and quantitative systems available.

Microbial Detection – Quantitative

Direct Laser Scanning

Visualization of colonies for slow growing microorganisms can require a number of days. Using lasers to scan microorganisms growing on membrane filters with optical imaging using a digital camera can greatly reduce this time. This technique allows detection and enumeration of microcolonies within a few days. An advantage of this technique is that the microorganisms remain viable for identification after colonies become visible.

ATP Bioluminescence

ATP bioluminescence can also enumerate the microorganisms present in a sample. After filtering samples in a liquid matrix onto a membrane and spraying the membrane with an ATP-releasing reagent and substrate, enumeration of fluorescent microorganisms occurs by capturing a digital image and processing the fluorescent data. These results can be available within hours. Historically, the staining process would render the microorganisms non-viable, so isolates would not be available for identification. Newer techniques are available allowing retention of microorganism viability for subsequent identification.

Autofluorescence

Immediate detection of viable microorganisms in our manufacturing environments is now possible by exploiting microorganism autofluorescence. Selective capture of the autofluorescence given off by viable microorganisms permits the instantaneous detection of changes in microbial levels during manufacturing operations.

Nucleic Acid-based Technologies

Quantitative nucleic acid-based detection technologies commonly use real-time PCR. After extraction and purification of the genomic DNA, broad-range (universal) primers or primer/probe sets amplify highly conserved specific regions of the bacterial, mycoplasmal, or fungal genomes. Real-time PCR uses kinetic detection with either a non-specific fluorescent stain or a specific probe-based detection system.

Microbial Identification

Previously, the identification of microorganisms was a multi-day activity. After initial detection of microorganisms following growth on nutrient medium, identification required subcultures and other classical microbiological techniques, such as colony isolation, Gram staining, and analyses of biochemical reactivity. Newer methods analyze the genetic or the biochemical structure of the microorganisms. All of these methodologies have advantages and disadvantages. An additional significant consideration has to be the database of known microorganisms available and validated or qualified for the system selected for use in the quality control microbiology laboratory.

Phenotypic and Biochemical Methods

Most laboratories still use the classical phenotypic / biochemical methods for routine microbial identifications. These methods require the growth and isolation of pure cultures, which can take a number of days. They may also require some preliminary characterization, such as Gram staining.

Genotypic Methods

Many laboratories have been shifting to genotypic characterization methods. The most prevalent methods use comparative DNA sequencing of the 16S rRNA gene in bacteria and a region associated with the 26S rRNA gene in fungi. After extraction and purification of the genomic DNA, PCR amplifies the gene or region of interest. A DNA sequencer automatically determines the nucleotide sequence using a process similar to PCR, but incorporating fluorescently labeled terminators to create DNA fragments spanning the length of the sequence. Comparison of the resulting DNA sequence to database library files provides identification data including taxonomy, data quality, and level of confidence.

Other genotypic methods include the analysis of restriction endonuclease patterns. Use of restriction mapping also allows strain differentiation and isolate comparisons. These genetic methods replace classical methods or they may supplement testing when the classical methods fail to yield an acceptable level of identity. Genetic methods can reduce identity testing to an overnight procedure.

Mass Spectrometry

A new identification methodology uses Matrix Assisted Laser Desorption/Ionization – Time of Flight (MALDI-TOF) mass spectrometry. This method simultaneously screens molecular ions and charged fragments by analyzing their mass-to-charge ratios. Comparing the patterns with the patterns from known microorganisms establishes identity. Identifications can be available in minutes rather than days for the classical methods.

Success Factor: Robust Validation

RMM for qualitative tests, quantitative tests, and identification tests have distinct validation requirements. USP <1223> [11] and EP 5.1.6 [12] give guidance on validating alternative microbiological methods; they require the alternative method to yield results equivalent or better than the compendial method. This requirement for equivalence is echoed by 21 CFR §610.9 [7] and JP XV General Notice 13 [17]. Specificity and sensitivity are the most critical validation parameters for qualitative tests, but robustness, precision, and ruggedness also require consideration. Quantitative tests additionally require assessment of accuracy, limit of quantitation, linearity, and range. The most critical validation parameter for rapid microbiological identification tests is accuracy, however precision and robustness may also apply. Prior to method validation, it is critical to validate any equipment, software, or databases (IQ/OQ/PQ) used in the test.

The accuracy of a quantitative test is the closeness of test results to the true value, demonstrated by comparison to well-characterized standards, and usually expressed as percent recovery. The accuracy of a microbiological identification test is identification of an organism to the desired taxonomic level, demonstrated by analysis of a wide variety of microorganisms, and usually expressed as percent agreement with standards of known identity or reference methods. The precision of a test procedure is the agreement among individual test results, demonstrated by testing multiple replicates of a homogeneous sample, and usually expressed as percent agreement for qualitative and identification tests or as coefficient of variation for quantitative tests. Repeatability refers to the intra-laboratory precision over a short time period with the same analyst and equipment [18]. The specificity of a test procedure is the ability to detect a range of microorganisms in the presence of components in the sample matrix expected to be present, demonstrated by testing both un-spiked samples and samples spiked with a wide variety of microorganisms, and usually expressed as percent false positive and percent false negative results. Detection limit and limit of quantitation are measures of sensitivity.

The detection limit of a qualitative or quantitative test is the lowest concentration of microorganisms detected, demonstrated using serial dilutions and a sufficient number of replicates for statistical analysis. The limit of quantitation of a quantitative test is the lowest concentration of microorganisms detected with acceptable accuracy and precision, demonstrated using a sufficient number of replicates at that concentration. The linearity of a quantitative test is its ability to produce results proportional to the concentration of microorganisms within a stated range, determined using a sufficient number of replicates at multiple concentrations, and usually expressed with statistical estimates of the degree of linearity such as correlation coefficient. The range of a quantitative test is the interval between the limit of quantitation and the maximum concentration of microorganisms exhibiting acceptable accuracy, precision, and linearity, determined from the evaluation of these parameters. The robustness of an RMM is its ability to remain unaffected by small deliberate variations in the method as an indication of its reliability during normal testing, but is perhaps a validation parameter best demonstrated during development. The ruggedness of an RMM is the precision of test results obtained by analysis using the same samples under a variety of normal test conditions, such as different analysts, instruments, reagent lots, and laboratories, demonstrated using the same samples under different conditions, and usually expressed as percent agreement for qualitative and identification tests or with equivalence statistics for quantitative tests [19].

Finally, replacing an official method with an alternative method requires demonstrating equivalence. Comparability protocols provide a mechanism to receive feedback from FDA on study design prior to execution. FDA typically assigns a reduced reporting category to changes submitted with a pre-approved protocol. Similarly, the European Medicines Agency (EMA) will provide feedback using the Scientific Advice procedure for European submissions. In addition, regulatory agencies may request analysis of routine test samples using the official method and the alternate method run in parallel as a condition of approval.

Success Factor: Analytical Methods Lifecycle Management (AMLM)

Developing an AMLM process is a regulatory expectation and promotes the application of appropriate analytical technologies in support of all phases of a pharmaceutical product life cycle. Selection and development of the appropriate method occurs during the discovery or preclinical phases. Qualification and validation in support of Biologics License Application (BLA) or New Drug Application (NDA) filings occurs during Phases 1, 2, or 3. Revalidation following changes or validation of new methods as technologies advance occurs post-licensure. AMLM requires support from senior management, technical expertise, financial resources, and internal procedures for change management.

Conclusion

Quality control organizations must understand their responsibilities in AMLM as a component of the product lifecycle. The evolution of microbiological testing from classical procedures to RMM will place new and unprecedented amounts of data into the hands of the microbiologist. One can then apply this information to aid in prompt decision-making in support of real-time release or perform timely root cause analysis for failure investigations, thus improving the safety of the product. As confidence grows in the use of these technologies the quality control microbiology laboratory will take advantage of these new tools to gain product and process knowledge and in so doing better perform its mission.

References

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Author Biographies

John Duguid is a Principal Process/Analytical Scientist at Genzyme, a Sanofi company, in Cambridge, MA. Genzyme is one of the world’s leading biotechnology companies with more than 12,000 employees working in countries throughout the world united by a common goal: to make a major positive impact on the lives of people with debilitating diseases. Mr. Duguid is currently responsible for developing, validating, and transferring molecular biology assays for rapid microbiology and cell differentiation applications, managing complex projects to implement process changes, and using statistical process control tools to implement process analytical technology for cell therapy products. He has been at Genzyme since 1995.

Before taking on his current technical role, Mr. Duguid managed quality control cell therapy operations at Genzyme for over 10 years, where he designed and implemented a comprehensive cGMP-compliant raw material program controlling 300-400 parts and participated in 16 vendor audits, directing 4 as lead auditor. He also represented QC during 10 FDA inspections and numerous audits from international regulatory authorities as a subject matter expert in material inspection and release, biopsy accessioning, endotoxin testing, Mycoplasma testing, analytical methods, flow cytometry, laboratory failure investigations, assay validation, and data management.

Mr. Duguid received his Bachelor of Science degree in chemistry in 1986 from the University of Michigan in Ann Arbor, MI and taught analytical chemistry in 2000 at Northeastern University in Boston, MA. Prior to joining Genzyme, he worked in analytical research at Abbott Laboratories’ Pharmaceutical Products Division in North Chicago, IL and then as a scientific consultant for Massachusetts biotechnology companies at Arthur D. Little in Cambridge, MA. Involved with the pharmaceutical and biotechnology industries for over 20 years, Mr. Duguid has technical and management experience spanning all phases of the product lifecycle from early research and development through cGMP quality operations.

Dr. Ed Balkovic is a Subject Matter Expert (SME) Microbiologist in Quality Control Technical Services at Genzyme - A Sanofi Company, Framingham, MA. His experience includes over 40 years in Microbiology & Virology, over 25 years in the Biologics & Biopharmaceutical industries, and over 15 years at Genzyme. He established the lab that is responsible for the identification of all microbial isolates obtained from Genzyme’s QC Microbiology testing labs. Dr. Balkovic now conducts investigations, special projects and training in the area of Pharmaceutical Microbiology, including numerous studies evaluating new and improved methods for microbial detection and identification.

He received his doctorate in Microbiology and Immunology from Baylor College of Medicine, Houston, TX. Prior to joining Genzyme, Dr. Balkovic has held various positions in Technical Support, Quality Control, Quality Assurance, Regulatory Affairs and Research & Development at both emerging biotechnology and established biologics companies. He has extensive experience as a Clinical Virologist and Microbiologist. Previously, he supervised the National Virology Reference Lab serving all of the U.S. Veterans Administration’s Medical Centers. He was also the Senior Research Virologist at a major vaccine manufacturer - Connaught Labs (now Sanofi Pasteur).

He serves on the Program Planning Committee for the Parenteral Drug Association’s (PDA) Global Pharmaceutical Microbiology Conference and he served as the Conference Co-Chair in 2009 and 2010.

Dr. Balkovic is also an Adjunct Associate Professor in the Department of Cell & Molecular Biology at the University of Rhode Island. He teaches in the areas of Clinical Microbiology & Virology, Vaccine Development, Emerging Infectious Diseases, Biowarfare & Bioterrorism, and Biotechnology Product Development & Evaluation.

Gary C. du Moulin, Ph.D., M.P.H. is Senior Director of Quality Aseptic Control for Genzyme (A Sanofi Company) where he participates in the development and execution of robust quality systems for Genzyme’s products. 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 on the faculty of Harvard Medical School in the Department of Anaesthesia at Beth Israel Hospital. He has more than 150 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 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 U.S. Pharmacopoeia’s expert committee for Biological Analysis and formally on the Gene Therapy, Cell Therapy, and Tissue Engineering Expert Committee and chaired the ad hoc advisory panel for fetal bovine serum. He serves on the editorial board of Regenerative Medicine and is RAC certified and past Chairman of the Editorial Board of the Regulatory Affairs Professionals Society Magazine, RAPS Focus and was appointed to the Grants Review Working Group of the California Institute for Regenerative Medicine. He is retired from the U.S. Army Reserve at the rank of Colonel after 38 years of service.

This article was printed in the November/December 2011 issue of American Pharmaceutical Review - Volume 14, Issue 7. Copyright rests with the publisher. For more information about American Pharmaceutical Review and to read similar articles, visit www.americanpharmaceuticalreview.com  and subscribe for free.