THE MICROBIAL ID BREAKTHROUGH: How DNA Sequencing Services Help Prevent Catastrophic Cleanroom Shutdowns

Transcription

1 A WHITE PAPER THE MICROBIAL ID BREAKTHROUGH: How DNA Sequencing Services Help Prevent Catastrophic Cleanroom Shutdowns By Dennis Champagne Director, Laboratory Services, Microtest, Inc.

2 A WHITE PAPER THE MICROBIAL ID BREAKTHROUGH: How DNA Sequencing Services Help Prevent Catastrophic Cleanroom Shutdowns INTRODUCTION Contamination by an unknown organism often calls for total shutdown of a cleanroom s operation until that organism can be identified and eliminated. Such shutdowns can have catastrophic effects on the schedules and productivity of medical device and pharmaceutical manufacturers, as well as other cleanroom operators. This report outlines some common sources of contamination. It discusses problems and delays associated with traditional methods of identifying those contaminants. It also highlights breakthrough solutions to these difficulties offered by a testing laboratory employing the latest genotyped-based microbial identification systems, in combination with other cutting-edge environmental services. URGENT PROBLEMS Modern cleanrooms used for the manufacture of medical devices, pharmaceuticals, or combination products face widespread, persistent threats of contamination. Much of this contamination comes from the people working in these environments. They carry in invasive organisms on their hands, their feet, and their clothes. They also bring contaminated equipment and materials into the environment. Risks of contamination may increase with the seasons, for instance in the prevalence of molds during spring blooms or summer flowerings. Other threats may exist in the cleanroom environments themselves. Water sources and drains are ready sources of foreign organisms, often due to broken, improperly installed, or clogged filtration devices. Heating, ventilation, and air conditioning systems may suffer similar problems with airborne contaminants. A HEPA filter charged with protecting against contaminants, when malfunctioning or filled to capacity, may instead do the opposite. Any cleanroom surface may contain a spore-forming organism allowed to grow due to improper disinfectant selection or procedures. As the U.S. FDA states, Characterization of recovered microorganisms provides vital information for the environmental monitoring program. Environmental isolates often correlate with the contaminants found in a media fill or product sterility testing failure, and the overall environmental picture provides valuable information for an investigation. Monitoring critical and immediately surrounding clean areas as well as personnel should include routine identification of microorganisms to the species (or, where appropriate, genus) level. 1 However, identifying the culprit is a challenging task indeed. Cumulatively, thousands of organisms pose plausible threats, including the following: Gram-positive bacteria numerous different species, including many that produce hard-to-kill spores Gram-negative bacteria including numerous pseudomonases Molds often during seasonal peaks Yeast Discovery of contaminants from any of these sources poses serious problems for quality managers, lab managers, and other executives responsible for cleanroom facilities. In almost all cases, serious contamination means the cleanroom must temporarily shut down. This brings production of pharmaceuticals or medical devices to a halt for days, weeks, or even months. Deadlines are missed. Schedules are thrown into chaos. Projects, contracts, and other relationships with the facility s customers can be imperiled, or terminated outright. Profits plummet. In some cases, personnel responsible for quality and productivity may lose their jobs. 1.

3 Fast action is vital. To get the cleanroom up and running as quickly as possible, the extent and location of areas affected must be determined, and the exact organism responsible identified. TRADITIONAL ANSWERS Conventional procedures for identification of unknown microorganisms are well established. Samples are gathered and submitted to a series of biochemical or phenotypic testing techniques. These traditional methods may still have some utility as part of a regular series of preventive measures. When coupled with minimal-contamination cleanroom design, properly designed operating procedures, and regular testing of surfaces and equipment, routine screening and identification with biochemical or phenolic-based techniques may provide some level of prophylaxis. Unfortunately, numerous problems are associated with these systems and methods. They must be based on the observed qualities of the target organism. Identification relies on observing a suspect organism s morphology, development, and behavior over time, as well as analyzing the structure and function of its cellular components. These tests are unsuitable for identifying such commonly encountered contaminants as mycoplasmas or molds. They demand the use of multiple isolated colonies of living organisms. Testers must make subjective decisions to attempt growth of the organism in media of a specific nature (aerobic or anaerobic) and Gram reaction (bacteria, mycobacteria, or yeast). If they suspect the organism is a bacteria, they must decide whether its source is environmental or clinical. Finally, testers must typically match the isolate against one of five libraries in the testing equipment s software: aerobic, environmental/clinical, anaerobic, yeast, or mycobacteria. Reproducibility is also a common problem. A suspect organism may be identified one way in one test, but another way in the next. Accuracy suffers likewise. A number of laboratories have discovered that, in this example, both identifications may subsequently prove incorrect. In addition, due to their frequently originating in medical settings, these tests may produce more accurate identifications of organisms common to clinical contexts, as opposed to isolates often encountered in the challenging environment of a large-scale manufacturing process. Perhaps most important, these tests take time. Attempting to regrow the organism for identification is far from an overnight process. In fact, with traditional methods, routine identification of a single sample may take as much as 1 month. Again, from a manufacturing viewpoint, this magnitude of delay can be catastrophic. When significant contamination has already occurred, traditional microbial identification techniques have thus proved increasingly unsatisfactory. Their results are too uncertain, and arrive much too slowly. These methods simply can t meet the urgent scheduling and production needs of a modern medical device or pharmaceutical manufacturing operation. Traditional identification methods Identify bacteria or yeast only Require live, isolated colonies Demand colonies grown in specific media to match library Deliver subjective results Must match results against appropriate library for accurate ID Provide library IDs based on multiple strains/species for each entry Require up to 4 weeks turnaround time 2.

4 NEW BREAKTHROUGHS When environmental microbial contamination poses problems up to and including catastrophic facility shutdowns quality control and production managers need their samples tested by a fast, accurate, highly reliable identification system. To provide that level of confidence, leading testers increasingly rely on DNA sequencing, a high-precision, gene-based method that represents a breakthrough in microbial identification. The U.S. FDA states: Genotypic methods have been shown to be more accurate and precise than traditional biochemical and phenotypic techniques. These methods are especially valuable for investigations into failures (e.g., sterility test; media fill contamination). 2 Experts agree that DNA sequencing is faster, more accurate, and more reproducible than phenotypic or biochemical identification methods. It provides the definitive information needed to control contamination and reduce risks associated with production downtime. That s why DNA sequencing has become the new gold standard in microbial identification, outpacing traditional methods. These benefits are exemplified in MicroSEQ microbial identification systems from Applied Biosystems. Such systems employ ribosomal DNA (rdna) sequencing to replace phenotypic microbial ID methods, fatty acid ID methods, traditional plate identification, ELISA, or antibody-based methods. Using a phylogenetic approach, the system sequences the stable 16S ribosomal RNA (rrna) gene present in all bacteria. For fungi, it sequences the D2 region of the large fungi sub-unit. (Note that a single isolated colony alive or dead is sufficient for identification purposes.) After sequencing the rrna gene, the system automatically compares the results to validated sequences in its customizable microbial libraries. It then delivers a list of the closest matches, ranked according to genetic distance from the sample. As opposed to traditional methods, DNA sequencing for microbial identification has proven highly accurate. Experienced users report positive identification rates over 99% for all suspect categories, including bacteria, mycoplasmas, yeast, and molds. Testers also observe that sequencing avoids the traditional bias toward clinical settings, showing strong results in identifying organisms widely encountered in environmental testing. Nor is accuracy impeded by subjective judgments. Unlike traditional methods, wherein testers must observe an organism s morphology and behavior or make best-guess estimates in choosing growth media or software libraries, testers can rely on straightforward procedures and objective criteria. They can easily decide between only two necessary libraries: bacterial or fungal. Reproducibility is also much improved. The system identifies the same microbe the same way from run to run, sample to sample. Identification can be tied to the unique DNA sequence of a target organism with extremely high confidence. Finally and most importantly, DNA sequence identification saves time. Where traditional methods may take weeks, a month, or longer to return results, DNA sequencing works in hours or at most days. Virtually all identifications can be completed within a week. On this count alone, DNA ID methodology recommends itself strongly to all manufacturing managers. DNA sequencing is ideal for testing applications including pharmaceutical quality assurance/quality control labs, finished product and in-process testing, media fill failure investigations, sterile medical products, opthalmics, medical devices, cosmetics, and nutritional supplements. Once the organism is positively identified, managers can much more easily deduce the source or sources of contamination, and take swift corrective action to enable the resumption of manufacturing production. 3.

5 Advanced DNA sequencing Identifies bacteria (including mycoplasmas), yeast, and molds Allows identification from a single isolated colony Allows identification whether colony is living or dead Provides definitive results requiring only bacterial or fungal library categories Delivers stable, reproducible results based on organism s unique DNA sequence Allows 1 week or less turnaround time THE MICROTEST SOLUTION One testing laboratory has been at the forefront of DNA sequencing for pharmaceutical, medical device, and combination product manufacturing since the introduction of this revolutionary methodology to this market. Microtest has been a leader in its field since the 1980s, offering a broad portfolio of laboratory testing and consulting services. The company has built an unequaled reputation based on up-to-date technology, process flexibility, scientific knowledge, proven quality, and dedicated client service. Clients affirm that Microtest s assistance provides critical competitive advantages such as shortest time to revenue. Microbial identification is a demanding specialty requiring special equipment and expertise. Many testing laboratories send out their identification work to subcontractors narrowly focused on this field. This can only delay the production of timely answers. Microtest has gone in the other direction. The company has recently expanded its commitment to this segment, doubling the resources devoted to its advanced microbial identification and analytical services. Added to its roster of state-of-the-art thermocyclers, centrifuges, water baths, and other equipment is a second MicroSEQ 3130 DNA sequencing ID unit the fastest and most accurate system of its type commercially available. So Microtest now devotes two MicroSEQ systems to these services. Genotypic-based technology enables identification of contaminating environmental organisms More quickly More accurately Reproducibly DNA sequencing allows Microtest to identify both viable and nonviable organisms. This ability becomes important, for example, in cases where clients need comparison of samples of dead organisms collected 4 weeks previously to living samples collected the day before. (If the organisms are identical, it may indicate a recurring problem.) It also enables clients to build libraries of common contaminants in their areas over time, developing systematic trending data. Access to two MicroSEQ systems simultaneously also doubles Microtest s testing throughput. The company can run about 60 organisms in a 24-hour period, normally utilizing two shifts. This can be vital when a client operation is down and every hour counts. For example, testers can run a yeast and mold plate on one instrument, and bacteria on the other; or put through two large batches of only mold, yeast, or bacteria (including mycoplasmas). Since other even relatively well-equipped laboratories usually possess only one DNA sequencer, the result is a distinct advantage for Microtest and its clients. Speed is the most obvious result of these advantages. Microtest can often produce preliminary microbial identification in 24 hours. Comprehensive final reports on a variety of samples from a given manufacturing area or an entire facility are typically available in 1 week or less. This contrasts strongly with traditional methodologies that can take 1 month or longer. 4.

6 Both remote and onsite services are available for clients anywhere in the U.S. At the client location, Microtest specialists can perform microbial sampling of water, surfaces, air, and compressed air/gases. For remote monitoring, Microtest supplies sampling plates and sterile water collecting vials everything needed for clients to collect samples in their own cleanrooms or manufacturing spaces. The company also furnishes full instructions for receipt, sampling, storage, and return to Microtest. The company produces quick, reliable results in failure investigations involving sterility testing, media fills, and more. It helps clients control contamination, both by taking immediate corrective action and by initiating long-term programs of prevention. And it reduces the loss and risk associated with manufacturing production downtime including delayed product releases, back orders, and recalls. Besides cutting-edge technology, the company brings to bear a highly technically qualified, experienced staff. In the area of environmental testing and microbial identification, Microtest microbiologists have received intensive training on MicroSEQ and other advanced equipment. They have provided literally thousands of successful identifications based on DNA sequencing in recent years. Each project is assigned an experienced team leader to facilitate effective communications and ensure that client objectives are clearly understood and quickly achieved. In addition, clients may feel free at any time to talk with the analysts performing their individual testing. Clients can be confident that the correct tests are carried out on the most accelerated possible schedule, with the most accurate results. Advanced microbial identification complements the full gamut of Microtest offerings available at competitive rates. These services include the following: Contract manufacturing Pharmaceutical testing and validation Medical device testing and validation Environmental control and testing Water validation Mold identification Biologics/virology Clients undertaking new pilot manufacturing lines or processes often call on Microtest for environmental testing and microbial identification on a biweekly or monthly basis. For established manufacturing operations, quarterly testing may provide adequate assurance. Note that maintaining its own manufacturing suite for contract projects helps the company better understand and meet the needs of its manufacturing clients. Microtest tries and verifies new methods in its own suite before recommending them to clients. Improvement in cleanroom equipment and procedures are implemented constantly, for optimum results. The recognized quality of these services has made Microtest an acknowledged leader in problem solving for life science testing and manufacturers of pharmaceuticals, medical devices, and combination products. Its use of breakthrough microbial identification methodologies only strengthens that position. Clients report that Microtest s scientific knowledge, process flexibility, and accelerated response combine to minimize supply chain disruption and provide the fastest time to revenue. 5.

7 REFERENCES 1, 2. Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing Current Good Manufacturing Practice; U.S. Department of Health and Human Services, Food and Drug Administration (FDA); September 2004; section X.B., Microbiological Media and Identification. About Microtest Microtest Laboratories, Inc. 104 Gold St. P.O. Box 848 Agawam, MA Microtest is a trademark of Microtest Laboratories, Inc. MicroSEQ is a registered trademark of Applied Biosystems. All other brands may be trademarks of their respective holders Microtest Laboratories, Inc. All rights reserved. Printed in U.S.A /08 Microtest is a leader in testing services and contract manufacturing for medical devices, pharmaceuticals, and biotechnology. The company was founded in Its expertise and flexible processes enhance product safety and security, speed time to market, and minimize supply chain disruption. Microtest s unique single-source capability to provide testing and manufacturing solutions allows the company to support a full pharmaceutical or medical device product release. Facilities in Agawam, Massachusetts, U.S.A. include state-of-the-art aseptic manufacturing areas; analytical chemistry, microbiological, and virological laboratories; Class 100 cleanrooms; onsite steam and ethylene oxide sterilization, plus depyrogenation capabilities; purified water systems; and voice/data systems. About the Author Dennis Champagne With more than 13 years experience in regulatory microbiology and contract laboratory operations, Dennis Champagne leads Microtest s microbiology, contract analytical chemistry, laboratory support, and virology departments. Mr. Champagne holds a B.S. degree in microbiology from Iowa State University, and is a nationally registered microbiologist. For more information, contact: info@microtestlabs.com (800)