How DNA Sequencing Services Help Prevent Catastrophic Cleanroom Shutdowns
Every minute of every day, 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 invasive organisms on their hands, feet, and clothes. They also bring contaminated equipment and materials into the environment. Risks of contamination also may increase with the seasons. For example, molds are prevalent 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 sporeforming organism allowed to grow due to improper disinfectant selection or procedures.
As the U.S. Food and Drug Administration (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 culprits is a challenging task indeed. Cumulatively, thousands of organisms pose plausible threats, including the following:
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 responsible organism identified.
TRADITIONAL ANSWERS
Conventional procedures for the 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 bacterium, 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 also suffers. A number of laboratories have discovered that, in this example, both identifications may subsequently prove incorrect. In addition, due to their frequent origination 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 one month. Again, from a manufacturing viewpoint, this magnitude of delay can be catastrophic.
When significant contamination has already occurred, traditional microbial identification techniques have 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.
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.
According to the FDA, “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).”1
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.
The new microbial identification systems, such as the MicroSEQ® instrument from Applied Biosystems, 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 systems sequence the stable 16S ribosomal RNA (rRNA) gene present in all bacteria. For fungi, they sequence 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 systems automatically compare the results to validated sequences in their customizable microbial libraries. They then deliver a list of the closest matches, ranked according to genetic distance from the sample.
In contrast 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 systems identify 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 for results. Virtually all identifications can be completed within a week. On this count alone, DNA ID methodology is highly attractive for 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.
Microbial identification is a demanding specialty requiring advanced equipment and expertise. Many testing laboratories send out their identification work to subcontractors narrowly focused on this field. But this can only delay the production of timely answers.
Pharmaceutical and medical device manufacturers should seek laboratory partners that have fully committed to the new microbial identification technologies. Look for labs that have recently expanded their resources by adding state-of-the-art thermocyclers, centrifuges, water baths, and DNA sequencing ID units. The genotypic-based identification technology enables identification of contaminating environmental organisms
Make sure your testing laboratory has at least two genotypic-based identification systems. This allows the lab to run about 60 organisms in a 24-hour period, normally utilizing two shifts. This capability can be vital when a client’s 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).
Speed is the most obvious result of the new testing technologies. A lab 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 one week or less. This contrasts strongly with traditional methodologies that can take one month or longer.
A reputable lab can perform both remote and onsite services for clients anywhere in the U.S. At the client’s location, their specialists can perform microbial sampling of water, surfaces, air, and compressed air/gases. For remote monitoring, the lab supplies sampling plates and sterile water collecting vials— everything needed for clients to collect samples in their own cleanrooms or manufacturing spaces. The lab also furnishes full instructions for receipt, sampling, storage, and return.
A high-quality lab with new microbial identification technology will produce quick, reliable results in failure investigations involving sterility testing, media fills, and more. It will help clients control contamination, both by taking immediate corrective action and by initiating long-term programs of prevention. And it will reduce the loss and risk associated with manufacturing production downtime—including delayed product releases, back orders, and recalls.
Besides cutting-edge technology, a laboratory with these capabilities will have a highly technically qualified, experienced staff. Their microbiologists are intensively trained on advanced testing equipment and will have performed literally thousands of successful identifications based on DNA sequencing in recent years. The lab will assign an experienced team leader to each project to facilitate effective communications and ensure that client objectives are clearly understood and quickly achieved. In addition, clients should feel free at any time to talk with the analysts performing their individual testing. Clients must be confident that the correct tests are carried out on the most accelerated possible schedule, with the most accurate results.
Some testing laboratories also have their own contract manufacturing suites. These labs can verify new methods in their own facilities before recommending them to clients. Improvement in cleanroom equipment and procedures are implemented constantly for optimum results.
REFERENCES
Dennis Champagne is Director, Laboratory Services, at Microtest Laboratories, Inc., Agawam, Mass., http://www.microtestlabs.com. 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. Contact Dennis at DChampagne@microtestlabs.com.
Every minute of every day, 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 invasive organisms on their hands, feet, and clothes. They also bring contaminated equipment and materials into the environment. Risks of contamination also may increase with the seasons. For example, molds are prevalent 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 sporeforming organism allowed to grow due to improper disinfectant selection or procedures.
As the U.S. Food and Drug Administration (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 culprits 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
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 responsible organism identified.
TRADITIONAL ANSWERS
Conventional procedures for the 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 bacterium, 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 also suffers. A number of laboratories have discovered that, in this example, both identifications may subsequently prove incorrect. In addition, due to their frequent origination 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 one month. Again, from a manufacturing viewpoint, this magnitude of delay can be catastrophic.
When significant contamination has already occurred, traditional microbial identification techniques have 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.
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.
According to the FDA, “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).”1
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.
The new microbial identification systems, such as the MicroSEQ® instrument from Applied Biosystems, 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 systems sequence the stable 16S ribosomal RNA (rRNA) gene present in all bacteria. For fungi, they sequence 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 systems automatically compare the results to validated sequences in their customizable microbial libraries. They then deliver a list of the closest matches, ranked according to genetic distance from the sample.
In contrast 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 systems identify 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 for results. Virtually all identifications can be completed within a week. On this count alone, DNA ID methodology is highly attractive for 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.
Microbial identification is a demanding specialty requiring advanced equipment and expertise. Many testing laboratories send out their identification work to subcontractors narrowly focused on this field. But this can only delay the production of timely answers.
Pharmaceutical and medical device manufacturers should seek laboratory partners that have fully committed to the new microbial identification technologies. Look for labs that have recently expanded their resources by adding state-of-the-art thermocyclers, centrifuges, water baths, and DNA sequencing ID units. The genotypic-based identification technology enables identification of contaminating environmental organisms
- More quickly
- More accurately
- Reproducibly
Make sure your testing laboratory has at least two genotypic-based identification systems. This allows the lab to run about 60 organisms in a 24-hour period, normally utilizing two shifts. This capability can be vital when a client’s 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).
Speed is the most obvious result of the new testing technologies. A lab 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 one week or less. This contrasts strongly with traditional methodologies that can take one month or longer.
A reputable lab can perform both remote and onsite services for clients anywhere in the U.S. At the client’s location, their specialists can perform microbial sampling of water, surfaces, air, and compressed air/gases. For remote monitoring, the lab supplies sampling plates and sterile water collecting vials— everything needed for clients to collect samples in their own cleanrooms or manufacturing spaces. The lab also furnishes full instructions for receipt, sampling, storage, and return.
A high-quality lab with new microbial identification technology will produce quick, reliable results in failure investigations involving sterility testing, media fills, and more. It will help clients control contamination, both by taking immediate corrective action and by initiating long-term programs of prevention. And it will reduce the loss and risk associated with manufacturing production downtime—including delayed product releases, back orders, and recalls.
Besides cutting-edge technology, a laboratory with these capabilities will have a highly technically qualified, experienced staff. Their microbiologists are intensively trained on advanced testing equipment and will have performed literally thousands of successful identifications based on DNA sequencing in recent years. The lab will assign an experienced team leader to each project to facilitate effective communications and ensure that client objectives are clearly understood and quickly achieved. In addition, clients should feel free at any time to talk with the analysts performing their individual testing. Clients must be confident that the correct tests are carried out on the most accelerated possible schedule, with the most accurate results.
Some testing laboratories also have their own contract manufacturing suites. These labs can verify new methods in their own facilities before recommending them to clients. Improvement in cleanroom equipment and procedures are implemented constantly for optimum results.
REFERENCES
- 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.”
Dennis Champagne is Director, Laboratory Services, at Microtest Laboratories, Inc., Agawam, Mass., http://www.microtestlabs.com. 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. Contact Dennis at DChampagne@microtestlabs.com.
