01. Rapid Microbiological Methods
Application of flow cytometry for rapid bioburden screening in vaccine virus production.- more Use of a real-time microbial air sampler for operational cleanroom monitoring.- more Guidance for Industry Comparability Protocols − Chemistry, Manufacturing, and Controls Information- more Guidance for Industry Formal Meetings between the FDA and Sponsors or Applicants of PDUFA Products - more In-process microbial testing: statistical properties of a rapid alternative to compendial enumeration methods.- more Overview of rapid microbiological methods evaluated, validated and implemented for microbiological quality control- more Electrochemical detection of bacterial cells using synthetic polymer "foot printing"- MICROPRINT - more A Fresh Look at USP <1223> Validation of Alternative Microbiological Methods and How the Revised Chapter Compares with PDA TR33 and the Proposed Revision to Ph. Eur. 5.1.6- more Rapid methods update: revisions to a United States Pharmacopeia chapter- more Alternative to Ph. Eur. pour-plate method for detection of microbial contamination in non-sterile pharmaceutical preparations- more Application of rapid microbiological methods for the risk assessment of controlled biopharmaceutical environments.- more Towards a Rapid Sterility Test?- more MALDI-ToF Mass Spectrometry: A valid alternative for microbial identification- more Books & Overview Articles- more A Preliminary Investigation into the Ability of Three Rapid Microbiological Methods To Detect Microorganisms in Hospital Intravenous Pharmaceuticals - more Development of an ethidium monoazide−enhanced internally controlled universal 16S rDNA real-time polymerase chain reaction assay for detection of bacterial contamination in platelet concentrates - more Sterility Testing of Stem Cell Products by Broad-Range Bacterial 16S Ribosomal DNA Polymerase Chain Reaction - more The Application of Noninvasive Headspace Analysis to Media Fill Inspection - more Comparison of Tunable Diode Laser Absorption Spectroscopy and Isothermal Micro-calorimetry for Non-invasive Detection of Microbial Growth in Media Fills - more Evaluation of the ScanRDI® as a Rapid Alternative to the Pharmacopoeial Sterility Test Method: Comparison of the Limits of Detection - more ! NEW ! Evaluation of an ATP-Bioluminescence Rapid Microbial Screening Method for In-Process Biologics - more ! NEW ! Total Laboratory Automation in Clinical Microbiology: a Micro-Comic Strip - more ! NEW ! Ensuring Data Integrity by Automated Microbiology Testing - more ! NEW ! Sterility Testing of Injectable Products: Evaluation of the Growth-based BacT/ALERT® 3D™ Dual T Culture System - more ! NEW ! Validation of a NAT-based Mycoplasma assay according European Pharmacopoiea - more ! NEW ! Pfizer Case Study: Rapid Microbial Methods For Manufacturing Recovery After Hurricane María - more ! NEW ! Method Verification Requirements for an Advanced Imaging System for Microbial Plate Count Enumeration - more ! NEW ! Real-Time PCR Detection of Burkholderia cepacia in Pharmaceutical Products Contaminated with Low Levels of Bacterial Contamination - more 1. Application of flow cytometry for rapid bioburden screening in vaccine virus production. Bhusari PK, Tabor DE, Yamagata R, Galinski MS PDA J Pharm Sci Technol. Abstract Sensitive and timely detection of bioburden in presterile filtration product in aseptic processing of biologics is a critical parameter for microbial control and assurance of final product sterility. An application of automated flow cytometry system was developed for rapid microbial assessment and in-process control in vaccine virus production. In order to minimize the background signal caused by the components of the chicken egg substrate sample matrix, a sample processing method to clear somatic cell debris was included. The sample processing and the automated analysis take approximately 5 to 7 min per test sample and the method provides objective results in real time, enabling uninterrupted processing. The flow cytometry method was compared with the standard aerobic plate count method using tryptic soy agar in a parallel study of 1566 independent production-scale samples. The method was further characterized by spike recovery of five model bacterial organisms in representative sample matrix. In comparison to the culture method, the flow cytometry method was shown to be 96.2% sensitive and 98.2% specific for the detection of bioburden at a level of sensitivity suitable for the process stage requirement with the advantage of a nearly instantaneous time to result. LAY ABSTRACT In-process bioburden control in the manufacturing of biopharmaceuticals is essential for final product sterility and integrity. In manufacturing contexts where an in-process hold time is infeasible or in cases where uninterrupted processing is desired, conventional culture-based bioburden detection methods cannot be used, as they require significant time to results that may not fit within the time constraints. In this case study we demonstrate the use of flow cytometry as an alternative rapid method that provides real-time results to enable uninterrupted processing.
2. Use of a real-time microbial air sampler for operational cleanroom monitoring. Eaton T, Wardle C, Whyte W PDA J Pharm Sci Technol. Abstract A sampler that detects and counts viable particles in the air of cleanrooms in real-time was studied. It was found that when the sampler was used to monitor airborne particles dispersed from a number of materials used in cleanrooms, including garments, gloves, and skin, the number of viable particles dispersed from these materials was greater than anticipated. It was concluded that a substantial proportion of these viables were of a non-microbiological origin. When the sampler was used to monitor a non-unidirectional airflow cleanroom occupied by personnel wearing cleanroom garments, it was found that the airborne viable concentrations were unrealistically high and variable in comparison to microbe-carrying particles simultaneously measured with efficient microbial air samplers. These results confirmed previously reported ones obtained from a different real-time sampler. When the real-time sampler was used in a workstation within the same cleanroom, the recorded viables gave results that suggest that the sampler may provide an effective airborne monitoring method, but more investigations are required. LAY ABSTRACT: The airborne concentrations measured by a real-time microbial air sampler within an operational, non-unidirectional airflow cleanroom were found to be unrealistically high due to a substantial numbers of particles of non-microbiological origin. These particles, which resulted in false-positive microbial counts, were found to be associated with a number of materials used in cleanrooms. When the sampler was used within a cleanroom workstation, the counts appeared to be more realistic and suggest that this type of real-time airborne microbial counter may provide a useful monitoring method in such workstations, but further investigations are required.
3. Guidance for Industry Comparability Protocols − Chemistry, Manufacturing, and Controls Information U.S. Department of Health and Human Services Food and Drug Administration Center for Drug Evaluation and Research (CDER) Center for Biologics Evaluation and Research (CBER) Center for Veterinary Medicine (CVM) February 2003 CMC https://www.fda.gov/media/70778/download This guidance provides recommendations to applicants on preparing and using comparability protocols for postapproval changes in chemistry, manufacturing, and controls (CMC). The guidance applies to comparability protocols that would be submitted in new drug applications (NDAs), abbreviated new drug applications (ANDAs), new animal drug applications (NADAs), abbreviated new animal drug applications (ANADAs), or supplements to these applications, except for applications for protein products. Well-characterized synthetic peptides submitted in these applications are included within the scope of this guidance. This guidance also applies to comparability protocols submitted in drug master files (DMFs) and veterinary master files (VMFs) that are referenced in these applications. The FDA is providing this guidance in response to requests from those interested in using comparability protocols.
4. Guidance for Industry Formal Meetings between the FDA and Sponsors or Applicants of PDUFA Products U.S. Department of Health and Human Services Food and Drug Administration Center for Drug Evaluation and Research (CDER) Center for Biologics Evaluation and Research (CBER) http://www.fda.gov/regulatory-information/search-fda-guidance-documents/... This guidance provides recommendations to industry on formal meetings between the Food and Drug Administration (FDA) and sponsors or applicants relating to the development and review of drug or biological drug products (hereafter products) regulated by the Center for Drug Evaluation and Research (CDER) and the Center for Biologics Evaluation and Research (CBER). This guidance does not apply to abbreviated new drug applications, applications for biosimilar biological products, or submissions for medical devices. For the purposes of this guidance, formal meeting includes any meeting that is requested by a sponsor or applicant (hereafter requester(s)) following the request procedures provided in this guidance and includes meetings conducted in any format (i.e., face to face, teleconference, videoconference, or written response).
5. In-process microbial testing: statistical properties of a rapid alternative to compendial enumeration methods. Friedman EM, Warner M, Shum SC, Adair F PDA J Pharm Sci Technol. Abstract In-process tests are used between manufacturing steps to avoid the cost of further processing material that is apt to fail its final tests. Rapid microbiological methods that return simple negative or positive results are attractive in this context because they are faster than the compendial methods used at product release. However, using a single such test will not reliably detect barely unacceptable material (sensitivity) without generating an undesirable number of false rejections (poor specificity). We quantify how to achieve a balance between the risks of false acceptance and false rejection by performing multiple rapid microbiological methods and applying an acceptance rule. We show how the end user can use a simple (and novel) graph to choose a sample size, the number of samples, and an acceptance rule that yield a good balance between the two risks while taking cost (number of tests) into account. LAY ABSTRACT: In-process tests are used between manufacturing steps to avoid the cost of further processing material that is apt to fail its final tests. Rapid microbiological methods that return simple negative or positive results are attractive in this context because they are faster than the compendial methods used at product release. However, using a single such test will not reliably detect barely unacceptable material (sensitivity) without generating an undesirable number of false rejections (poor specificity). We quantify how to achieve a balance between the risks of false acceptance and false rejection by performing multiple rapid microbiological methods and applying an acceptance rule. We show how the end user can use a simple (and novel) graph to choose a sample size, the number of samples, and an acceptance rule that yield a good balance between the two risks while taking cost (number of tests) into account.
6. Overview of rapid microbiological methods evaluated, validated and implemented for microbiological quality control Oliver Gordon, Jennifer C. Gray, Hans-Joachim Anders, Alexandra Staerk & Oliver Schlaefli, Novartis Pharma Stein AG Gunther Neuhaus University of Freiburg http://cdn2.hubspot.net/hub/54227/file-14536665-pdf/docs/2011-novartisrmm-epr-trimmedandcroped-final.pdf The risk for patients through spoiled or otherwise adulterated pharmaceuticals has been acknowledged for many centuries and led to the establishment of Good Manufacturing Practice (GMP) and pharmacopoeial guidelines. Besides chemical purity, pharmaceuticals also have to meet microbiological standards, the latter primarily depending on the administration route. Drug products which are injected directly into blood vessels or tissues or that are applied directly into eyes and ears represent a greater infection risk than products which are administered orally or onto intact healthy skin. While parenteral drug products are required to be free from any viable microorganism (USP <71>, Ph. Eur. 2.6.1), oral and topical products are not required to be sterile, but are subject to strict guidelines limiting the number and types of acceptable microorganisms (USP <61> and <62>, Ph. Eur. 2.6.12 and 2.6.13).
7. Electrochemical detection of bacterial cells using synthetic polymer "foot printing"− MICROPRINT Santhosh Padmanabhan, Marc Kelly, Lokesh Bhadravathi Eswara, Brian Seddon, James Hayes, Niall O'Reilly, Eithne Dempsey http://micra.ie MICROPRINT BIO-CARD is a bacterial detection system based on cell-enzyme profiling. The device encompasses a cell-capture membrane and a complement of electrodes which quantifies the electrons derived from enzyme reactions. This technology intends to replace current practice for the quantification of total bio-burden or individual species which is both labour intensive and time-consuming; testing typically requires 18-24 hours for a confirmatory result. Here we present a selective polymer "foot-printing" based bio-assay, involving imprinting of cells on a polyurethane film. Electrochemical method is used for the detection of enzymes expressed from the captured bacteria. Escherichia coli (a gram-negative bacterium) was selected as a model organism and a range of enzymes arising from major metabolic pathways were targeted and screened as candidates for detection.
8. A Fresh Look at USP <1223> Validation of Alternative Microbiological Methods and How the Revised Chapter Compares with PDA TR33 and the Proposed Revision to Ph. Eur. 5.1.6 Michael J. Miller, Ph.D. http://www.americanpharmaceuticalreview.com/Featured-Articles/... The validation and implementation of rapid and alternative microbiological methods has gained significant momentum over the past decade, with multinational firms validating new technologies for a wide range of applications including finished product release testing (e.g., sterility), environmental monitoring, in-process control, Wfi analysis and microbial identification. When applicable, companies have submitted validation data to regulatory agencies and received approval to implement these same technologies for essential quality control uses. For example, Novartis obtained regulatory approval from more than 50 different countries for releasing their vaccine products using a rapid ATP bioluminescence sterility test.
9. Rapid methods update: revisions to a United States Pharmacopeia chapter Michael J. Miller, PhD Microbiology Consultants, LLC http://www.europeanpharmaceuticalreview.com/34483/... From 2010 to 2013, European Pharmaceutical Review published a very successful series on rapid methods (RMM) that included hot topics such as the European Medicines Agency's and US Food and Drug Administration's expectations, implementation strategies, scientific principles behind the technologies and validation. The final article of the 2012 series introduced the United States Pharmacopeia's (USP's) plan to revise informational chapter <1223>, Validation of Alternative Microbiological Methods. On June 1, 2015, a substantially modified chapter <1223> was published in the second supplement to USP38/NF33 with an official date of 1st December 2015. Because the original USP chapter was published almost 10 years ago, this article will review the most notable changes and compare them with what is recommended in the Parenteral Drug Association (PDA) Technical Report Number 33 and the proposed revision to European Pharmacopoeia (Ph. Eur.) chapter 5.1.6.
10. Alternative to Ph. Eur. pour-plate method for detection of microbial contamination in non-sterile pharmaceutical preparations A.Palicz, A. Paul, A. Hofmann, K. Denzel https://pubmed.ncbi.nlm.nih.gov/27506140/ ABSTRACT The SimPlate method was developed and validated for determining microbial count in order to record possible microbial contamination in non-sterile pharmaceutical preparations according to the European Pharmacopoeia (Ph. Eur.). In blank solutions, the validation results showed that the performance of the SimPlate method was in a similar range (between 1 cell to over 106 cells) or better than that obtained with the European Pharmacopoeia pour-plate method. According to the data, the SimPlate method was sufficiently accurate, specific, linear, repeatable and robust to determine the total aerobic microbial count (TAMC, e.g. Pseudomonas aeruginosa, Staphylococcus aureus) and total combined yeast/mould count (TYMC, e.g. Candida albicans, Aspergillus brasiliensis) in the solutions compared to the pour-plate method. Further development demonstrated the interchangeability of the SimPlate method and the pharmacopoeial pour-plate method for the determination of TAMC and TYMC in non-sterile pharmaceutical preparations. A dilution of 1:100 of Mycobacterium phlei e volumine cellulae in NaCl-peptone buffer showed comparable results between the SimPlate method and the pour-plate method for the detection of TAMC and TYMC with a detection limit of 100 CFU/g. Optimal incubation time was found to be between 24-28 h for TAMC and 3 days for TYMC. The microbial count of samples with and without Mycobacterium phlei e volumine cellulae differed by not more than a factor of 2 in accordance with the European Pharmacopoeia. Compared with the pharmacopoeial pour-plate method, the recovery of micro-organisms with the SimPlate method was mostly higher, and never lower (from factor 1 to factor 3). The selected method for the determination of microbial counts was suitable to record possible microbial contamination in Mycobacterium phlei e volumine cellulae extract and showed good correlation with the European Pharmacopoeia pour-plate method.
11. Application of rapid microbiological methods for the risk assessment of controlled biopharmaceutical environments Sandle T, Leavy C, Jindal H, Rhodes R http://www.ncbi.nlm.nih.gov/pubmed/24575809 Abstract AIMS: To assess the different operational states within a biopharmaceutical grade clean room, using a rapid microbiological method. The method was a novel system, based on spectrometry, designed for sampling, discriminating, and enumerating airborne particles. Central to the study was the aim to determine the microbiological levels as a clean room went from standard use through maintenance and shutdown, disinfection, and then back to standard use. The objective was to evaluate whether a rapid method could replace conventional environmental monitoring using growth-based media. METHODS AND RESULTS: The instrument evacuated was a BioVigilant IMD-A(®) System, which is a real-time and continuous monitoring technology based on optical spectroscopy that can differentiate between biological particles and inert ones (biological particles expressed as bio-counts based on the detection of microbial metabolites). The results indicated that certain activities lead to a high generation of biological particles and in showing an increase over the baseline, would be regarded as presenting a microbiological risk to the cleanroom. These activities include removing HEPA filter grilles, turning off an air handing unit, and tasks which requires an active personnel presence, such as cleaning and disinfection. CONCLUSIONS: The optical instrument can be used to process sufficient information, so that clean rooms can be returned to use following a period of unexpected downtime or following maintenance without the need to wait for the results from growth-based methods. As such, this type of rapid microbiological method is worth exploring further for clean room air monitoring. SIGNIFICANCE AND IMPACT OF THE STUDY: Few studies have been undertaken which examine air-monitoring devices that can both enumerate and discriminate particulates, in a volume of air as 'inert' or 'biological'. This study extends this limited field. Furthermore, the data collected in relation to cleanrooms is of interest in helping microbiologists understand that risks posed by different activities in relation to clean air-handling systems and personnel particle shedding.
12. Towards a Rapid Sterility Test? Tim Sandle http://www.omicsonline.org/open-access/towards-a-rapid-sterility-test-1948-5948-1000209.pdf Introduction Sterility test is an established method for detecting the presence of viable forms of microorganisms in or on finished pharmaceutical products. Sterility, in this sense, means that a product is free from viable microorganisms (although not necessarily metabolic by-products or toxins). The classic form sterility test examines a pharmaceutical product in contact a culture medium, as a way of detecting the possible presence of viable microorganisms. The test is mandatory for all aseptically filled products. In recent years a number of new technology platforms have emerged. This has been facilitated by a change in policy by the U.S. Food and Drug Administration (FDA), opening the door to alternatives to the pharmacopeia methods. This short review assesses some of these technologies.
13. MALDI-ToF Mass Spectrometry: A valid alternative for microbial identification Wickham Laboratories Ltd https://www.pharmig.org.uk/en/product/.../ Rapid and accurate identification of pathogenic microorganisms in areas such as pharmaceutical and medical device manufacturing are of paramount importance. Traditional identification techniques often require subcultures on selective medium, colony isolation, Gram staining or biochemical testing, which are labour intensive and time consuming.
14. Books & Overview Articles
2013 Encyclopedia of Rapid Microbiological Methods, Volume I - VI (2006/2013). Michael J. Miller Abstract:The Encyclopedia of Rapid Microbiological Methods is a culmination of many years of research, development and implementation of new technologies by a number of industry sectors, including pharmaceuticals, medical device, cosmetic and personal care, health and clinical, food and beverage, and municipal water, as well as government agencies and their subsidiaries, including bio-defense laboratories, first responders and homeland security. Furthermore, support for novel ways in which to conduct microbiological assays is becoming the norm for both regulatory agencies and pharmacopoeias, as demonstrated in recent initiatives and guidance documents provided by the FDA, EMEA, USP and Ph. Eur. The encyclopedia attempts to pull together the opinions of these organizations, suppliers of new microbiology platforms, and the laboratories and endusers of the technologies that will be discussed within its pages. Volume 1 provides an overview of microbiological methods and opportunities for industry, regulatory and pharmacopoeial perspectives, and validation strategies. Topics include the history of microbiological methods, risk-based approaches to pharmaceutical microbiology, the realities and misconceptions of implementing rapid methods in the manufacturing environment, the use of rapid methods in bio-defense and the food industry, PAT, comparability protocols, 21 CFR Part 11 and practical guidance on RMM validation and implementation. Volumes 2 and 3 explore specific rapid microbiological methods, technologies and associated instrumentation, from both a supplier and an end-user viewpoint. Volume 2 concentrates on growth-based and viability-based rapid microbiological technologies, including flow and solid phase cytometry, ATP bioluminescence, impedance microbiology, and a variety of microbial identification platforms relying on physiological responses. Volume 3 concentrates on artifact-based and nucleic acid-based technologies, the detection of Mycoplasma, and the use of microarrays, biochips and biosensors. Some of the platforms that are discussed include fatty acid analysis, MALDI and SELDI-TOF mass spectrometry, portable endotoxin testing, 16S rRNA typing, DNA sequencing, PCR, advances in Micro-Electro-Mechanical Systems (MEMS) including Lab-On-A-Chip systems, and a novel instantaneous and real-time optical detection technique for airborne microorganisms. 2013 RMMs & Environmental Monitoring: In-depth focus 2013. Emanuele Selvaggio, Chris Delaney (2013). European Pharmaceutical Review. Microbiology was officially born in 1676 when a Dutch tradesman and scientist from Delft, the Netherlands, observed bacteria and other microorganisms for the first time using a single-lens microscope of his own design. Almost two centuries later, a German biologist called Robert Koch founded modern bacteriology and microbiology. In the 1850s at the University of Breslau, Ferdinand Cohn's main research tool was a large and expensive microscope that his father had bought for him. In the 1850s, he studied the growth and division of plant cells and he proved that the use of liquid media was disadvantageous for isolating pure culture. He was determined to find an alternative technique and introduced the gelatine liquid culture media to be poured on sterilised glass plates to solidify for the first time. 2012 Rapid micro methods and EMA's post approval change management protocol. M. J. Miller (2012). European Pharmaceutical Review. This is the second paper in our continuing series on Rapid Microbiological Methods that will appear in European Pharmaceutical Review during 2012. In my last article, we discussed a number of myths or misconceptions associated with the validation and implementation of rapid microbiological methods (RMMs). In fact, most RMM myths that have been circulating throughout our industry are not true or have been exaggerated to the point that many companies continue to be hesitant in exploring what RMMs have to offer. One of the most prominent myths is that the regulators do not understand, accept or even encourage the use of rapid methods. I submit to you that the regulators want to see RMMs implemented, as their use is directly aligned with the future state of pharmaceutical manufacturing, QbD, PAT and continuous process and product improvement. Further − more, recent changes to regulatory guidance and proposed policy have made it easier to implement RMMs than ever before. In my last article, I introduced a relatively new process that the European Medicines Agency (EMA) launched that allows for the review and approval of RMM validation strategies before testing is initiated. A more thorough review of this process, better known as the Post Approval Change Management Protocol (PACMP), is presented herein. 2011 A regulators view of rapid microbiology methods. Riley B.S. (2011). European Pharmaceutical Review, Volume 16, Issue 5, Rapid Methods Supplement p. 3-5 2011 Challenges and strategies for the application of Rapid Microbiological Methods in the Pharmaceutical Industry. Pan Y (2011) European Pharmaceutical Review, Volume 16, Issue 5, Rapid Methods Supplement p. 10-13 A regulators view. Microbiology series: Nucleic acid and gene amplification-based technologies. Challenges and strategies for application in the pharmaceutical industry. 2011 Overview of Rapid Microbiological Methods Evaluated, validated and Implemented for Microbiological Quality Control. Gordon O, Gray JC, Anders HJ, Staerk A, Schlaefli O, Neuhaus G (2011). European Pharmaceutical Review. 16(2): 9-13. The risk for patients through spoiled or otherwise adulterated pharmaceuticals has been acknowledged for many centuries and led to the establishment of Good Manufacturing Practice (GMP) and pharmacopoeial guidelines. Besides chemical purity, pharmaceuticals also have to meet microbiological standards, the latter primarily depending on the administration route. Drug products which are injected directly into blood vessels or tissues or that are applied directly into eyes and ears represent a greater infection risk than products which are administered orally or onto intact healthy skin. While parenteral drug products are required to be free from any viable microorganism (USP <71>, Ph. Eur. 2.6.1), oral and topical products are not required to be sterile, but are subject to strict guidelines limiting the number and types of acceptable microorganisms (USP <61> and <62>, Ph. Eur. 2.6.12 and 2.6.13). It is the responsibility of the pharmaceutical industry that these microbiological standards are maintained until secondary packaging of the drug product. Knowledge of the microbiological quality of the used excipients and active ingredients, microbiological monitoring of the environment in which the pharmaceuticals are produced, as well as release-testing of the final drug product contribute to maximising patient safety. Testing for microbiological quality requirements relies on traditional methods based on visual detection of a large enough population of microorganisms, either as a colony on solid nutrient medium or as turbidity in liquid nutrient medium. The duration until microbial growth can be detected visually is dictated by the generation time of the microorganisms present; whilst fast-growing microorganisms like E. coli can be seen within hours, visual detection of slow-growing microorganisms can take days or even weeks. Therefore, microbiological quality control often represents the bottleneck for release of drug products after manufacturing. In addition, the late detection of a microbiological quality issue complicates subsequent investigations for the root cause of the contamination. Accordingly, there is high interest throughout the pharmaceutical industry to replace traditional test methods by faster alternative methods. The encouragement by several health authorities to implement such alternative microbiological test methods, as well as official validation guidance documents for the pharmaceutical industry (USP <1223>, Ph. Eur 5.1.6,1) heralded a start to the transition to the use of alternative, faster test methods. In this article, several Rapid Microbiological Methods which were evaluated or validated by Novartis will be presented. 2011 Validating your RMM technology - Understanding the process and developing your approach. David Jones, Kevin Walsh (2011). Biosciences Quality Testing Forum. If you have kept up with prior spotlights, you have gained a great understanding of the RMM solutions that are available and the various benefits of each approach. The next logical question may now be, "what does it take to validate these solutions". Much like the different types of RMM approaches, the validation process can vary. Validation of the RMM solution requires understanding both internal and external requirements to appropriately validate the RMM solution. In addition, time and resource budgeting for validation is an important consideration. First, let's look at the requirements. 2010 Developing a Validation Strategy for Rapid Microbiological Methods. Michael J. Miller. 2010. American Pharmaceutical Review. 13(3): 28-33 Method validation is the process used to confirm that an analytical procedure employed for a specific test is reliable, reproducible and suitable for its intended purpose. All analytical methods need to be validated prior to their introduction into routine use, and this is especially true for novel technology platforms, such as rapid microbiological methods (RMMs). Because many RMM technologies consist of a combination of instrumentation, software, consumables and reagents, in addition to specific detection, quantitative or identification methodologies, it is important to develop a comprehensive and holistic approach to the validation process to ensure that the entire RMM system is suitable for its intended use. The following sections provide an overview of how to design a meaningful validation program in order to effectively demonstrate that the new RMM is equivalent to, or better than, the existing method you intend to replace. 2010 The implementation of rapid microbiological methods. M.J. Miller (2010). European Pharmaceutical Review. This is the sixth and final paper in a series of articles on rapid microbiological methods that have appeared in European Pharmaceutical Review during 2010. Over the past year, we have explored the world of rapid microbiological methods (RMMs), focusing on validation strategies, regulatory expectations, and the technical and quality benefits of these novel systems as compared with conventional techniques. It should be obvious by now that RMMs will significantly impact the future of microbiology within the pharmaceutical and biotech industries. But don't just take my word for it. 2009 Ensuring ROI from your RMM. Miller MJ (2009). Pharmaceutical Manufacturing. Vol. 8(6): p. 32-35. The implementation of rapid microbiological methods (RMMs) has been gaining momentum for years. For the most part, the pharmaceutical industry has acknowledged that regulatory agencies are accepting of RMMs and encourage their use, and that PDA Technical Report #33, USP <1223> and EP 5.1.6 all provide sufficient guidance on validation strategies. Although the industry has the necessary tools for putting alternative microbiology technologies in place, a perception that the long-term benefits will not outweigh the short-term costs persists among manufacturing site heads and senior management teams. For many companies, this has resulted in a significant delay or abandonment of meaningful RMM implementation plans. Therefore, it is imperative that the industry understands how to develop a comprehensive business case and economic analysis of the proposed RMM, and link this information to the long-term technical benefits that the new method will provide. This paper will provide an overview of the financial components that should go into an RMM business strategy, and practical examples of how to calculate the return on investment and payback period for a real-time RMM. 2009 Rapid Microbiological Methods and Demonstrating a Return on Investment: It's Easier Than You Think! M. J. Miller (2009). American Pharmaceutical Review. These are tough times for the introduction of new technologies. When proposing a change in the manufacturing environment, today's economic climate forces us to debate between scientific opportunity and validation costs, improving product quality versus return on investment (ROI), and moving toward a model of continuous improvement without impacting the bottom line. When it comes to the implementation of rapid microbiological methods (RMMs), pharmaceutical microbiologists and QC managers have literally run into a wall with financial planners and manufacturing site heads over the potential costs associated with the evaluation, qualification and installation of these novel technology platforms. If the industry is going to move into the 21st Century with respect to the implementation of RMMs in Process Analytical Technology (PAT) and Quality by Design (QbD)-driven surroundings, then we must be prepared to use appropriate financial models to economically justify the time and expense in qualifying and utilizing these new approaches. 2008 Implementation, validation and registration of rapid microbiological methods. Newby, P (2008). European Pharmaceutical Review. Vol. 13(3): p. 67-73. Over the last decade, interest in rapid microbiological methods (RMMs) in the pharmaceutical sector has grown considerably. Technologies such as ATP bioluminescence, solid phase laser cytometry and genetic-based identification systems are being vigorously investigated. Validation and regulatory requirements for such new technologies are beginning to emerge. However, there is a lot of confusion and considerable hesitancy associated with the introduction of these methods into the pharmaceutical sector. The aim of this article is to help clear up some of these issues in light of recent published literature. These new methods rely increasingly on complex technology platforms. The "black box" approach to validation of such technologies is not acceptable. Instrument qualification, computer validation and computer system validation plus microbiological performance testing must all be considered. 2007 Food Pathogen Detection. Batt, C. A. (2007). Science 316 (5831): 1579-1580. The detection of food pathogens is crucial for food safety; detection methods must be fast, sensitive, and accurate. Yet, almost all techniques used today to identify specific pathogens in foods take at least 48 hours, and some take as long as a week. Further confounding the challenge is the need to address "zero tolerance," a standard that mandates that no viable pathogens are allowed in certain foods. To meet zero-tolerance levels, detection methods need to be sensitive down to a single pathogen in a prescribed sample. Current methods require several days to achieve this standard, because they rely on culturing the pathogen to increase its numbers to detectable levels. 2007 The Advent of Rapid Microbiological Methods: Background, Applications, and Validation. Ball, P. R., L. Arbizzani, et al. (2007). White Paper Microbial contamination poses enormous risks to consumers of pharmaceuticals. There is also the associated financial liabilities and potential for damage to the pharmaceutical manufacturer's reputation. To guard against these risks, pharmaceutical manufacturers have typically collected hundreds of samples per week, incubated them on agar plates for 7 to 14 days, and then counted the colonies to judge for the presence or absence of bacteria. This approach is very time-consuming and runs the risk that by the time a problem is discovered, a large amount of money may have been invested in manufacturing the product. An even worse scenario is that the product may have already been shipped, creating a potential risk to consumers of the drug. These challenges help to explain the increasing interest by pharmaceutical manufacturers in rapid microbiological methods (RMMs) that provide the ability to detect microbial contamination in a fraction of the time of traditional methods. RMM instruments have been on the market for a number of years, but recent developments, such as performance improvements and cost reductions in the technology, have made them more attractive than in the past. The U.S. Food and Drug Administration (FDA) has also helped to spur the introduction of RMMs through its Process Analytical Technology (PAT) initiative which encourages real-time process monitoring. The result is that a recent survey shows that more than 70% of biopharmaceutical manufacturers either are currently using or plan to introduce RMM technology within three years. 2006 Alternative microbiology methods and pharmaceutical quality control. Hussong D and Mello R (2006). Amer. Pharm. Rev., Vol. 9(1), p. 62-9 This paper discusses microbiology in the pharmaceutical quality control environment and the opportunities for development and application of new microbiology methods. Many new methods use technologies developed for space research [1], clinical studies [2], and the food industry [3]. While it may seem odd that the pharmaceutical industry lags behind in implementing new microbiological technologies, it can be readily explained as a resistance to change spawned, in part, by assay complexity and regulatory pressures. In a regulated environment, once a method is accepted, there is significant corporate pressure to maintain procedures the same, thereby avoiding delays associated with regulatory scrutiny. As such, "accepted methods" are used repetitively, and often without question. Therefore, unfortunately, without critical evaluation, pharmaceutical microbiology and process understanding fail to advance and serve only to satisfy regulatory requirements. 2006 Microbiological quality of non-sterile drugs, cosmetics, personal consumer products, foods and nutritionals. Cooper (2006). The Microbiological Update 23: 1-4 Regulatory and Compendial Aspects of Developing "Rapid" Microbiological Analytical Methologies for Detecting, Speciating and Enumerating Microorganisms in Samples. 2005 Rapid Microbiological Methods and the PAT Initiative (2005). J. Moldenhauer, BioPharm International The methods used in most microbiological test laboratories originated in the laboratories of Koch, Lister, and Pasteur. While numerous changes have occurred in the chemistry laboratory, there have been limited improvements in methods used for microbiological testing. In the past decade, many researchers have focused on the study and implementation of improved methods for isolation, early detection, characterization, and enumeration of microorganisms and their products. This translates into better methods, automated and miniaturized methods, methods that require less time or those that are less costly. All of these changes are collectively grouped into the category known as rapid microbiological methods (RMM). In some compendia, these are also called alternative microbiological methods. Although these methods are called rapid microbiological test methods, many of them have their roots in other sciences, e.g., chemistry, molecular biology, biochemistry, immunology, immunochemistry, molecular electronics, and computer-aided imaging. 2004 Rapid Microbiology Methods in the Pharmaceutical Industry. B.S. Riley (2004), American Pharmaceutical Review Rapid microbiology methods have long been essential tools of the clinical and food industry microbiology laboratories. Swift diagnosis of infectious diseases by clinical labs and the need for prompt test results from perishable food items have been strong incentives for the use of rapid methods. The pharmaceutical industry, however, has not been as quick to embrace rapid microbiology despite the potential advantages. Faster microbiology test results would provide better control over the manufacturing process. More rapid microbiology assays would also allow for earlier release of product. One of the explanations offered by the pharmaceutical industry for not using rapid microbiology methods is the uncertainty over regulatory acceptance. The process of evaluating, validating and implementing rapid microbiology test methods can be an expensive and time consuming task. Industry has been reluctant to expend precious resources when regulatory approval of the new method may be in doubt. This article will provide an overview of microbiology testing in the pharmaceutical industry and will look at where rapid microbiological testing methods could fit into the manufacturing process. The issues involved with validation of rapid microbiology methods will be considered with regard to CDER review expectations. Finally, current FDA initiatives that could facilitate the use of rapid microbiology will be described. 2003 Rapid Microbiological Methods in the Pharmaceutical Industry. M.C. Easter (2003), Hygiena International, Ltd., Hertsfordshire, UK In recent years there has been increased interest in the possibility of rapid microbiological methods offering enhanced potential error detection capabilities. However, these methods raise a number of questions, such as how to validate new methods, will they be accepted by the pharmacopoeias, and, most importantly, how will the regulators respond? Rapid Microbiological Methods in the Pharmaceutical Industry answers these questions and more.
15. A Preliminary Investigation into the Ability of Three Rapid Microbiological Methods To Detect Microorganisms in Hospital Intravenous Pharmaceuticals Sarah Hiom, Stephen Denyer, Catherine Talbot, et al. https://www.researchgate.net/publication/250923939_.../ Abstract This study investigated the capability of three rapid microbiological methods to detect microorganisms in aseptically prepared pharmacy preparations at National Health Service hospitals in the United Kingdom. BacT/ALERT 3D (bioMerieux), AKuScreen (Celsis), and BactiFlow ALS (AES Chemunex) technologies were used to detect levels of microorganisms in pharmaceutical products. Four products selected to represent the range of pharmaceuticals prepared in National Health Service hospital pharmacy departments were spiked with known levels of microorganisms. The presence of microorganisms in these products was then determined using each of the rapid microbiological methods and compared to the number determined by traditional total aerobic microbial count methodology. An evaluation of the performance parameters associated with each of the methods, including cost analysis, was also undertaken. There was good correlation between rapid microbiological methods and total aerobic microbial count for heparin and parenteral nutrition products. The rapid microbiological methods had difficulty recovering Gram-positive organisms from vancomycin and methotrexate products; however, protocol developments demonstrated that this was surmountable. The main differences between the rapid microbiological method systems were time-to-result, the initial equipment cost, and the skill required to operate the instruments. The main finding from this work is that rapid microbiological methods can detect microbial contamination of hospital pharmaceutical products in a reduced time when compared to traditional microbiological techniques. The instrument comparison showed that Celsis AKuScreen provided the most rapid result for detecting bacteria; BacT/ALERT was the least expensive instrument and the simplest system to use; and BactiFlow ALS was the most expensive and more complex to use and gave intermediate time to results.
16. Development of an ethidium monoazide−enhanced internally controlled universal 16S rDNA real-time polymerase chain reaction assay for detection of bacterial contamination in platelet concentrates Poorvi Patel, Jeremy A. Garson, Kate I. Tettmar, Siobhan Ancliff, Carl McDonald, Tyrone Pitt, Juliana Coelho, and Richard S. Tedder http://onlinelibrary.wiley.com/doi/10.1111/j.1537-2995.2011.03484.x/abstract Abstract BACKGROUND: Bacterial contamination of platelet (PLT) concentrates remains a problem for blood transfusion services. Culture-based bacterial screening techniques are available but offer inadequate speed and sensitivity. Alternative techniques based on polymerase chain reaction (PCR) amplification have been described but their performance is often compromised by traces of bacterial DNA in reagents. STUDY DESIGN AND METHODS: Universal 16S rDNA primers were used to develop a real-time PCR assay (TaqMan, Applied Biosystems) and various reagent decontamination strategies were explored. Detection sensitivity was assessed by spiking PLT concentrates with known concentrations of 13 different organisms. RESULTS: Restriction enzyme digestion, master mix ultrafiltration, and use of alternative Taq polymerases all reduced the level of reagent DNA contamination to some extent but all proved unreliable. In contrast, ethidium monoazide (EMA) treatment of the PCR master mix followed by photoactivation was reliable and effective, permitting a full 40 amplification cycles, and totally eliminated contamination without compromising assay sensitivity. All 13 organisms were efficiently detected and the limit of detection for Escherichia coli−spiked PLTs was approximately 1 colony-forming unit/mL. Coamplification of human mitochondrial DNA served to confirm efficient nucleic acid extraction and the absence of PCR inhibition in each sample. One of five automated extraction platforms evaluated was found to be contamination free and capable of high-throughput processing. CONCLUSION: Cross-linking of EMA to DNA via photoactivation solved the previously intractable problem of reagent contamination and permitted the development of a high-sensitivity universal bacterial detection system. Trials are ongoing to assess the suitability of the system for high-throughput screening of PLT concentrates.
17. Sterility Testing of Stem Cell Products by Broad-Range Bacterial 16S Ribosomal DNA Polymerase Chain Reaction Tokuno O, Hayakawa A, Yanai T, Mori T, Ohnuma K, Tani A, Minami H, Sugimoto T. http://www.ncbi.nlm.nih.gov/pubmed/25617390 Abstract OBJECTIVE: To evaluate broad-range 16S ribosomal DNA (rDNA) polymerase chain reaction (PCR) as a rapid screening tool to detect bacterial contamination of stem-cell products. METHODS: We performed the evaluation using whole blood spiked with serially diluted bacterial-type strains. Detection sensitivity was defined as the bacterial concentration for which all replicates were positive at each concentration (100% detection). We tested the sterility of 29 bags of autologous peripheral blood stem cell (PBSC) products harvested at our facility using the 16S rDNA PCR method. RESULTS: The detection sensitivity of 16S rDNA PCR in spiked whole blood was 101 to 102 colony-forming units (CFU) per mL, depending on the bacterial strain. We detected no amplified 16S rDNA among the PBSCs we used in this study. The BacT/ALERT automated bacterial culture system that we used also showed no positive signals in any of the PBSCs tested. CONCLUSIONS: Our data indicate that bacterial 16S rDNA PCR is a useful alternative for rapid sterility testing, not only for blood products used in transfusion medicine but also for stem-cell products used in regenerative medicine.
18. The Application of Noninvasive Headspace Analysis to Media Fill Inspection Duncan D, Cundell T, Levac L, Veale J, Kuiper S, Rao R. http://www.ncbi.nlm.nih.gov/pubmed/26865678 Abstract The results of a proof-of-principle study demonstrating a new analytical technique for detecting microbial growth directly in pharmaceutical containers are described. This analytical technique, laser-based headspace analysis, uses tunable diode laser absorption spectroscopy to nondestructively determine gas concentrations in the headspace of a media-filled pharmaceutical container. For detecting microbial growth, the levels of headspace oxygen and carbon dioxide are measured. Once aerobic microorganisms begin to divide after the lag phase and enter the exponential growth phase, there will be significant consumption of oxygen and concomitant production of carbon dioxide in the sealed container. Laser-based headspace analysis can accurately measure these changes in the headspace gas composition. The carbon dioxide and oxygen measurement data for the representative microorganisms Staphylococcus aureus, Bacillus subtilis, Candida albicans, and Aspergillus brasiliensis were modeled using the Baranyi-Roberts equation. The mathematical modeling allowed quantitative comparisons to be made between the data from the different microorganisms as well as to the known growth curves based on microbial count. Because laser-based headspace analysis is noninvasive and can be automated to analyze the headspace of pharmaceutical containers at inspection speeds of several hundred containers per minute on-line, some potential new applications are enabled. These include replacing the current manual human visual inspection with an automated analytical inspection machine to determine microbial contamination of media fill and pharmaceutical drug product vials.
19. Comparison of Tunable Diode Laser Absorption Spectroscopy and Isothermal Micro-calorimetry for Non-invasive Detection of Microbial Growth in Media Fills David Brueckner, David Roesti, Ulrich Georg Zuber, Rainer Schmidt, StefanKraehenbuehl, Gernot Bonkat & Olivier Braissant http://www.nature.com/articles/srep27894 Abstract Two methods were investigated for non-invasive microbial growth-detection in intact glass vials as possible techniques for automated inspection of media-filled units. Tunable diode laser absorption spectroscopy (TDLAS) was used to determine microbially induced changes in O2 and CO2 concentrations within the vial headspaces. Isothermal microcalorimetry (IMC) allowed the detection of metabolic heat production. Bacillus subtilis and Streptococcus salivarius were chosen as test organisms. Parameters as robustness, sensitivity, comparability and time to detection (TtD) were evaluated to assess method adequacy. Both methods robustly detected growth of the tested microorganisms within less than 76 hours using an initial inoculum of <10CFU. TDLA SO2 turned out to be less sensitive than TDLA SCO2 and IMC, as some false negative results were observed. Compared to the visual media-fill examination of spiked samples, the investigated techniques were slightly slower regarding TtD. Although IMC showed shorter TtD than TDLAS the latter is proposed for automating the media-fill inspection, as larger throughput can be achieved. For routine use either TDLA SCO2 or a combination of TDLA SCO2 and TDLA SO2 should be considered. IMC may be helpful for replacing the sterility assessment of commercial drug products before release.
20. Evaluation of the ScanRDI® as a Rapid Alternative to the Pharmacopoeial Sterility Test Method: Comparison of the Limits of Detection Ron Smith, Mark Von Tress, Cheyenne Tubb, et al. http://www.ncbi.nlm.nih.gov/pubmed/21502036 Abstract Two sterility test methods, the ScanRDI® rapid sterility test and the United States Pharmacopeia/European Pharmacopoeia/Japanese Pharmacopoeia (USP/EP/JP) compendial sterility test, were compared with respect to the limits of detection for the presence of viable microorganisms in aqueous solutions at low inoculation levels. The ScanRDI® system employs a combination of direct fluorescent labeling techniques and solid-phase laser scanning cytometry to rapidly enumerate viable microorganisms from aqueous samples, whereas the compendial sterility test is a qualitative, growth-based method that uses a visual assessment of turbidity to indicate microbial contamination. Eight microorganisms were evaluated, seven compendial microorganisms (Clostridium sporogenes, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis, Aspergillus niger, Candida albicans) and the Gram-positive anaerobe Propionibacterium acnes. The number of viable organisms was estimated using the ScanRDI® method and the conventional sterility test method using most probable number methodology. The mean difference between the methods was computed and 95% confidence intervals around the mean difference were estimated. The ScanRDI® method was found to be numerically superior and statistically non-inferior to the compendial (USP/EP/JP) sterility test with respect to the limits of detection for all organisms tested.
21. Evaluation of an ATP-Bioluminescence Rapid Microbial Screening Method for In-Process Biologics Spaeth S, Tran O, Liu Z PDA J Pharm Sci and Tech 2018, 72 574-583 https://www.ncbi.nlm.nih.gov/pubmed/29954921 Abstract: This study compared an adenosine triphosphate (ATP)-based bioluminescence rapid microbial method (RMM) with a conventional sterility method for biologics sample testing. The RMM is based on a comparison of ATP levels in inoculated and uninoculated microbiological growth medium samples following growth enrichment incubation. The biologics samples qualified in this study were recombinant monoclonal antibodies and hybridoma cell culture supernatants. Initially, the lot-to-lot variation in background ATP of these samples posed significant challenges. Two strategies to increase the signal-to-noise ratio (positive result/background ATP) were evaluated: enzyme-based signal amplification and reduction of the broth-based noise through broth selection. Following qualification of the RMM for antibody and cell culture samples, the RMM was also utilized for rapid screening of several sources of purified water. This ATP-based RMM has proved invaluable in routine testing of diverse biologics samples at our discovery research site and plays a key role in the investigation of contaminated samples.
22. Total Laboratory Automation in Clinical Microbiology: a Micro-Comic Strip McAdam AJ Journal of Clinical Microbiology, 2018 Volume 56 Issue 4 e00176-18 https://www.ncbi.nlm.nih.gov/pubmed/29581314 Abstract: Laboratory automation in clinical microbiology has the potential to revolutionize laboratory operations (1, 2). A number of clinical microbiology laboratories have automated part or most of their work and found that testing can be performed accurately, with reduced turnaround times, improvements in laboratory efficiency, and increased flexibility in the level of skill required to perform work in the laboratory (3−7). Even highly complex tasks such as visual interpretation of Gram stains, of culture results, and of susceptibility tests can be automated (4, 8−14). Use of total laboratory automation has the potential to allow staff to perform more-complex tasks that will take advantage of their expertise (1, 15). It also has the potential to affect laboratory needs for expert technologists. How might clinical technologists view the possible effects of total laboratory automation? Read the comic strip to find out.
23. Ensuring Data Integrity by Automated Microbiology Testing Gay S, Samson Y, McDowall RD PHARMACEUTICAL MICROBIOLOGY WHITE PAPER SERIES, 2017 Abstract: Microbiology testing in pharmaceutical development and manufacture is used for environmental monitoring, sterility testing and detection and identification of microorganisms and the applicable regulations for this work are Good Manufacturing Practices (GMP) [Refs 1, 2]. Currently, data integrity is a major issue in the pharmaceutical industry and citations from FDA warning letters for microbiology laboratories can be classified as: Failure to perform actual testing Falsification of data e.g. reporting failed tests as passes or modifying records The reasons are that microbiological testing is often manual and data often relies on observation that can be manipulated, often without photographs of the plates. Alternatively, if blank paper worksheets are used they can be photocopied and passing results substituted. Some typical warning letter citations can be seen in Table 1. Microbiological testing can take between 3 − 7 days for environmental monitoring and 14 days for pharmacopoeial sterility testing. Data integrity could be compromised if there is a need to release a batch before testing is completed but also if there are large numbers of EM tests conducted manually.
24. Sterility Testing of Injectable Products: Evaluation of the Growth-based BacT/ALERT ® 3D™ Dual T Culture System Kaiser SJ, Mutters NT, Backhaus J, et al. PDA J Pharm Sci and Tech 2016, 70 568-576 https://www.ncbi.nlm.nih.gov/pubmed/27325593 Abstract: Sterility testing as described in the European Pharmacopoeia Chapter 2.6.1 as well as the United States Pharmacopeia Chapter 71 requires a 14 day incubation period of the test product in two different media and at two different temperatures. Because of extensive personnel requirements for test performance and quality assurance, alternative and partially automated methods for product sterility testing are of interest. The study objective was to evaluate the applicability of the BacT/ALERT® 3D™ Dual T system (Biomérieux, Nürtingen, Germany) for detection of microbial contaminants according to current pharmacopoeia standards. In addition, we compared the BacT/ALERT® 3D™ Dual T system to conventional pharmacopoeia sterility testing using the direct inoculation method. The results showed no significant disadvantages of sterility testing by BacT/ALERT® 3D™ Dual T compared to the direct inoculation method regarding the ability to detect microbial contamination. Furthermore, product testing using the BacT/ALERT® 3D™ Dual T system met the compendia requirements for method qualification. Altogether, our data provide evidence that the BacT/ALERT® 3D™ Dual T system is a promising alternative for sterility testing of injectable products of sample volume below 10 mL and without antimicrobial activity.
25. Validation of a NAT-based Mycoplasma assay according European Pharmacopoiea Deutschmann SM, Kavermann H, Knack Y Biologicals 38 (2010) 238−248 https://www.ncbi.nlm.nih.gov/pubmed/20207553 Abstract: Eucaryotic expression systems are widely used to produce biologicals for human use, e.g. vaccines, recombinant proteins and monoclonal antibodies. As part of the safety testing the current U.S. Food and Drug Administration (FDA) regulatory guidelines as well as several European Pharmacopoiea monographs requests the demonstration of the absence of Mycoplasma in the cell culture in the bioreactors prior to harvest and further downstream processing. In recent years progress has been made in the development of a sensitive NAT-based method for the detection of Mycoplasma species in CHO cells, e.g. Eldering et al. This method is based on a nucleic acid amplification technique using a very sensitive touch-down PCR-profile. The presence of mollicutes DNA in the test specimens is determined by an approx. 450 bp target sequence which is amplified and this amplicon is finally detected by polyacrylamide gel electrophoresis. Based on this method a ready-to-use test kit was developed. In this report the validation of both method variants according the European Pharmacopoiea monograph 2.6.7 "Mycoplasmas" is described. The validation demonstrated the robustness and precision as well as a sufficient specificity of both assay formats. The validated sensitivity fulfills the requirements of the European Pharmacopoiea for a PCR-based method proposed as an alternative to the time consuming indicator cell culture and the culture method for the detection of Mollicutes (requested sensitivity of at least 10 colony-forming-units/mL).
26. Pfizer Case Study: Rapid Microbial Methods For Manufacturing Recovery After Hurricane María Montenegro-Alvarado JM, Salvas J, Weber J, Mejías S, Arroyo R www.pharmaceuticalonline.com Abstract: Good manufacturing practices (GMPs) are a prerequisite for commercial production in the pharmaceutical industry. They are a basic set of requirements to ensure patient safety. For different reasons, GMP conditions may be lost, including, for example, as part of a planned shutdown for maintenance or construction or due to an unplanned disruptive event. This article shares a case study in which rapid microbiological methods (RMMs) were used to evaluate risk and expedite recovery of GMP conditions after the devastation of Hurricane María in Puerto Rico.
27. Method Verification Requirements for an Advanced Imaging System for Microbial Plate Count Enumeration Jones D, Cundell T PDA J Pharm Sci and Tech 2018, 72 199-212 Abstract: The Growth Direct™ System that automates the incubation and reading of membrane filtration microbial counts on soybean-casein digest, Sabouraud dextrose, and R2A agar differs only from the traditional method in that micro-colonies on the membrane are counted using an advanced imaging system up to 50% earlier in the incubation. Based on the recommendations in USP _1223_ Validation of New Microbiological Testing Methods, the system may be implemented in a microbiology laboratory after simple method verification and not a full method validation.
28. Real-Time PCR Detection of Burkholderia cepacia in Pharmaceutical Products Contaminated with Low Levels of Bacterial Contamination RoJimenez L, Jashari T, Vasquez J, et al. PDA J Pharm Sci and Tech 2018, 72 73-80 Abstract: A real-time polymerase chain reaction (RT-PCR) assay was developed to detect Burkholderia cepacia in pharmaceutical products contaminated with low levels of bacteria. Different pharmaceutical suspensions were artificially contaminated with B. cepacia, Escherichia coli, Staphylococcus aureus, and Bacillus megaterium. After a 24 h incubation in trypticase soy broth with Tween 20, samples were streaked on mannitol salt, phenyl ethyl alcohol, eosin methylene blue, MacConkey, and pseudomonas isolation agar. Microbial DNA was extracted from each sample by using a Tris-EDTA, proteinase K, Tween 20 buffer. Regular PCR targeting the 1.5 kilobases 16S rRNA eubacterial gene and cloning showed the predominant DNA in the extracted mix belonged to E. coli. Selective media isolation of bacterial contamination showed B. cepacia only detected on pseudomonas isolation while eosin methylene blue and MacConkey detected only E. coli. RT-PCR using primers PSL1 and PSR1 amplified a 209 bp 16S rRNA fragment using a Roche LightCycler 96® system with SYBR green I, a common double-stranded binding dye. The cycle at which fluorescence from amplification exceeds the background fluorescence was referred to as quantification cycle.