Journal of Laboratory Automation http://jla.sagepub.com/
Validation of Rapid Microbiological Methods Juan Peris-Vicente, Samuel Carda-Broch and Josep Esteve-Romero Journal of Laboratory Automation published online 10 October 2014 DOI: 10.1177/2211068214554612 The online version of this article can be found at: http://jla.sagepub.com/content/early/2014/10/07/2211068214554612
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JLAXXX10.1177/2211068214554612Journal of Laboratory AutomationPeris-Vicente et al.
Review Articles
Validation of Rapid Microbiological Methods
Journal of Laboratory Automation 1–6 © 2014 Society for Laboratory Automation and Screening DOI: 10.1177/2211068214554612 jala.sagepub.com
Juan Peris-Vicente1, Samuel Carda-Broch1, and Josep Esteve-Romero1
Abstract Classical microbiological methods currently have unacceptably long cycle times. Rapid microbiological methods have been available on the market for decades and have been applied by the clinical and food industries. However, their implementation in the pharmaceutical industry has been hampered by stringent regulations on validation and comparison with classical methods. To encourage the implementation of these methodologies, they must be validated to assess that the results are straightforward. A comparison with traditional methods should be also performed. In this review, information about the validation of rapid microbiological methods reported in the literature is provided as well as an explanation of the difficulty of validation of these methods. A comparison with traditional methods is also discussed. This information is useful for industries and laboratories that can potentially implement these methods. Keywords analysis, guideline, laboratory, microorganism, validation
Introduction Gene therapy products and human somatic cell therapy are emerging and present multiple challenges to safety, purity, and potency. Gene therapy products include vectors (such as nucleic acid, virus, or genetically modified microorganisms) that are directly administered to patients and cells that are transduced with a vector ex vivo prior to administration to the patient. Certain genetically modified microorganisms (bacteria and yeast), cellular therapy products, and cells transduced with a gene therapy vector present similar challenges to sterility assurance and can be considered cellbased products. These products cannot be cryopreserved or otherwise stored without affecting viability and potency. Most cell-based products are manufactured using aseptic manipulations because they can undergo sterile filtration or sterilization.1 However, the incubation times needed for these methods is usually too long.2 Thus, rapid and effective testing is needed because many cell-based products have a potential short dating period, which often necessitates administration of the final product to a patient before sterility test results. Because of the challenges associated with cell-based products, there is a significant need to develop, validate, and implement sterility test methods.3 Traditional methods, described by Title 21 of the Code of Federal Regulations, 610.12 (21 CFR 610.12),4 are used for the isolation of microorganisms. However, the present limitations related to cell-based products include low production volumes, limited manufacturing time, short dating periods, requirement for large sample volumes, and the need for manual and visual examination of cultures to detect
growth. These limitations have motivated sponsors and manufacturers to develop rapid microbiological methods (RMMs) based on techniques that reportedly yield accurate and reliable test results in less time and often with less operator intervention. Therefore, the use of RMMs for cellbased products might provide rapid results that could be applied for product release.5 RMMs are methods designed to provide performance equivalent to the sterility traditional methods while providing results in significantly less time. In general, RMMs involve technologies that can be growth-based, viabilitybased, or surrogate-based cellular markers for a microorganism (e.g., nucleic acid based, fatty acid based). RMMs are usually automated, and many have been used in clinical laboratories to detect viable microorganisms in patient specimens. These methods show improved sensitivity in detecting changes in the sample matrix (e.g., by-products of microbial metabolism), under conditions that favor the growth of microorganisms. The RMMs can be applied in the manufacturing of cell-based products to test the components (raw material, excipients), for in-process testing, for drug substance testing, and to test the drug product in its final container.6 1
QFA, ESTCE, Universitat Jaume I, 12071, Castelló, Spain
Received May 13, 2014. Corresponding Author: Juan Peris-Vicente, Universitat Jaume I, QFA, ESTCE, UJI, Campus del Riu Sec, Av/Sos Banyat s/n, Castelló, 12071, Spain. Email: [email protected]
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To apply these methods, the operator must prove that RMMs provide the same assurance of effectiveness of the biological product as traditional methods do. A validation guide should be followed to indicate the protocol is needed to assess the usefulness of the method. Several guides have been suggested and can be applied to validation of RMMs, such as the Guides for Industry: Validation of Growth-Based Rapid Microbiological Methods for Sterility Testing of Cellular and Gene Therapy Products, described by the Food and Drug Administration (FDA)3; Guidance for Industry PAT—A Framework for Innovative Pharmaceutical Development, Manufacturing, and Quality Assurance, September 20046; and Presidential Task Force on Best Practices in Microbiological Methodology performed by AOAC International, 2006.7 These guides require detailed information on all of the validation parameters: accuracy, precision, ruggedness, specificity, limit of quantification (LOQ), limit of detection (LOD), linearity, range, equivalence, and quality by design. An analytical methodology is accepted if the validation parameters are under those guideline requirements. Although RMMs are now routinely used in limited settings, such as clinical testing, they have not been comprehensively validated for use in a variety of manufacturing settings. Cell-based products present especially challenging manufacturing issues, as these products often introduce additional product or process variables that would likely affect the test outcome. These variables should be considered in a validation study to demonstrate that the method is suitable for its intended product and application. The aim of this work is to discuss the two main guides proposed to validate RMMs: the Guidance for Industry: Validation of Growth-Based RMMs for Sterility Testing and Gene Therapy Products and the Guidance for Industry PAT—A Framework for Innovative Pharmaceutical Development, Manufacturing, and Quality Assurance focusing on the description of the applied validation protocol. The ability to recover and detect organisms from the product and demonstrate their viability by multiplication in liquid media must be evaluated. The guidance has been developed for RMMs with qualitative results (e.g., detection of microorganisms). Reliance on validated sterility testing methods is a critical element in assuring the safety of a product. Validation of most methods for final product testing, including those assessing sterility, is required under 21 CFR 211.165(e) and 21 CFR 11.194(a).4 Validation of critical methods proves that the methods are suitable for their intended purpose, to measure the quantity of microorganisms in the sample, and provides assurance about the accuracy and reproducibility.
Description of the Guides Guidance for Industry:Validation of Growth-Based Rapid Microbiological Methods for Sterility Testing and Gene Therapy Products has been developed by the Center for
Biologics Evaluation and Research/FDA, from the U.S. Department of Health and Human Services. FDA guidance exposes a recommendation to test the suitability of the validation of the RMMs, but it has not law level.3 This guide proposes recommendations for manufacturers and users for validation and sterility testing of therapy and gene therapy products, as well as growth-based RMMs. However, the guideline is not directly applicable to human cells, tissues, or cellular and tissue products.3 The guidance applies to the following products. 1. Somatic cell therapy products: Autologous or allogeneic cells that have been propagated, expanded, selected, pharmacologically treated, or otherwise altered in biological characteristics ex vivo, to be administered to humans and applicable to the prevention, treatment, cure, diagnosis, or mitigation of disease or injuries.8 2. Gene therapy products: All products that mediate their effects by transcription and/or translation of transferred genetic material and/or by integrating into the host genome and that are administered as nucleic acids, viruses, or genetically engineered microorganisms. The products may be used to modify cells in vivo or transferred to cells ex vivo prior to administration to the recipient.9 Guidance for Industry PAT—A Framework for Innovative Pharmaceutical Development, Manufacturing, and Quality Assurance was developed by the U.S. Department of Health and Human Services, FDA, in September 2004. The guide is intended to describe a regulatory framework (process analytical technology [PAT]) that will encourage industry and research laboratories to develop and implement innovative pharmaceutical development, manufacturing, and quality assurance. Working with existing regulations, the agency has developed an innovative approach for helping the pharmaceutical industry address anticipated technical and regulatory issues and questions.6 The Presidential Task Force on Best Practices in Microbiological Methodology was performed by AOAC International in September 2006.7 This guide is devoted to the validation microbiological methods for the industry and refers to laboratories working in the field of food safety, quality assurance, clinical diagnostics, veterinary diagnostics, and engineering.7
Validation Procedure The final objective of validation of an analytical method is to demonstrate that it is suitable for its intended purpose. Thus, RMMs must be compared with traditional methods to prove that they are equivalent or even that they provide less uncertainty.10
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Contamination Studies During the production of cell-based products and microorganisms, several mistakes, such as incorrectly controlled process streams and failures in aseptic processing techniques, can cause the introduction of microorganisms. Thus, a serious assessment of the production must be performed to identify and, eventually, avoid potential routes of microbial contamination8. The main risk of contamination comes from the following: source material, level manipulation, processing path, cell culture, incubation duration, and cell culture vessel.11
Validation Parameters The validation parameters that must be tested during validation are described below.10,12 •• Limit of detection (LOD): The lowest number of microorganisms detectable in the sample matrix. LOD is essential to define what is considered to be contaminated. The LOD is determined by inoculating samples with serial dilutions of viable contaminants, with colony-forming units (CFUs) confirmed at inoculation by plate counts. Challenge testing is performed with each challenge microorganism using an amount between 10 and 100 CFU. Any growth is recovered and identified to confirm identity. •• Incubation time robustness: Time range required for detection of microorganisms at a determinate level. •• Accuracy: Closeness between measured value and true value of microorganism amount. •• Precision: Agreement among individual test results for multiple samplings from a homogeneous sample. Repeatability refers to using the test procedure within a laboratory over a short time period. •• Linearity: The linearity is the ability (within a given range) to obtain a detection signal that is directly proportional to the CFU of a microorganism in a sample, with suitable accuracy and precision. The evaluation of linearity is assessed by the determination coefficient (r2), which should be greater than 0.95 to consider that linearity is adequate. •• Limit of quantification (LOQ): The lowest CFU of microorganisms quantifiable in cell culture with adequate accuracy and precision. •• Calibration range: Minimal and maximal amount of CFU, which could be reliably quantified. •• Recovery: Ratio between the measured CFU and the CFU really incubated in the cell culture. The acceptance criterion is more than 70%. •• Viability recovery: Ratio between the CFU count after reincubation and traditional method count. The acceptance criterion is more than 70%.
•• Specificity: The ability of the test method to detect a panel of organisms within the sample matrix established in the validation protocol. Specificity is demonstrated by challenging the RMMs to detect variations in microbial growth characteristics and concentrations. Challenge testing is performed with organisms, including those recovered in the manufacturing environment and from sterility test positives, as well as common technical and operator contaminants. Known quantities of organisms inoculated into product samples need to be detected within the detection limit (see LOD), recovered, and identity confirmed. •• Ruggedness: Degree of reproducibility of results obtained by analysis of the same sample under a variety of normal test conditions, such as different analysts, different instruments, and different reagent lots. Ruggedness is the lack of influence on the test results of operational and environmental variables of the microbiological method and should be assessed by being prepared by different analysts, as specified in the procedures. Positive and negative controls should be included and should test true to their characteristics. Ruggedness is evaluated by tabulating the mean time to detection for each inoculate and determining the difference between the means for each analyst. •• Robustness: Capacity of a method to remain unaffected by slight, but deliberate, variations in method parameters.
Selection of Microorganisms for Validation The RMMs must be tested using the microorganism more relevant to the product and manufacturing method. The guide suggests the inclusion of microorganisms belonging to several categories: gram-negative bacteria, gram-positive bacteria, aerobic bacteria, anaerobic bacteria, yeast, fungi, isolates detected in starting materials, isolates detected by in-process testing or during preliminary product testing, isolates detected by environmental monitoring of your manufacturing facility, isolates from production areas that represent low-nutrient and high-stress environments, and microorganisms from commercial sources that have continually been exposed to high-nutrient growth media and slow-growing bacteria.3 The same microorganism can show strong differences in growth-rate kinetics, depending on the source, significantly affecting the needed incubation time and the temperature to detect growth. Thus, the validation should include the study of growth under aerobic and anaerobic conditions, especially for products manufactured in closed systems.3
Quality by Design of Method Validation The composition of the test sample should be representative of the product samples that RMMs would test. The parameters to
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consider are cell concentration, media, and additives, as well as preservation and antimicrobial agents, with a bacteriostatic or fungistatic effect. Several matrix compositions should be tested to validate the method for multiple sample types of the same cellular product. The application of RMMs must be validated for in-process and final product in a cell culture production. The cell culture medium can contain antibiotics in different stages of the in-process, which might contain a preservation agent as well as a higher cell concentration. Thus, the different steps of cell production must be separately validated because of their different compositions. To validate the performance of a commercially available testing system, studies using characteristic organisms under prescribed assay conditions should be performed, and the results should be compared with those provided by the supplier. In the validation study design, the potential for the materials being tested to generate false-positive or false-negative results should be evaluated using the appropriate controls. This will depend on the product matrix, additives and preservatives, and unique characteristics of the product. For example, if a freshly isolated cellular product is tested for sterility using a detection method based on CO2 production, then the same freshly isolated cellular product should serve as a control to determine whether uncontaminated cells generate levels of CO2 that would produce a false-positive result. The use of additional positive and negative controls containing product components, but not cells, is also recommended.3
Comparison between RMMs and Traditional Methods To compare RMMs and traditional methods, the same amount of potential microbial contaminants as microorganisms indicated in the previous section are inoculated in cell cultures (measured in CFU/volume). The ability of both methods to identify the microorganisms is tested, together with recovery, detection limit, and assay performance. Several dilutions of microorganisms are tested (10–99 CFU/sample), in order to evaluate the usefulness of RMMs at the main working concentration levels of microorganisms.3
Revalidation Method Any changes in product manufacturing, including formulation, or changes in the RMMs that can potentially inhibit or enhance detection of viable microorganisms need to revalidate the RMMs. Changes in cell types, harvesting procedures, culture media, additives, critical processing steps, or postprocessing handling can potentially affect the detection of viable microorganisms. Initial validation can be designed to minimize the effect of some changes by designing assay conditions that encompass known or proposed changes
(e.g., worst case, matrix approach). Revalidation of the RMM should be performed whenever there are changes in the process that could potentially inhibit (e.g., the addition of antimicrobials) or enhance detection of viable microorganisms. Verification of critical parameters of the test method postprocess, or product changes using the microorganisms most difficult to detect, can serve as an indication of the need to revalidate the method. Validation of the sterility test should be performed on all new products.3
Advantages of RMMs The implementation of RMMs also provides interesting economic features, because of the significant reductions in time to result over conventional methods. Normally, the production cycle is stopped or a product is kept until checking the absence of contamination. If the time to result is shortened, the product would be more rapidly released. Moreover, RMMs allow continuous sampling and data collection and provide time-sensitive outputs and real-time data analysis. Therefore, more screenings and repeat tests could be performed in all stages of production, improving the surveillance process.13 Real-time control and early warning of contamination are essential before introducing raw materials and other product components to the process, validating manufacturing, or distributing finished products.14 RMMs involves an easy-to-handle protocol, with a negligible production of waste, minimal operator variability, reduced staff training requirement, and possibility of automation. They also facilitate research through generating earlier results. Moreover, the shorter analysis time reduces the probability of contamination, improving the robustness. A shorter time to result facilitates replication of an analysis if a contamination or a strange result occurs. Thus, RMMs can improve manufacturing consistency by permitting faster implementation of corrective actions and fostering opportunities to improve the safety of the process. Moreover, RMMs also show improved sensitivity, efficiency, selectivity, accuracy, high throughput, and low false-positive rate, leading to higher efficiency.13 These features of RMMs provide an opportunity to assess the significance of viable but nonculturable or stressed microorganisms.15,16
Application of Validation of RMMs The Milliflex Quantum method is an RMM based in fluorescence detection for the quantification of microorganisms. The culture media are prefilled tryptic soy agar plates, R2A plates, and Sabouraud dextrose agarplates. The authors have validated this methodology for the analysis of contaminants in water. The tested microorganisms were the following American Type Culture Collection (ATCC) strains (TM): Candida albicans (10231D-5), Aspergillus brasiliensis
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Peris-Vicente et al. (16404D-2), Staphylococcus epidermidis (12228D-5), Ralstonia pickettii (27511), Brevundimonas diminuta (19146), Staphylococcus aureus (700699D-5), Pseudomonas aeruginosa (47085D-5), Bacillus subtilis (23857D-5), and Escherichia coli (10798D-5). The experimental procedure was described Meder et al.12 This method was compared with a compendial method. •• Incubation time robustness was tested at 10 CFU and was found to be between 8 and 28 h, depending on the microorganism. •• Ruggedness was evaluated using different media lots, membrane lots, and reagent lots and treating the results by analysis of variance. No significant differences were detected in fluorescence or viability recovery. •• Fluorescence recovery was 98% to 102%, and validation recovery was 97% to 102%. Thus, no significant differences were found between the Milliflex Quantum and traditional method. •• Accuracy was 90% to 112%. •• Linearity was found at r2 > 0.96 between 4 to 10 CFU and 100 CFU. •• The LOD was 1 CFU. •• Specificity: the challenged microorganisms were correctly identified. The validated RMM and the compendial method were applied to the detection of microorganisms in purified water sampled in pharmaceutical plants. Milliflex Quantum provides results in 24 to 40 h, whereas the compendial method takes 5 to 7 d. The obtained results were similar in both cases.12 RMMs offer evident improvements in comparison with traditional methods, and they especially provided results in a shorter time. This review outlines the basic principles of validation and verification in an implementation process for microbiological methods. The main validation parameters have been described. Thus, the implementation of these methodologies is encouraged. According to the literature, RMMs provide significant improvements with the same results as traditional methods. This encourages their implementation in laboratories carrying out microbiological studies for clinical or food safety purposes. Declaration of Conflicting Interests The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This
work has been supported by a project from the University Jaume I (project P1.1B2012-36).
References 1. Food and Drug Administration. Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing— Current Good Manufacturing Practice (September 2004). http://www.fda.gov/cber/guidelines.html (accessed May 14, 2014). 2. Verdonk, G. P. H. T.; Willemse, M. J.; Hoefs, S. G. G.; et al. The Most Probable Limit of Detection (MPL) for Rapid Microbiological Methods. J. Microbiol. Meth. 2010, 82, 193–197. 3. Food and Drug Administration. Guidance for Industry: Validation of Growth-Based Rapid Microbiological Methods for Sterility Testing of Cellular and Gene Therapy Products (February 2008). http://www.fda.gov/downloads/BiologicsBloodVaccines/ GuidanceComplianceRegulatoryInformation/Guidances/ CellularandGeneTherapy/ucm078696.pdf (accessed May 14, 2014). 4. United States Pharmacopeia 28 and National Formulary 23 (USP 28 NF 23), Sterility Tests, General Chapter 71, 2005; pp 1–13. 5. Miller, M. J. Case Study of a New Growth-Based Rapid Microbiological Method (RMM) That Detects the Presence of Specific Organisms and Provides an Estimation of Viable Cell Count. Am. Pharmaceut. Rev. 2012, 15(2). 6. Food and Drug Administration. Guidance for Industry PAT—A Framework for Innovative Pharmaceutical Development, Manufacturing, and Quality Assurance (September 2004). http://www.fda.gov/downloads/Drugs/ Guidances/ucm070305.pdf (accessed May 14, 2014). 7. Food and Drug Administration; AOAC International. Final Report and Executive Summaries, Presidential Task Force on Best Practices in Microbiological Methodology (August 2006). http://www.fda.gov/Food/FoodScienceResearch/Labora toryMethods/ucm124900.htm (accessed May 14, 2014). 8. Food and Drug Administration. Application of Current Statutory Authorities to Human Somatic Cell Therapy Products and Gene Therapy Products (58 FR 53248) (October 1993). http://www.fda.gov/downloads/BiologicsBloodVaccines/ SafetyAvailability/UCM148113.pdf (accessed May 14, 2014). 9. Food and Drug Administration. Guidance for Industry: Gene Therapy Clinical Trials—Observing Participants for Delayed Adverse Events (November 2006). http://www .fda.gov/BiologicsBloodVaccines/GuidanceCompliance RegulatoryInformation/Guidances/CellularandGeneTherapy/ ucm072957.htm (accessed May 14, 2014). 10. ICH Expert Working Group. The International Conference on Harmonisation (ICH) Guidance; Q2(R1), Validation of Analytical Procedures: Text and Methodology (November 2005). http://www.ich.org/fileadmin/Public_Web_Site/ICH_ Products/Guidelines/Quality/Q2_R1/Step4/Q2_R1__ Guideline.pdf (accessed May 14, 2014). 11. ICH Expert Working Group. The International Conference on Harmonisation (ICH) Guidance; Q9, Quality Risk Management (June 2006). http://www.ich.org/fileadmin/
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Public_Web_Site/ICH_Products/Guidelines/Quality/Q9/ Step4/Q9_Guideline.pdf (accessed May 14, 2014). 12. Meder, H.; Baumstummler, A.; Chollet, R.; et al. FluorescenceBased Rapid Detection of Microbiological Contaminants in Water Samples. Sci. World J. 2012, 234858. 13. Miller, M. J., , ed. Encyclopedia of Rapid Microbiological Methods; DHI Publishing, LLC: River Grove, IL; 2005.
14. Cundell, A. M. Opportunities for Rapid Microbial Methods. Eur. Pharm. Rev. 2006, 1, 64–70. 15. Newby, P. The Significance and Detection of VBNC Microorganisms. Eur. Pharm. Rev. 2007, 3, 87–92. 16. Sandle, T. A Review of Cleanroom Microflora: Types, Trends, and Patterns. PDA J. Pharm. Sci. Technol. 2011, 65, 392–403.
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Validation of Rapid Microbiological Methods Juan Peris-Vicente, Samuel Carda-Broch and Josep Esteve-Romero Journal of Laboratory Automation published online 10 October 2014 DOI: 10.1177/2211068214554612 The online version of this article can be found at: http://jla.sagepub.com/content/early/2014/10/07/2211068214554612
Published by: http://www.sagepublications.com
On behalf of:
Society for Laboratory Automation and Screening
Additional services and information for Journal of Laboratory Automation can be found at: Email Alerts: http://jla.sagepub.com/cgi/alerts Subscriptions: http://jla.sagepub.com/subscriptions Reprints: http://www.sagepub.com/journalsReprints.nav Permissions: http://www.sagepub.com/journalsPermissions.nav
>> OnlineFirst Version of Record - Oct 10, 2014 What is This?
Downloaded from jla.sagepub.com at TEXAS SOUTHERN UNIVERSITY on November 29, 2014
554612
research-article2014
JLAXXX10.1177/2211068214554612Journal of Laboratory AutomationPeris-Vicente et al.
Review Articles
Validation of Rapid Microbiological Methods
Journal of Laboratory Automation 1–6 © 2014 Society for Laboratory Automation and Screening DOI: 10.1177/2211068214554612 jala.sagepub.com
Juan Peris-Vicente1, Samuel Carda-Broch1, and Josep Esteve-Romero1
Abstract Classical microbiological methods currently have unacceptably long cycle times. Rapid microbiological methods have been available on the market for decades and have been applied by the clinical and food industries. However, their implementation in the pharmaceutical industry has been hampered by stringent regulations on validation and comparison with classical methods. To encourage the implementation of these methodologies, they must be validated to assess that the results are straightforward. A comparison with traditional methods should be also performed. In this review, information about the validation of rapid microbiological methods reported in the literature is provided as well as an explanation of the difficulty of validation of these methods. A comparison with traditional methods is also discussed. This information is useful for industries and laboratories that can potentially implement these methods. Keywords analysis, guideline, laboratory, microorganism, validation
Introduction Gene therapy products and human somatic cell therapy are emerging and present multiple challenges to safety, purity, and potency. Gene therapy products include vectors (such as nucleic acid, virus, or genetically modified microorganisms) that are directly administered to patients and cells that are transduced with a vector ex vivo prior to administration to the patient. Certain genetically modified microorganisms (bacteria and yeast), cellular therapy products, and cells transduced with a gene therapy vector present similar challenges to sterility assurance and can be considered cellbased products. These products cannot be cryopreserved or otherwise stored without affecting viability and potency. Most cell-based products are manufactured using aseptic manipulations because they can undergo sterile filtration or sterilization.1 However, the incubation times needed for these methods is usually too long.2 Thus, rapid and effective testing is needed because many cell-based products have a potential short dating period, which often necessitates administration of the final product to a patient before sterility test results. Because of the challenges associated with cell-based products, there is a significant need to develop, validate, and implement sterility test methods.3 Traditional methods, described by Title 21 of the Code of Federal Regulations, 610.12 (21 CFR 610.12),4 are used for the isolation of microorganisms. However, the present limitations related to cell-based products include low production volumes, limited manufacturing time, short dating periods, requirement for large sample volumes, and the need for manual and visual examination of cultures to detect
growth. These limitations have motivated sponsors and manufacturers to develop rapid microbiological methods (RMMs) based on techniques that reportedly yield accurate and reliable test results in less time and often with less operator intervention. Therefore, the use of RMMs for cellbased products might provide rapid results that could be applied for product release.5 RMMs are methods designed to provide performance equivalent to the sterility traditional methods while providing results in significantly less time. In general, RMMs involve technologies that can be growth-based, viabilitybased, or surrogate-based cellular markers for a microorganism (e.g., nucleic acid based, fatty acid based). RMMs are usually automated, and many have been used in clinical laboratories to detect viable microorganisms in patient specimens. These methods show improved sensitivity in detecting changes in the sample matrix (e.g., by-products of microbial metabolism), under conditions that favor the growth of microorganisms. The RMMs can be applied in the manufacturing of cell-based products to test the components (raw material, excipients), for in-process testing, for drug substance testing, and to test the drug product in its final container.6 1
QFA, ESTCE, Universitat Jaume I, 12071, Castelló, Spain
Received May 13, 2014. Corresponding Author: Juan Peris-Vicente, Universitat Jaume I, QFA, ESTCE, UJI, Campus del Riu Sec, Av/Sos Banyat s/n, Castelló, 12071, Spain. Email: [email protected]
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To apply these methods, the operator must prove that RMMs provide the same assurance of effectiveness of the biological product as traditional methods do. A validation guide should be followed to indicate the protocol is needed to assess the usefulness of the method. Several guides have been suggested and can be applied to validation of RMMs, such as the Guides for Industry: Validation of Growth-Based Rapid Microbiological Methods for Sterility Testing of Cellular and Gene Therapy Products, described by the Food and Drug Administration (FDA)3; Guidance for Industry PAT—A Framework for Innovative Pharmaceutical Development, Manufacturing, and Quality Assurance, September 20046; and Presidential Task Force on Best Practices in Microbiological Methodology performed by AOAC International, 2006.7 These guides require detailed information on all of the validation parameters: accuracy, precision, ruggedness, specificity, limit of quantification (LOQ), limit of detection (LOD), linearity, range, equivalence, and quality by design. An analytical methodology is accepted if the validation parameters are under those guideline requirements. Although RMMs are now routinely used in limited settings, such as clinical testing, they have not been comprehensively validated for use in a variety of manufacturing settings. Cell-based products present especially challenging manufacturing issues, as these products often introduce additional product or process variables that would likely affect the test outcome. These variables should be considered in a validation study to demonstrate that the method is suitable for its intended product and application. The aim of this work is to discuss the two main guides proposed to validate RMMs: the Guidance for Industry: Validation of Growth-Based RMMs for Sterility Testing and Gene Therapy Products and the Guidance for Industry PAT—A Framework for Innovative Pharmaceutical Development, Manufacturing, and Quality Assurance focusing on the description of the applied validation protocol. The ability to recover and detect organisms from the product and demonstrate their viability by multiplication in liquid media must be evaluated. The guidance has been developed for RMMs with qualitative results (e.g., detection of microorganisms). Reliance on validated sterility testing methods is a critical element in assuring the safety of a product. Validation of most methods for final product testing, including those assessing sterility, is required under 21 CFR 211.165(e) and 21 CFR 11.194(a).4 Validation of critical methods proves that the methods are suitable for their intended purpose, to measure the quantity of microorganisms in the sample, and provides assurance about the accuracy and reproducibility.
Description of the Guides Guidance for Industry:Validation of Growth-Based Rapid Microbiological Methods for Sterility Testing and Gene Therapy Products has been developed by the Center for
Biologics Evaluation and Research/FDA, from the U.S. Department of Health and Human Services. FDA guidance exposes a recommendation to test the suitability of the validation of the RMMs, but it has not law level.3 This guide proposes recommendations for manufacturers and users for validation and sterility testing of therapy and gene therapy products, as well as growth-based RMMs. However, the guideline is not directly applicable to human cells, tissues, or cellular and tissue products.3 The guidance applies to the following products. 1. Somatic cell therapy products: Autologous or allogeneic cells that have been propagated, expanded, selected, pharmacologically treated, or otherwise altered in biological characteristics ex vivo, to be administered to humans and applicable to the prevention, treatment, cure, diagnosis, or mitigation of disease or injuries.8 2. Gene therapy products: All products that mediate their effects by transcription and/or translation of transferred genetic material and/or by integrating into the host genome and that are administered as nucleic acids, viruses, or genetically engineered microorganisms. The products may be used to modify cells in vivo or transferred to cells ex vivo prior to administration to the recipient.9 Guidance for Industry PAT—A Framework for Innovative Pharmaceutical Development, Manufacturing, and Quality Assurance was developed by the U.S. Department of Health and Human Services, FDA, in September 2004. The guide is intended to describe a regulatory framework (process analytical technology [PAT]) that will encourage industry and research laboratories to develop and implement innovative pharmaceutical development, manufacturing, and quality assurance. Working with existing regulations, the agency has developed an innovative approach for helping the pharmaceutical industry address anticipated technical and regulatory issues and questions.6 The Presidential Task Force on Best Practices in Microbiological Methodology was performed by AOAC International in September 2006.7 This guide is devoted to the validation microbiological methods for the industry and refers to laboratories working in the field of food safety, quality assurance, clinical diagnostics, veterinary diagnostics, and engineering.7
Validation Procedure The final objective of validation of an analytical method is to demonstrate that it is suitable for its intended purpose. Thus, RMMs must be compared with traditional methods to prove that they are equivalent or even that they provide less uncertainty.10
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Contamination Studies During the production of cell-based products and microorganisms, several mistakes, such as incorrectly controlled process streams and failures in aseptic processing techniques, can cause the introduction of microorganisms. Thus, a serious assessment of the production must be performed to identify and, eventually, avoid potential routes of microbial contamination8. The main risk of contamination comes from the following: source material, level manipulation, processing path, cell culture, incubation duration, and cell culture vessel.11
Validation Parameters The validation parameters that must be tested during validation are described below.10,12 •• Limit of detection (LOD): The lowest number of microorganisms detectable in the sample matrix. LOD is essential to define what is considered to be contaminated. The LOD is determined by inoculating samples with serial dilutions of viable contaminants, with colony-forming units (CFUs) confirmed at inoculation by plate counts. Challenge testing is performed with each challenge microorganism using an amount between 10 and 100 CFU. Any growth is recovered and identified to confirm identity. •• Incubation time robustness: Time range required for detection of microorganisms at a determinate level. •• Accuracy: Closeness between measured value and true value of microorganism amount. •• Precision: Agreement among individual test results for multiple samplings from a homogeneous sample. Repeatability refers to using the test procedure within a laboratory over a short time period. •• Linearity: The linearity is the ability (within a given range) to obtain a detection signal that is directly proportional to the CFU of a microorganism in a sample, with suitable accuracy and precision. The evaluation of linearity is assessed by the determination coefficient (r2), which should be greater than 0.95 to consider that linearity is adequate. •• Limit of quantification (LOQ): The lowest CFU of microorganisms quantifiable in cell culture with adequate accuracy and precision. •• Calibration range: Minimal and maximal amount of CFU, which could be reliably quantified. •• Recovery: Ratio between the measured CFU and the CFU really incubated in the cell culture. The acceptance criterion is more than 70%. •• Viability recovery: Ratio between the CFU count after reincubation and traditional method count. The acceptance criterion is more than 70%.
•• Specificity: The ability of the test method to detect a panel of organisms within the sample matrix established in the validation protocol. Specificity is demonstrated by challenging the RMMs to detect variations in microbial growth characteristics and concentrations. Challenge testing is performed with organisms, including those recovered in the manufacturing environment and from sterility test positives, as well as common technical and operator contaminants. Known quantities of organisms inoculated into product samples need to be detected within the detection limit (see LOD), recovered, and identity confirmed. •• Ruggedness: Degree of reproducibility of results obtained by analysis of the same sample under a variety of normal test conditions, such as different analysts, different instruments, and different reagent lots. Ruggedness is the lack of influence on the test results of operational and environmental variables of the microbiological method and should be assessed by being prepared by different analysts, as specified in the procedures. Positive and negative controls should be included and should test true to their characteristics. Ruggedness is evaluated by tabulating the mean time to detection for each inoculate and determining the difference between the means for each analyst. •• Robustness: Capacity of a method to remain unaffected by slight, but deliberate, variations in method parameters.
Selection of Microorganisms for Validation The RMMs must be tested using the microorganism more relevant to the product and manufacturing method. The guide suggests the inclusion of microorganisms belonging to several categories: gram-negative bacteria, gram-positive bacteria, aerobic bacteria, anaerobic bacteria, yeast, fungi, isolates detected in starting materials, isolates detected by in-process testing or during preliminary product testing, isolates detected by environmental monitoring of your manufacturing facility, isolates from production areas that represent low-nutrient and high-stress environments, and microorganisms from commercial sources that have continually been exposed to high-nutrient growth media and slow-growing bacteria.3 The same microorganism can show strong differences in growth-rate kinetics, depending on the source, significantly affecting the needed incubation time and the temperature to detect growth. Thus, the validation should include the study of growth under aerobic and anaerobic conditions, especially for products manufactured in closed systems.3
Quality by Design of Method Validation The composition of the test sample should be representative of the product samples that RMMs would test. The parameters to
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consider are cell concentration, media, and additives, as well as preservation and antimicrobial agents, with a bacteriostatic or fungistatic effect. Several matrix compositions should be tested to validate the method for multiple sample types of the same cellular product. The application of RMMs must be validated for in-process and final product in a cell culture production. The cell culture medium can contain antibiotics in different stages of the in-process, which might contain a preservation agent as well as a higher cell concentration. Thus, the different steps of cell production must be separately validated because of their different compositions. To validate the performance of a commercially available testing system, studies using characteristic organisms under prescribed assay conditions should be performed, and the results should be compared with those provided by the supplier. In the validation study design, the potential for the materials being tested to generate false-positive or false-negative results should be evaluated using the appropriate controls. This will depend on the product matrix, additives and preservatives, and unique characteristics of the product. For example, if a freshly isolated cellular product is tested for sterility using a detection method based on CO2 production, then the same freshly isolated cellular product should serve as a control to determine whether uncontaminated cells generate levels of CO2 that would produce a false-positive result. The use of additional positive and negative controls containing product components, but not cells, is also recommended.3
Comparison between RMMs and Traditional Methods To compare RMMs and traditional methods, the same amount of potential microbial contaminants as microorganisms indicated in the previous section are inoculated in cell cultures (measured in CFU/volume). The ability of both methods to identify the microorganisms is tested, together with recovery, detection limit, and assay performance. Several dilutions of microorganisms are tested (10–99 CFU/sample), in order to evaluate the usefulness of RMMs at the main working concentration levels of microorganisms.3
Revalidation Method Any changes in product manufacturing, including formulation, or changes in the RMMs that can potentially inhibit or enhance detection of viable microorganisms need to revalidate the RMMs. Changes in cell types, harvesting procedures, culture media, additives, critical processing steps, or postprocessing handling can potentially affect the detection of viable microorganisms. Initial validation can be designed to minimize the effect of some changes by designing assay conditions that encompass known or proposed changes
(e.g., worst case, matrix approach). Revalidation of the RMM should be performed whenever there are changes in the process that could potentially inhibit (e.g., the addition of antimicrobials) or enhance detection of viable microorganisms. Verification of critical parameters of the test method postprocess, or product changes using the microorganisms most difficult to detect, can serve as an indication of the need to revalidate the method. Validation of the sterility test should be performed on all new products.3
Advantages of RMMs The implementation of RMMs also provides interesting economic features, because of the significant reductions in time to result over conventional methods. Normally, the production cycle is stopped or a product is kept until checking the absence of contamination. If the time to result is shortened, the product would be more rapidly released. Moreover, RMMs allow continuous sampling and data collection and provide time-sensitive outputs and real-time data analysis. Therefore, more screenings and repeat tests could be performed in all stages of production, improving the surveillance process.13 Real-time control and early warning of contamination are essential before introducing raw materials and other product components to the process, validating manufacturing, or distributing finished products.14 RMMs involves an easy-to-handle protocol, with a negligible production of waste, minimal operator variability, reduced staff training requirement, and possibility of automation. They also facilitate research through generating earlier results. Moreover, the shorter analysis time reduces the probability of contamination, improving the robustness. A shorter time to result facilitates replication of an analysis if a contamination or a strange result occurs. Thus, RMMs can improve manufacturing consistency by permitting faster implementation of corrective actions and fostering opportunities to improve the safety of the process. Moreover, RMMs also show improved sensitivity, efficiency, selectivity, accuracy, high throughput, and low false-positive rate, leading to higher efficiency.13 These features of RMMs provide an opportunity to assess the significance of viable but nonculturable or stressed microorganisms.15,16
Application of Validation of RMMs The Milliflex Quantum method is an RMM based in fluorescence detection for the quantification of microorganisms. The culture media are prefilled tryptic soy agar plates, R2A plates, and Sabouraud dextrose agarplates. The authors have validated this methodology for the analysis of contaminants in water. The tested microorganisms were the following American Type Culture Collection (ATCC) strains (TM): Candida albicans (10231D-5), Aspergillus brasiliensis
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Peris-Vicente et al. (16404D-2), Staphylococcus epidermidis (12228D-5), Ralstonia pickettii (27511), Brevundimonas diminuta (19146), Staphylococcus aureus (700699D-5), Pseudomonas aeruginosa (47085D-5), Bacillus subtilis (23857D-5), and Escherichia coli (10798D-5). The experimental procedure was described Meder et al.12 This method was compared with a compendial method. •• Incubation time robustness was tested at 10 CFU and was found to be between 8 and 28 h, depending on the microorganism. •• Ruggedness was evaluated using different media lots, membrane lots, and reagent lots and treating the results by analysis of variance. No significant differences were detected in fluorescence or viability recovery. •• Fluorescence recovery was 98% to 102%, and validation recovery was 97% to 102%. Thus, no significant differences were found between the Milliflex Quantum and traditional method. •• Accuracy was 90% to 112%. •• Linearity was found at r2 > 0.96 between 4 to 10 CFU and 100 CFU. •• The LOD was 1 CFU. •• Specificity: the challenged microorganisms were correctly identified. The validated RMM and the compendial method were applied to the detection of microorganisms in purified water sampled in pharmaceutical plants. Milliflex Quantum provides results in 24 to 40 h, whereas the compendial method takes 5 to 7 d. The obtained results were similar in both cases.12 RMMs offer evident improvements in comparison with traditional methods, and they especially provided results in a shorter time. This review outlines the basic principles of validation and verification in an implementation process for microbiological methods. The main validation parameters have been described. Thus, the implementation of these methodologies is encouraged. According to the literature, RMMs provide significant improvements with the same results as traditional methods. This encourages their implementation in laboratories carrying out microbiological studies for clinical or food safety purposes. Declaration of Conflicting Interests The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This
work has been supported by a project from the University Jaume I (project P1.1B2012-36).
References 1. Food and Drug Administration. Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing— Current Good Manufacturing Practice (September 2004). http://www.fda.gov/cber/guidelines.html (accessed May 14, 2014). 2. Verdonk, G. P. H. T.; Willemse, M. J.; Hoefs, S. G. G.; et al. The Most Probable Limit of Detection (MPL) for Rapid Microbiological Methods. J. Microbiol. Meth. 2010, 82, 193–197. 3. Food and Drug Administration. Guidance for Industry: Validation of Growth-Based Rapid Microbiological Methods for Sterility Testing of Cellular and Gene Therapy Products (February 2008). http://www.fda.gov/downloads/BiologicsBloodVaccines/ GuidanceComplianceRegulatoryInformation/Guidances/ CellularandGeneTherapy/ucm078696.pdf (accessed May 14, 2014). 4. United States Pharmacopeia 28 and National Formulary 23 (USP 28 NF 23), Sterility Tests, General Chapter 71, 2005; pp 1–13. 5. Miller, M. J. Case Study of a New Growth-Based Rapid Microbiological Method (RMM) That Detects the Presence of Specific Organisms and Provides an Estimation of Viable Cell Count. Am. Pharmaceut. Rev. 2012, 15(2). 6. Food and Drug Administration. Guidance for Industry PAT—A Framework for Innovative Pharmaceutical Development, Manufacturing, and Quality Assurance (September 2004). http://www.fda.gov/downloads/Drugs/ Guidances/ucm070305.pdf (accessed May 14, 2014). 7. Food and Drug Administration; AOAC International. Final Report and Executive Summaries, Presidential Task Force on Best Practices in Microbiological Methodology (August 2006). http://www.fda.gov/Food/FoodScienceResearch/Labora toryMethods/ucm124900.htm (accessed May 14, 2014). 8. Food and Drug Administration. Application of Current Statutory Authorities to Human Somatic Cell Therapy Products and Gene Therapy Products (58 FR 53248) (October 1993). http://www.fda.gov/downloads/BiologicsBloodVaccines/ SafetyAvailability/UCM148113.pdf (accessed May 14, 2014). 9. Food and Drug Administration. Guidance for Industry: Gene Therapy Clinical Trials—Observing Participants for Delayed Adverse Events (November 2006). http://www .fda.gov/BiologicsBloodVaccines/GuidanceCompliance RegulatoryInformation/Guidances/CellularandGeneTherapy/ ucm072957.htm (accessed May 14, 2014). 10. ICH Expert Working Group. The International Conference on Harmonisation (ICH) Guidance; Q2(R1), Validation of Analytical Procedures: Text and Methodology (November 2005). http://www.ich.org/fileadmin/Public_Web_Site/ICH_ Products/Guidelines/Quality/Q2_R1/Step4/Q2_R1__ Guideline.pdf (accessed May 14, 2014). 11. ICH Expert Working Group. The International Conference on Harmonisation (ICH) Guidance; Q9, Quality Risk Management (June 2006). http://www.ich.org/fileadmin/
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Public_Web_Site/ICH_Products/Guidelines/Quality/Q9/ Step4/Q9_Guideline.pdf (accessed May 14, 2014). 12. Meder, H.; Baumstummler, A.; Chollet, R.; et al. FluorescenceBased Rapid Detection of Microbiological Contaminants in Water Samples. Sci. World J. 2012, 234858. 13. Miller, M. J., , ed. Encyclopedia of Rapid Microbiological Methods; DHI Publishing, LLC: River Grove, IL; 2005.
14. Cundell, A. M. Opportunities for Rapid Microbial Methods. Eur. Pharm. Rev. 2006, 1, 64–70. 15. Newby, P. The Significance and Detection of VBNC Microorganisms. Eur. Pharm. Rev. 2007, 3, 87–92. 16. Sandle, T. A Review of Cleanroom Microflora: Types, Trends, and Patterns. PDA J. Pharm. Sci. Technol. 2011, 65, 392–403.
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