The manufacture of biologic products is a complex process and requires the use of living cells. These processes and products are prone to contamination by adventitious agents such as bacteria, fungi and viruses. Microbial contaminations have a huge impact on biologic product manufacture as they introduce product variability and can cause loss of potency due to degradation or modification of product by microbial enzymes, changes in impurity profiles, and an increase in the levels of bacterial endotoxins. In addition, the investigations of microbial contaminations can result in lengthy shutdown periods and delays in manufacturing operations that in turn, may sometimes result in shortages of essential drug products. Strict microbial production controls are essential to ensure the manufacture of a drug product with consistent quality. This article discusses elements of a microbial control strategy, recent cases of microbial contamination in specified biologic products, the need to perform risk assessments on a periodic basis, and additional areas of improvement in the management of risks.
Biologic products are manufactured using living cells such as bacteria, yeast, and mammalian cells. These include specified biologics such as monoclonal antibodies and therapeutic recombinant DNA-derived products licensed under Section 351 of the Public Health Service Act  and currently regulated by the Center of Drug Evaluation and Research (CDER). These biological products are also regulated as drugs under the Federal Food, Drug, and Cosmetic Act . The upstream process in the manufacture of monoclonal antibodies and therapeutic recombinant proteins typically involves cell expansion, cell culture, and recovery steps. The downstream process involves multiple purification steps. The purified protein is ultrafiltered/diafiltered with formulation buffer to provide a formulated bulk drug substance. The formulated bulk drug substance is sterile-filtered and filled to provide a final drug product. Because of the consequences of microbial contamination on product safety and quality, there is continued interest in understanding the root causes of microbial contamination and controlling these risks in biologic product manufacture. This article discusses some of the bacterial contamination cases reported to the Agency or identified during pre-license/pre-approval inspections of biologic drug substance manufacturers in the past two years. The cases highlight areas for improvement in risk management and the need for developing a robust microbial control strategy for biologic products.
Figure 1- Sources of microbial contamination.
Sources of Microbial Contamination
Microorganisms are ubiquitous in nature. Microorganisms can adapt and survive under a variety of conditions and can pose a significantrisk to biologic products. An understanding of the microbial entry points and implementation of measures to prevent microbial contamination is critical for manufacture of safe, pure and potent biologic products. As shown in Figure 1, microorganisms can gain entry into a production process stream from several sources: the facility, equipment, process operations, raw materials, column resins, filter membranes, water, process gases, and personnel. All sources of microbial contamination should be considered when developing a microbial control strategy and performing an investigation for a microbial contamination deviation.
Regulation and Guidance
The minimum current good manufacturing practice (CGMP) requirements for preparation of finished human drug products are described in 21CFR§211 . These include the use of suitable protective apparel (21CFR§211.28), appropriate facility design and placement of equipment (21CFR§211.42), equipment cleaning, sterilization, and maintenance (21CFR§211.67), and production and process controls (21CFR§211.100). All these preventive measures and precautions are implemented to protect product and prevent contamination. Therapeutic recombinant products and monoclonal antibodies are also subject to applicable regulation in 21CFR parts 600-610 . The guidance on CGMP for active pharmaceutical ingredients, Q7A, provides general CGMP guidance for biologic drug substance manufactured by cell culture or fermentation under section XVIII . Additional guidance documents cover prevention or control of adventitious agents in cell-derived biologic products and address the quality concerns originating from cell substrates used for manufacture of these products. These documents include (a) Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use published in February 1997, (b) Guidance on Viral Safety Evaluation of Biotechnology Products Derived From Cell Lines of Human or Animal Origin (Q5A), (c) Guidance on Quality of Biotechnological/Biological Products: Derivation and Characterization of Cell Substrates Used for Production of Biotechnological/Biological Products (Q5D), and (d) Guidance on Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products (Q6B) [6,7,8,9]. The Q6B guidance states that contaminants should be strictly avoided and/or suitably controlled with appropriate in-process acceptance criteria or action limits for the drug substance or drug product to meet specifications. The 1994 FDA Guidance for Industry on the Submission Documentation for Sterilization Process Validation in Applications for Human and Veterinary Drug Products provides guidance on sterilization process validation for final drug product . The 2004 FDA Guidance for Industry on “Sterile Drug Products Produced by Aseptic Processing – Current Good Manufacturing Practice” provides guidance on personnel qualifications, clean room design, process design, and aseptic processing of final drug products . These regulations and guidance documents provide the backbone for the development of an appropriate microbial contamination control strategy.
Elements of a Microbial Control Strategy
A microbial control strategy should be developed once a comprehensive risk assessment has been performed for all possible microbial entry points into the manufacturing process. This requires a good understanding of the manufacturing process and product attributes. In general, the design of the facilities should allow for proper operations and prevention of contamination. The flow of personnel, material and waste should be from clean to dirty areas and critical upstream open operations liable to microbial contamination should be performed in designated biosafety hoods or areas with ISO 5 classification. Depending on the risks to the process, areas should be appropriately segregated. Segregation of pre-viral and post-viral clearance steps in processes using mammalian host cells is important to prevent cross-contamination of process intermediates and the facility. Segregation of areas, appropriate changeover procedures, and other procedural controls should be in place to prevent crosscontamination in a multi-product facility. Environmental monitoring of manufacturing areas should be performed routinely at appropriate intervals. Process gases and water should be tested and monitored to ensure adequate microbial control. The design of equipment (singleuse disposable versus multi-use), validated cleaning and sterilization processes along with a comprehensive preventative maintenance plan are critical components of the microbial control strategy. Microbial control for the lifetime use of membranes and resins should be demonstrated. In addition, it is critical to identify and establish processing steps that decrease bioburden and bacterial endotoxin levels as the process intermediates are processed through sequential purification steps. Bioburden reducing filters should be used at critical steps in the process. This is critical for buffer solutions and in process intermediates conducive to microbial growth. Minimizing the number of open operations reduces the risk to product from external (personnel and environmental) microbial contamination sources. Biologic products are usually rich in carbon sources that favor microbial growth. Hold conditions (time, temperature) for a process should be validated to control and prevent potential microbial growth. Bioburden and endotoxin alert and action limits should be set for process steps based on process capability. Raw materials should be screened for microbial quality and should behandled and stored in a manner to prevent contamination and cross-contamination. Personnel are important contributors to microbial contaminations. Appropriate gowning should be implemented to prevent contamination. All personnel performing open operations should be trained adequately and evaluated periodically in such operations.
n the last two years, several contamination events were reported to the Agency. They included viral or bacterial contamination of upstream cell culture or fermentation processes. Viral contamination events were extensively covered in the recent 2010 PDA/FDA Adventitious Viruses in Biologics: Detection and Mitigation Strategies Workshop. Only bacterial contaminations are discussed in this article. One case involved contamination of a fermentor used in the manufacture of a protein product secreted by a bacterial host. The contaminant was identified as Bacillus cereus (a Gram positive spore forming rod). A second case involved the contamination of a fermentor used in the manufacture of a recombinant protein by Paenibacillus curdlanolyticus (a Gram variable spore-forming rod). A systematic approach was used during the investigations to identify the root cause of the contamination and included several media simulations to aid in identifying the point of entry into the fermentor. In addition, the investigations involved the manufacture of engineering batches. After a lengthy investigation in both case studies, problems with the sampling devices, addition valves, incorrectly fitted components, missing O-rings, incorrect installation and deformation of an air filter after sterilization, and/or inadequate slope of a condensate line were identified. Immediate corrective actions included the replacement of valve diaphragms in fermentor addition ports, replacement of a membrane valve in the sampling device, and replacement of O-rings on the measuring probes. Enhancements were also made to the sterilization processes of fermentor and associated transfer lines. A preventative maintenance plan was developed for all fermentor valves. All valves were tagged using a detailed checklist to ensure correct installation. All SOPs were updated and employees were trained on the revised versions. The investigations and corrective actions addressed all possible causes of contamination as an unequivocal root cause could not be assigned. In most cases, it is very difficult to identify a definitive assignable cause. It is highly recommended that a systematic approach be followed to determine the root cause. Media simulations help in demonstrating that sterility of the fermentor is not compromised. Recent microbial contamination events at several manufacturing facilities point to breaches in the sterile boundary caused by damaged vent filters, damaged O-rings, diaphragms, and elastomers, and improperly sloped condensate lines.
When bacterial hosts are used, microscopic examinations of the fermentation culture for contamination is difficult. A culture purity test should be perfomed using appropriate media and culture conditions. It is crucial to have a comprehensive preventative maintenance plan for fermentor and tank agitators, probes, gaskets, O-rings, valves, and filters. The design of piping and valves should prevent steam condensate from collecting and leading to contamination by back-flow. After periods of shutdown or maintenance, it is important to perform media simulations on sterile equipment that has remained idle for a period of time. Procedural details on assembly and set-up of fermentors/bioreactors should be clear and very detailed. Training in this area can reduce inadvertent leaks and contamination of the systems. Continuous assessments of change control, work orders, and other process improvements should be conducted to ensure that the microbial control strategy is not impacted. Of note in both cases, the contaminating microorganism was a facultative anaerobic Gram positive spore-forming rod. Risk mitigation strategies based on microbial environmental flora should be considered. The areas for improvement identified in the case studies were in preventative maintenance plans for all fermentor valves including valves on sampling devices and in the documentation for correct assembly of components.
Two cases of microbial contamination of the downstream process were identified during pre-approval/pre-license inspections of drug substance manufacturing facilities. Bioburden deviations were observed in several batches at the ultra-filtration/diafiltration (UF/DF) step. The contaminants identified were Sphingomonas species, Stenotrophomonas maltophilia, Ralstonia pickettii, and Staphylococcus species suggesting probable water and human sources of contamination. Presence of repeated high bioburden counts in several batches suggested development of biofilm and inadequate contamination control procedures for the UF/DF steps. After extensive investigations, several corrective actions were implemented in terms of cleaning, storage and re-use of UF/DF systems, sterilization/sanitization of buffer tanks, assessment of the water for injection (WFI) system and transfer lines, introduction of in-process bioburden reducing filters (in cases where there were no filters before the UF/DF steps), validation of hold times and storage conditions of process intermediates and revisions to bioburden limits based on process capability. Demonstration of microbial control over the lifetime use of membranes and validation of in-process hold times are essential for ensuring the consistent quality of biologic products. All WFI piping locations with stagnant water should be assessed and eliminated. Microbial trend reports for water systems should be reviewed regularly.
The investigations of microbial contaminations are challenging due to the ubiquitous nature of the microorganisms, multiple points of microbial entry, growth promoting properties of biological process streams, limitations of sampling and detection methods, and the time and resources involved in performing complex investigations. All microbial entry points should be systematically evaluated. For fermentor contaminations, seed fermentors and associated additions and transfer lines should be included in the investigations. A hazard analysis and critical control point assessment for bioburden control throughout the manufacturing process is useful for the design of a microbial control strategy and the performance of a systematic investigation. In addition, failure data should be tracked to gain a better understanding of root causes. The information should be used to continuously evaluate risks and implement process and/or equipment improvements to mitigate and prevent microbial contaminations.
Microbial contamination is a risk to biologic product quality and safety. The cost of inadequate microbial control in biologic product manufacture is enormous as facilities or bioreactor production trains may have to be shut down for lengthy periods of time in order to conduct investigations and identify the root cause to prevent reoccurrence. The recent cases of bacterial contamination of biologic products suggest that preventative maintenance plans for fermentor and associated valves, types of materials used for diaphragms and O-rings, and understanding of microbial control at certain process steps need further attention. Contamination control requires an understanding of the microbial entry points and risks to the process as well as the microbial growth potential of the product, media and buffer solutions. Microbial contamination control requires appropriate design of facility and equipment, validated cleaning and sterilization cycles for equipment, detailed and robust preventative maintenance plans for equipment, measures to reduce bioburden and bacterial endotoxins at appropriate steps in the process, and routine monitoring of these process steps for bioburden and endotoxin with defined alert and action limits. A contamination remediation plan should be established. Such a plan is beneficial for meeting CGMP and has the advantage of reducing facility downtime. Investigations should be comprehensive and include assessment of all microbial entry points. Corrective actions should address all possible identified causes in the absence of a known assignable root cause. The information gathered during these investigations should feed into the overall risk management plan. The quality risk management plan should be integrated into the quality system and allow for continuous improvement.
- Public Health Service Act, Biological Products; as amended
- Federal Food Drug and Cosmetic Act; as amended.
- FDA, “Current Good Manufacturing Practices for Finished Pharmaceuticals,” 21 CFR part 211.
- FDA, “Biologics,” 21 CFR parts 600-610.
- U.S. Department of Health and Human Services, Food and Drug Administration. Guidance for Industry: Q7A Good Manufacturing Practice Guidance for Active Pharmaceutical Ingredients. Rockville, MD; 2001.
- U.S. Department of Health and Human Services, Food and Drug Administration. Centre for Biologics Evaluation and Research. Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use. February 1997.
- U.S. Department of Health and Human Services, Food and Drug Administration. Guidance for Industry: Q5A Viral Safety Evaluation of Biotechnology Products Derived From Cell Lines of Human or Animal Origin. Rockville, MD; 1998.
- U.S. Department of Health and Human Services, Food and Drug Administration. Guidance for Industry: Q5D Guidance on Quality of Biotechnological/Biological Products: Derivation and Characterization of Cell Substrates Used for Production of Biotechnological/Biological Products. Rockville, MD; 1998. Federal Register Vol. 63, No. 182, 1998.
- U.S. Department of Health and Human Services, Food and Drug Administration. Guidance for Industry: Q6B Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products, FDA, 1999.
- U.S. Department of Health and Human Services, Food and Drug Administration. Guidance for Industry for the Submission Documentation for Sterilization Process Validation in Applications for Human and Veterinary Drug Products. Rockville, MD; 1994.
- U.S. Department of Health and Human Services, Food and Drug Administration. Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing – Current Good Manufacturing Practice. Rockville, MD; 2004.
Kalavati Suvarna, Ph.D. is a Microbiologist with the Biotech Manufacturing Team in the Division of Manufacturing and Product Quality in the Office of Compliance, CDER, FDA. She has over nine years of experience as a microbiology reviewer at the FDA. Kalavati holds a Ph.D. in Biological Sciences from Northern Illinois University. Prior to joining the Agency, she worked in an academic and pharmaceutical setting.
Anastasia G. Lolas is a Microbiologist with the Biotech Manufacturing Team in the Division of Manufacturing and Product Quality in the Office of Compliance, CDER, FDA. She has over 5 years of experience as a microbiology reviewer of drug applications at the FDA. Anastasia holds a B.S. in Biology from Virginia Polytechnic Institute and State University and a M.S. in Food Science from the University of Illinois at Urbana-Champaign.
Patricia F. Hughes, Ph.D. is the Team Leader in the Biotech Manufacturing Team in the Division of Manufacturing and Product Quality in the Office of Compliance in CDER, FDA. She has over twenty years experience in the Pharmaceutical/Biotech industry in fermentation & cell culture process development and manufacturing. In addition, she has over twelve years of experience as a microbiology reviewer at the FDA, in CDER and CBER. Patricia holds a Ph.D. in Microbiology from Georgetown University.
Richard Friedman is the Director of the Division of Manufacturing & Product Quality in the Center for Drug Evaluation and Research (CDER), Office of Compliance. In this position, he directs the interpretation and development of CGMP policy, review of inspectional recommendations and determination of manufacturing site acceptability. He has been employed by FDA since 1990, including prior positions as New Jersey District Drug Specialist, CDER Senior Compliance Officer and Team Leader of Guidance and Policy. Mr. Friedman has authored several publications on topics including sterile drugs and quality management systems, and was awarded The George M. Sykes Award by the Parenteral Society for outstanding journal paper for the year 2005. Mr. Friedman is also an adjunct faculty member of Temple University School of Pharmacy in their QA/RA graduate program. Prior to joining FDA, Mr. Friedman worked in the toxicology research division of an innovator pharmaceutical company. Mr. Friedman received his B.S. in Biology with honors from Montclair State University in 1989 and his M.S. in Microbiology from Georgetown University School of Medicine in May, 2001.
This article was printed in the January/February 2011 issue of American Pharmaceutical Review - Volume 14, Issue 1. Copyright rests with the publisher. For more information about American Pharmaceutical Review and to read similar articles, visit www.americanpharmaceuticalreview.com and subscribe for free.