Microbial Control Strategies in Bioprocessing Falling Short of Assuring Product Quality and Satisfying Regulatory Expectations

• Visionary Pharma Consulting, LLC

Introduction

Recent bioprocessing contamination events, warning letters and consent decrees have demonstrated yet again that microbial control is indispensable in pharmaceutical drug manufacturing operations. Biopharmaceuticals or therapeutic protein products are derived from recombinant DNA and hybridoma technology. The protein is produced by fermentation of media supplemented with nutrients using mammalian cells, bacterial or yeast organisms. The protein is then harvested and purified. The resulting bulk drug substance is stored refrigerated or frozen. Additional modifications or purification may be performed to the protein drug substance prior to sterile filtration and aseptic fill that produce a sterile drug product for patient administration. Low bioburden must be maintained from cell harvest and recovery up to sterile filtration to assure product quality and satisfy regulatory commitments and compliance requirements [1, 2]. The fermenting cell culture must remain pure to produce the desired protein and yield. Bioburden and other contaminants must be prevented in subsequent harvest/recovery and purification operations to meet the low bioburden claim and assure purity, potency and safety.

Bioprocessing is a biological process. Raw materials, media, buffer solutions, in-process intermediates and the drug substance itself are generally growth promoting. The facility, equipment and personnel contribute to bioburden through manipulations, interventions and sampling. Maintaining low bioburden can be a challenge. A robust microbial control strategy is the solution to preventing contamination events and maintaining process consistency. Such a strategy is successful when it does not rely only on testing but identifies and implements measures to: control the source of microorganisms and the conditions that promote microorganism survival, growth and persistence; monitor the facility, equipment, process and product; and, intervene when there is a drift from established controls. Remediation may be necessary when lack of control has progressed considerably to place the facility, equipment and operations at risk and operations cannot continue.

Keys for successful implementation of microbial control in bioprocessing are robust quality, facility and equipment, production, material and laboratory systems. Successful implementation must be highly proactive. Risk assessment is indispensable for identifying, analyzing and reducing or mitigating the risks of microbial contamination.

Control strategy Defi nition

Control strategy, in the context of the pharmaceutical industry, was recently defi ned in ICH guidelines Q8 and Q11 [3, 4] as a “planned set of controls, derived from current process and product understanding that assures process performance and product quality”. The target is to achieve product quality and process consistency. The terms “control strategy” and “control theory” are not new and has its origins in basic science fi elds and other industries. Breaking down the ICH defi nition into workable parts, control strategy can be defi ned as the use of measures and tools to:

• Prevent process and product variability (usually by design)
• Reduce the probability or frequency of variability, and
• Minimize the impact of variability when it occurs

The target is to minimize variability in process performance and product quality and eliminate product non-conformance at minimal cost to achieve process control and process consistency [5]. Control of variability is also discussed in the ICH Q7 guideline [6].

Microbial Control strategy

A microbial control strategy establishes adequate measures to prevent growth of microorganisms in the facility, equipment and product that can potentially result in compromised product quality. Not only does it address the product’s microbiological attributes, but it also addresses the impact of the microorganisms and their by-products on the product’s purity, potency and safety at release and throughout shelflife. The measures are not isolated to testing plans but encompass all other systems to assure prevention of microbial contamination and a consistent process and product from batch to batch.

In fact, regulatory authorities are highly concerned about the impact of bioburden on purity and potency as it is well known that microorganisms produce enzymes that can degrade proteins. There have been cases where batches with high bioburden counts in fi nal drug substance and/ or during purifi cation were discarded at a huge cost. Unfortunately, it is diffi cult to understand the impact of microorganisms on protein purity and potency, as there are relatively few published scientifi c data. In addition, the correlation between types and numbers of microorganisms and their eff ect on protein degradation is not known; and most likely, it will vary depending on the microorganism. However, biopharmaceutical companies must develop tools to assess product impact. Until that time, the bar on bioburden will continue to be raised higher and higher by regulatory authorities, particularly FDA, causing delays in BLA approvals and resulting in Form 483 observations.

A microbial control strategy also addresses bioreactor contamination events caused by microorganisms. Such events have caused submission approval and facility inspection delays as well as signifi cant use of resources [7-12]. Even apparent contamination has caused signifi cant use of resources as well as submission and inspection delays [13, 14]. Most importantly, contamination events have caused drug shortages, limiting the supply of critical medicine to patients [15, 16]. Regulators have presented case studies of microbial contamination and elements of a microbial control strategy based on their experience from reviewing applications and conducting facility inspections [17].

A microbial control strategy comprises 3 parts:

• Identifi cation and implementation of measures to control the sources of microorganisms and the conditions that promote their survival and persistence;
• Monitoring of the facility, equipment, personnel, process, product and their interactions; and,
• Intervening or remediating when there is a drift from established controls.

The quality system, facility and equipment system, production system, raw materials system, and laboratory controls system are the enablers of an eff ective microbial control strategy because they must work eff ectively to fulfi ll the objectives of the strategy. In addition, risk management is a critical enabler and must be integrated well within the quality system and the other systems to identify, reduce, and/or mitigate microbial risks as well as assess product and process impact. In addition, knowledge, science, and experience are prerequisites to the development and implementation of microbial controls. All these elements converge and interact to create the microbial control strategy (Figure 1).

Figure 1. Enablers and elements of a microbial control strategy (provided with permission from the author)

identification and implementation of Measures

The most eff ective measures for microbial control are those that are established by design: facility, equipment and process design. The means by which personnel interact with the facility, equipment and process is also part of the design element. In addition, the design of the cleaning and sterilization processes, the validation, qualifi cation, calibration and maintenance programs are critical for implementing and maintaining microbial control. Risk assessment for the identifi cation of microbial risks is indispensable for identifying problem areas. Heat inactivation and use of barrier technologies are process design measures to reduce the risk of contamination [7, 18, 19].

Monitoring

Once measures or controls are implemented, the monitoring phase includes observing, noting and trending of these measures to verify performance. Monitoring encompasses in-process and final drug substance sampling and testing for microbiological attributes to meet acceptance criteria and specifications, but it also includes monitoring of all elements that help to assure and maintain process consistency and product quality. These elements are calibration and maintenance of equipment, revalidation and requalification, and monitoring and trending of cleaning and sterilization processes. Additionally, the quality system is in full force as excursions and deviations must be assessed. Corrective and preventive actions (CAPA) must be created and implemented to maintain microbial and process control. Changes must be assessed for their potential to affect the established microbial controls. Data and trends must be analyzed for opportunities to improve process control for a consistent, quality product.

Remediation

The remediation phase commonly begins with a serious event that compromises the process, facility, and equipment. Such events are bioreactor contamination, equipment malfunction, and utility problems (e.g., biofilm in the water system, mold in the facility) among others that require a plan with specific actions to return the facility, equipment and processes under control. Such events require extensive investigations. It is important to note that these events are usually caused by design and monitoring faults as well as faults in the original risk assessment. The faults could also be due to limited scientific knowledge and information. Most of the time, they are due to wrong assumptions or questions in the identification of microbial risks, faulty design of equipment that does not allow for appropriate cleaning and sterilization, faulty design of cleaning and sterilization processes, and faulty monitoring where the wrong parameters are monitored and controlled. Remediation has regulatory and compliance implications that must be considered. Furthermore, a risk review must be performed to improve the risk management process as it was unsuccessful at reducing and mitigating the microbial risks.

Examples of Faulty or Incomplete Microbial Control Strategies

No microbial control strategy?

One could argue that a microbial control strategy is completely amiss at a manufacturing facility when things go wrong. However, in reality, most of the time, there are control elements embedded within systems or records but may not be referred to as a microbial control strategy and may not have been considered as elements of the control strategy. However, most of the time, there is not a single overarching document that outlines the company’s policy and approach regarding microbial control in bioprocessing. Such a document is more likely to be present for a drug product aseptic fill process, but not so for other processes. The microbial control strategy must be well defined, implemented and documented. There must be provisions to revisit and update the control strategy as more knowledge and experience is gained.

Risk Management Shortcomings

Risk management is relatively new in the pharmaceutical industry with several guidelines published in recent years [20, 21]. While tremendous progress has been achieved, risk management is not fully integrated within the pharmaceutical quality system. Risk assessments are not conducted appropriately and the appropriate tools are not being used. The analysis of risk is not complete. Reduction and mitigation controls are not sufficient. Even when the risks are assessed and controls placed for their reduction or mitigation, the rationale is not documented. The process of risk review is almost nonexistent. It is very difficult to identify and assess microbial risks without a systematic risk assessment process. Control measures cannot be implemented if there is not a proper assessment of microbial risk.

For example:

• There are facilities that operate without appropriate segregation between animal-derived and non animalderived materials. Has the risk been assessed and documented? How is this risk reduced or mitigated?
• How is risk assessment performed and documented when bioburden or other microorganism by-products are found in purification operations? What are the measures or controls implemented to reduce or mitigate this risk? Are these controls working effectively? Is there a systematic process for reviewing the risks and controls?

Facility, Utilities, and Equipment Design Shortcomings

There are many design shortcomings regarding facility, utilities, and equipment that cannot be possibly all described in this section. Some common shortcomings are presented.

In one example, a facility is not designed optimally to allow for appropriate flow of personnel, dirty and clean equipment. There is inadequate segregation among activities, operations, materials and product. Utilities are not designed optimally and are frequently the cause of contamination. For example, the water system is the primary culprit of biofilm and presence of Gram negative rods that can eventually find their way into the process and product. The HVAC air handling system has been the cause of mold contamination. There have been cases where poor design itself has caused contamination. There have also been cases where poor controls such as ineffective preventive maintenance program have been the root cause.

The inappropriate design of equipment for interventions and sampling that does not allow for effective microbial control is another example. When sampling locations are not placed optimally and require additional interventions or difficult maneuvers, contamination can result. Another common example is improper installation of equipment, (particularly filters), that does not perform as described due to improper installation. Wrong installation has also led to damaged filters and sampling valves. Design of equipment that does not allow for adequate cleaning and draining, such as improperly sloped piping, has also resulted in incomplete removal of residue and incomplete draining.

Process Design Shortcomings

Microbial control problems are also the result of poor or faulty process design. The quality of the raw materials is critical for biotechnology operations. Variability in the materials and changes of suppliers have led to contamination events [10]. Lack of adequate filtration steps, particularly before UF/DF or chromatography steps or lack of appropriate filter porosity have led to elevated bioburden counts in purification operations [17]. Lack of filtration of media and buffer solutions has also contributed to microbial problems.

Other very common examples are faulty designs of cleaning processes and lack of or inadequate sterilization of equipment. Inadequate cleaning has led to corrosion in tanks and purification columns, biofilm problems and bioburden problems, particularly in chromatography resins and ultrafiltration membranes. Crevices, plates, diaphragms, and elastomers offer ample opportunities for the attachment of microorganisms. Lack of sterilization of equipment used in harvest, recovery and purification operations as well as inadequate sterilization have also led to insufficient microbial control. The combination of inadequate cleaning and sterilization can be especially detrimental as the high temperature of sterilization can make any leftover residues difficult to remove.

Hold times of in-process intermediates, media and buffer solutions have not been validated appropriately for microbial control. This is a regulatory expectation and a frequent review and inspection issue [22]. Process hold times must be designed and validated so as to assure microbial control, and should not be determined based on the manufacturing schedule. For example, a 3-day hold time for in-process intermediates to allow for hold during the weekend may not be appropriate for some processes and must be validated to assure purity, potency and safety of the final drug substance.

Media simulations are not performed for upstream inoculum operations to verify that they can be performed aseptically from cell bank vial thaw to bioreactor inoculation simulating all transfers. It is only after bioreactor contamination events that such simulations may be performed to find the root cause of the contamination.

Lifetime studies are not performed for chromatography resins and membranes. Frequently, resins and membranes are the causes of elevated bioburden counts either due to inadequate cleaning or inability to drain completely. Resins and membranes can be stored for long periods of time, while bacteria are protected in the crevices of the pores. If process residues have not been removed completely, microorganisms can survive and grow contaminating the next batch.

An important shortcoming that is not commonly afforded much attention is that of old and outdated processes that do not meet today’s standards and scientific knowledge. These processes are at great risk for contamination and their design must be improved to safeguard product quality and patient safety. Examples are mammalian cell culture processes that are not designed to reduce and mitigate the risk of viral contamination and processes that employ too many fermentation and purification cycles to arrive at an acceptable yield and obtain one batch of drug substance.

Monitoring Shortcomings

Appropriate monitoring controls for the facility, equipment, utilities, raw materials and process are often not established. Monitoring is not only testing but also analysis and trending of operational parameters that help assure product quality. This is particularly critical for bioprocessing operations that are so dependent on process control to obtain the expected yield, purity and potency. Parameters that lead to microbial control are not always microbiological in nature. For example, water conductivity, and TOC are critical parameters used in water monitoring and cleaning operations that also serve to indirectly monitor and control microorganisms.

Other frequent examples of shortcomings in monitoring for microbial control are poor equipment maintenance, instrument calibration and inadequate requalification/revalidation programs. Periodic reviews are not performed appropriately. Equipment maintenance and calibration frequencies are not appropriate and/ or the manufacturer’s instructions are not followed. Deadlines are missed increasing the possibility that an instrument or equipment is used in the process out of calibration and out of maintenance.

Changes, particularly changes to equipment and instruments, are not assessed properly leading to failed or questionable batches. Deviations and excursions are not risk assessed based on a systematic process. Frequently, the root cause of contamination is not identified. CAPAs are being implemented without finding the root cause and are therefore ineffective. As a result, the same deviations occur again.

A frequent shortcoming encountered in testing and monitoring of microbiological attributes is that method suitability studies have not been performed for testing in-process intermediates, media and buffer solutions. Alert and action limits have not been set based on process capability and product impact.

Remediation Shortcomings

There are many facilities today that do not have a remediation plan to address a contamination event. This is so critical as such events can at times incapacitate the whole facility for weeks and even months, particularly in the case of virus contamination.

Another shortcoming is that the regulatory and compliance aspects of microbial issues have not been fully considered and addressed. For example, can a drug substance batch with high bioburden counts during its purification be released for drug product manufacture? If yes, why? If the facility is affected by virus contamination or biofilm in the water system, what is the effect on previously released batches and on batches awaiting disposition?

A specific plan must be developed and implemented to assure that the changes are adequate to re-initiate production operations, and return the facility and process under control.

Contamination events are commonly caused by faulty design, wrong assumptions and incomplete knowledge. The ultimate goal of the remediation phase is to correct the design flaws but also the monitoring flaws as the established controls did not identify the problem early enough. The initial risk assessment and the risk review process must be revisited as they lacked the necessary elements to prevent and control the risk and event.

Weak Enablers

A microbial control strategy cannot be successful if its enablers are weak. Lack of or weak risk management cannot enable complete identification and understanding of microbial risks to establish a microbial control strategy. Neither can it lead to appropriate risk analysis and risk reduction or mitigation. Product impact cannot be assessed without a sound risk assessment process.

A weak quality system cannot manage deviations, CAPA and changes effectively and efficiently. Complex batch records, standard operating procedures (SOPs) and document control procedures cannot enable personnel adherence and consistency in operations. Lack of adherence and consistency lead to microbial control problems. Another element of the quality system is the training program. Incomplete and unsatisfactory training has contributed to contamination control problems (e.g., incorrect filter installation). Personnel must understand the basics of microbial and contamination control and why this is critical for product quality.

Summary

A microbial control strategy is necessary for low bioburden biotech operations to maintain a low number of microorganisms and safeguard product quality. Multiple elements and systems converge to develop and implement an effective strategy. The preferred method is to establish controls by design to avoid unnecessary manipulations and/or interventions. When this is not possible, other types of controls must be established. Once controls are established, they must be monitored for their effectiveness. Being proactive is key to preventing problems before they occur. Having a remediation plan is also important as even in the best cases, microbial contamination can and will occur, especially in mammalian cell culture operations. This article has presented some common examples of ineffective microbial control that have led to bioburden and contamination problems. It has also presented a few ideas for developing strategies for the mitigation of microbial risks.

Author biography

Anastasia Lolas is an independent consultant and the owner of Visionary Pharma Consulting LLC. She provides consulting services to the pharmaceutical industry regarding regulatory and compliance issues related to product quality microbiology of small molecules and therapeutic proteins. Previously, she was a Microbiologist with the Biotech Manufacturing Team in the Division of Manufacturing and Product Quality at CDER/FDA and a Microbiology Reviewer with the New Drug Microbiology Staff in the Office of Pharmaceutical Science at CDER/FDA. In her 6 years at the FDA, Anastasia evaluated numerous new drug applications and biologics license applications and conducted prelicense and pre-approval inspections of therapeutic protein manufacturing facilities. 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. Prior to joining FDA in 2005, Anastasia was employed by Nestlé Waters North America and was a member of the Corporate Quality Assurance Group.

References

1. U.S. Code of Federal Regulations, Title 21, Part 601.20 Biologics Licenses; issuance and conditions.
2. Lolas, A.G., Chi, B., Hughes, P.F., and Suvarna, K. (2010), “CMC Microbiology Review of Biologics License Applications and Pre-License/Pre-Approval Inspections: Therapeutic Biological Proteins,” Amer. Pharm. Rev. 13(2): 56-60.
3. International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH), (2009), Pharmaceutical Development Q8(R2).
4. International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH), (2012), Development and Manufacture of Drug Substances (Chemical Entities and Biotechnological/Biological Entities) Q11.
5. Petrovich, M.V. Strategies for Improvement of Process Control. Luftig & Warren International.
6. International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH), (2000), Good Manufacturing Practice Guide for Active Pharmaceutical Ingredients Q7.
7. Chen, J., Bergevin, J., Kiss, R., Walker, G., Battistoni, T., Lufburrow, P., Lam, H., and Vinther, A. (2012), “Case Study: A Novel Bacterial Contamination In Cell Culture Production - Leptospira licerasiae,” PDA J. Pharm. Sci. and Tech. 66: 580-591.
8. Jones, N. (2011), “Identification and Remediation of a Cell Culture Virus Contamination,” PDA J. Pharm. Sci. and Tech. 65: 615.
9. Lutgen, M. (2011), “Chlorine Dioxide Remediation of a Virus-Contaminated Manufacturing Facility,” PDA J. Pharm. Sci. and Tech. 65: 620-624.
10. Moody, M., Alves, W., Varghese, J., and Khan, F. (2011), “Mouse Minute Virus (MMV) Contamination - A Case Study: Detection, Root Cause Determination, and Corrective Actions,” PDA J. Pharm. Sci. and Tech. 65: 580-588.
11. Nims, R.W., Chun, A., Marino, A., and Dieringer Boyer, S. (2011), “Nontuberculosis Mycobacterium Contamination of a Mammalian Cell Bioreactor Process,” BioPharm International 24(6): 30-34.
12. Skrine, J. (2011), “A Biotech Production Facility Contamination Case Study - Minute Mouse Virus,” PDA J. Pharm. Sci. and Tech. 65: 599-611.
13. Hendricks, L.C., and Vacante, D.A. (2011), “Case Study of Apparent Virus Contamination In Biopharmaceutical Product at Centocor,” PDA J. Pharm. Sci. and Tech. 65:612-613.
14. Hendricks, L.C., Jordan, J., Yang, T.Y., Driesprong, P., Haan, G.J., Viebahn, M., Mikosch, T., Van Drunen, H., and Lubiniecki, A.S. (2010), “Apparent Virus Contamination In Biopharmaceutical Product at Centocor,” PDA J. Pharm. Sci. and Tech. 64: 471-480.
15. Thomson Reuters (2011), “Timeline-Key Dates in Genzyme’s Manufacturing Crisis,” http:// www.reuters.com/article/2010/08/13/genzyme-idUSN1321252820100813
16. Bethencourt, V. (2009), “Virus Stalls Genzyme Plant,” Nature Biotechnology 27: 681.
17. Suvarna, K., Lolas, A., Hughes, P., and Friedman, R.L. (2011), “Case Studies of Microbial Contamination in Biologic Product Manufacturing,” Amer. Pharm. Rev. 14(1): 50-56.
18. Kiss, R.D. (2011), “Practicing Safe Cell Culture: Applied Process Designs for Minimizing Virus Contamination Risk,” PDA J. Pharm. Sci. and Tech. 65: 715-729.
19. Gauvin, G., and Nims, R. (2010), “Gamma-Irradiation of Serum for the Inactivation of Adventitious Contaminants,” PDA J. Pharm. Sci. and Tech. 64: 432-435.
20. International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH), (2005). Quality Risk Management Q9.
21. Parenteral Drug Association (PDA) Paradigm Change in Manufacturing Operations (PCMO) Technical Report 54: Implementation of Quality Risk Management for Pharmaceutical and Biotechnology Manufacturing Operations. 2012.
22. Suvarna, K. (2012), “Hold Time Studies in Therapeutic Biological Product Manufacturing,” Presentation at PDA’s 7th Annual Global Conference on Pharmaceutical Microbiology.