Process maps and risk assessments are among the valuable tools operators can apply to reduce the risk of microbial contamination.
By Randi Hernandez
There are multiple variables to consider in the reduction of bioburden, including the evaluation of microbes in a facility, in the manufacturing process, and in the materials used and produced in the process stream. Kathleen Souza, senior microbiologist in the Department of Virology and Microbiological Sciences at EMD Millipore, and Nick Hutchinson, market development manager, pharma and biotech at Parker domnick hunter, discuss the use of a combination of orthogonal approaches in the assessment of bioburden.
Also included are comments from Marc Mittelman, PhD, senior managing scientist, Exponent, Inc. and Vince Anicetti, executive director of quality, Coherus Biosciences. Mittelman and Anicetti are co-chairs of the Parenteral Drug Association task force that developed the bioburden and biofilm technical report titled, PDA Technical Report No. 69, (TR 69) Bioburden and Biofilm Management in Pharmaceutical Manufacturing Operations, published June 2015.
BioPharm: What type of planning is necessary to target all aspects of bioburden reduction?
Mittelman and Anicetti (PDA task force): The most important consideration in bioburden control is staff training at all levels within an organization. The planning process needs to involve representatives from QA/QC, manufacturing, and facilities engineering; an effective communication strategy needs to be developed to ensure that any bioburden excursions are immediately addressed. Understanding potential sources of contamination for a given facility and manufacturing process is key to planning for design, preventative maintenance, bioburden monitoring, and remedial actions. Effective bioburden control plans involve development of detailed, written standard operating procedures (SOPs) that address both preventative and remedial actions at each unit operation.
Hutchinson: A suitable risk assessment tool should be used to assess the likelihood of bioburden becoming problematic. Process maps showing process inputs can be constructed and the risk of these contributing to bioburden loads assessed. Chromatography resins, for example, are not typically provided sterile or capable of being sterilized, so they are a potential source of contaminations unless cleaned, sanitized, and stored correctly. Failure mode effect analysis (FMEA) and fishbone diagrams are useful tools to use.
Souza: Planning the design of a comprehensive bioburden reduction strategy should start with risk assessments for each of the entry routes. The identified risks and severity levels inform planning of raw material selection, facility design, process elements and conditions, cleaning and sanitization requirements, establishment of alert/action levels, selection of monitoring and detection methods, operator training requirements, and quality system oversight.
BioPharm: What procedure is used to identify the various points in which bioburden can be introduced? Is the identification process performed for each variety of biologic product?
Hutchinson: The creation of a process map will depend on each process. Indeed, some processes, like a cell therapy process, will need to be performed in a sterile manner, so no level of bioburden would be acceptable. My view is that if a good risk assessment has been performed on one variety of biologic, then the framework would be suitable for a second type with the relevant adaptations.
Mittelman and Anicetti (PDA task force): Identification of potential bioburden entry points begins with effective communications between QA/QC, manufacturing, and facilities engineering staff. FDA guidance documents, PDA TRs, and the peer-reviewed literature all provide descriptions of potential point-sources of contamination. Bioburden may be associated with personnel, incoming raw materials (excipients, APIs, filters, etc.), compendial and non-compendial waters, and ambient air and associated airborne detritus. The potential point-sources for contamination are often common among different products, but the nature of the bioburden is influenced by the product. For example, mammalian cell lines or formulation constituents may require routine monitoring for mycoplasma or viruses. Open process operations are more susceptible to contamination than are closed processes.
Souza: Bioburden may enter a manufacturing process or product through multiple routes, including cell banks, raw materials, utilities, equipment, the facility, or personnel. A risk assessment of each entry route using tools such as an FMEA will determine the risk levels and help target the highest risk operations or materials in the development of a comprehensive strategy. During a risk analysis, the process or environment is assessed for potential types of contamination. If components or equipment are different for processes, such as microbial fermentation and mammalian culture, a separate risk analysis is performed for each process.
BioPharm: Does operating personnel need specific training on bioburden reduction?
Souza: The manufacture of biologics requires operators trained in aseptic techniques, with specific training for relevant unit operations and cleanroom classification. Operators in the critical final fill area require training in practices that minimize product contact. Operators are trained on appropriate gowning procedures, required hygiene practices, and self-reporting of personnel conditions that could contribute to high levels of bioburden or potentially pathogenic contamination. Finally, operator training should stress compliant behaviors and commitment to established procedures.
Hutchinson: Training of personnel will be very important and will vary depending on roles and processes. In my experience, this is typically covered in GMP training, for example, gowning procedures, good aseptic practices, and specific operating procedures for operations, such as taking samples.
Mittelman and Anicetti (PDA task force): All staff should be aware of the importance of bioburden control in both unit operations and in the finished product. Understanding the basics of microbial growth and routes of contamination is key to effective management independent of the products manufactured. Bioburden problems often result from a lack of understanding of personnel actions that result in a process or product contamination event.
BioPharm: How do endemic bacteria affect the type of filter needed for filtration?
Mittelman and Anicetti (PDA task force): While bacteria (and fungi) may be present in all in-process formulations, viruses and mycoplasma are typically only encountered in formulations containing serum or animal, plant, or insect cell lines. The selection of specific microporous membrane filters or ultrafilters for a given product should also be based upon experience with the formulation process and the required bioburden reduction at each step. Considerations such as membrane porosity, charge, or protein binding may also influence the selection process.
Hutchinson: Undoubtedly, understanding the organisms endemic within the facility plays a role. I have described previously the impact of mycoplasma species on cell cultures. There are a variety of approaches to controlling these organisms. I advocate the use of 0.1-micron rated filters that have been tested to demonstrate their effectiveness at removing these organisms for cell culture media filtration.
Despite normal flow filtration being an established operation within the industry, we continue to see some relatively poor practices being performed. Confusion remains amongst bioproducers who believe that, if they are using a sterilizing-grade filter, they will automatically get a sterile filtrate. This is not necessarily true, and I think this can lull people into a false sense of security. How a filter is challenged and used in operation is extremely important.
Souza: Biologics may be at risk for contamination from fungus, bacteria, mycoplasma, and virus in the bioreactor depending on raw material source. Risk of mycoplasma contamination in raw materials may warrant the use of a 0.1 um-rated filter, while risk of virus contamination may warrant the use of an upstream virus barrier filter. Virus nanofiltration is typically implemented downstream for removal of adventitious viruses from raw materials or endogenous retroviruses expressed by cells.
Methods and Processes
BioPharm: Is there a specific method or process that you find to be the most reliable to ensure maximum bioburden reduction of biologics?
Souza: A comprehensive approach—combining bioburden removal methods, process design elements, operator training, cleaning and sterilization methods, process monitoring, and quality oversight—is essential. A combination of orthogonal methods is most effective for minimizing bioburden during production. Culture media treatment by high-temperature–short-time (HTST) or UV-C will reduce microbial load. Filtration may be used for cell-culture media or buffers. Chromatographic steps, low pH, and chemical inactivation provide virus reduction during processing. Virus and sterile filtration prior to final fill ensure maximum bioburden reduction. This comprehensive approach will lead to greater assurance of patient safety.
Mittelman and Anicetti (PDA task force): There is no single process that will ensure effective bioburden control in a biologics manufacturing process. Point-source recognition, then employing effective controls at each unit operation, is key to effective bioburden management. Effective bioburden reduction begins with raw material and supplier controls. The PDA TR repeatedly notes that personnel are the most important source of bioburden--either through direct contamination or improper application of bioburden control practices. Ensuring that compendial and non-compendial waters that are used in the manufacturing process are maintained under good microbiological control is important: water is typically the single-largest raw material used and [one of] the most common sources of bacterial contaminants.
Hutchinson: Bioburden considerations will vary even if the same process is being performed in different ways. For example, when processing a small number of batches for clinical trials in multi-product facilities, there is a tendency to have long intermediate product hold times in order to account for uncertainties in process timings, equipment availability, and set-up times. If this is the case, then in-process bioburden filtrations and aseptic techniques become more important. The batch might be being processed over many weeks. As the volumes of intermediate product pools are typically small, they can be moved into cold rooms between processing steps to prevent microbial growth. At large scale, when campaigns are being manufactured to supply the market, it is more likely campaigns of a single-product are being run at a higher run rate in order to achieve operation efficiency. In these cases, the hold times are typically shorter, but there is less of an opportunity to reduce the temperature of intermediate product pools. As such, the approach that might be adopted for bioburden control will vary.