Biologic drugs, whether administered parenterally or subcutaneously, must be sterile to ensure the safety of the patient. Prevention and control of contamination from microorganisms, microbial toxins, viruses, and particulates begins with appropriate process design, and includes monitoring and an established decontamination strategy for addressing any contamination events. The challenges to contamination control are often magnified for cell therapies, including both patient-specific autologous and allogeneic products, because of the time criticality. Automated decontamination is more robust and reliable than manual approaches and, when combined with the use of isolator technology, particularly for autologous cell therapies, can ensure the production of safe and effective therapeutic products.
Many Contamination Threats in Biomanufacturing
Biologic drugs, whether more conventional recombinant proteins and monoclonal antibodies or new modalities, such as cell therapies, can be administered parenterally and thus directly into the bloodstream. Sterile manufacturing is therefore essential to ensure that biopharmaceutical drug products are free of contaminants, including microorganisms (bacteria and fungi), microbial toxins (e.g., endotoxin), viruses, and particulates.
Contamination can enter biomanufacturing operations through a number of sources, most prominently human operators, water, material surfaces, and air. Humans shed hair and skin cells on a continual basis and can carry microorganisms and viruses on their skin and clothes. Water used in cleanrooms can enable the spread and growth of microorganisms. Equipment can be contaminated with not just microorganisms and viruses but dust and particles from the air.
Materials used in biopharmaceutical processes — cells, media, supplements, oxygen and other gases, and so on — can also contain undesired adventitious agents. For cell therapy manufacturing in particular, the donor cells for autologous treatments present a lower risk of contamination, because they are ultimately administered to the same patients from which they are derived. The risk of viral or other contamination will be much greater for allogeneic cell therapies produced in large quantities using cells from a single donor and administered to multiple patients.
The greatest risk for contamination exists when materials are transferred in or out of an aseptic production process. Where possible, manufacturing is performed in closed environments, but at some point those systems must be opened to allow the introduction of cells, media, and other reagents and materials. The air in the environment can carry and therefore deposit contaminants during bioreactor filling and the removal of bioprocess fluids for downstream purification. While sterile connection technology is well established, any connections also present an opportunity for contamination.
From a holistic viewpoint, it is therefore essential to never take for granted that everything in a cleanroom is contaminant-free. Control of gowning and the flow of operators in and out of a cleanroom is only one aspect of preventing contamination. Engineering controls must be constantly monitored to ensure that they provide effective contamination control.
Comprehensive Contamination Control Strategy Essentials
The control of biocontamination in biomanufacturing requires a comprehensive strategy that includes prevention, monitoring, and an established process for addressing any contamination events that occur. It begins with understanding the risks for contamination, which informs and leads to the design of process systems that take those risks into account. Such designs typically include the use of proper gowning procedures; isolators or other enclosed production systems; appropriate air filtration, flows, and pressures; and regular disinfection.
These efforts are supplemented with ongoing environmental monitoring (EM) activities, including trending of data, to identify potential contamination concerns before they impact the manufacturing process. Such monitoring programs are risk-based with respect to location of sampling sites, the frequency and timing of sampling (surface and air), and the choice of culture media, incubation times, and temperatures for evaluating the samples.
In cases where contamination or microbial data deviations occur, comprehensive investigations are necessary to address root cause analysis, process or product impact, risk, and suitable corrective and preventative action. Beyond the cost associated with the loss of product, these investigations are themselves costly, and as such contamination or excursion events should be avoided where possible.
Several Key Pain Points for Cell Therapy Manufacturing
As with any biological process, the biggest challenge in cell therapy manufacturing with respect to contamination control is preventing the introduction of contaminants by the operators and from the equipment or materials. Although contaminants can enter throughout the supply chain, this article will focus on production-related challenges.
This challenge is magnified by the fact that, as an emerging field, there are few people with extensive experience in proper contamination control procedures working in cell therapy manufacturing. Most understand that, according to good manufacturing practice (GMP), closed systems should be used. However, small-scale cell therapy production generally occurs in biosafety cabinets, the vast majority of which are open systems. Although the technicians are partially or fully gowned and follow procedures designed to minimize human intervention, there is no physical barrier between the operator and the process, so the potential for contaminated air entering these biosafety cabinets remains. Even versions of cabinets that have been modified to introduce gloves on the glass sashes are still not truly closed systems. The way to address these issues is to perform cell therapy manufacturing operations in isolators or other fully closed barrier systems.
Allogeneic cell therapy products present another level of contamination concern beyond those associated with autologous products. These cell therapies are produced on a larger scale and then frozen in a stabilized medium (or lyophilized) so that they can be stored for extended periods of time and thawed before use, during which time the growth of adventitious agents may occur. Consequently, the need for isolators for the production of these therapies is even more critical.
There are few systems on the market that can support the entire cell therapy process. Even with these systems, the need for humans to make aseptic connections is necessary and introduces risk of contamination.
The end result of contaminated cell therapy products is the need to discard the product and start manufacturing anew, possibly from the point of apheresis from the patient or donor, with significant additional manufacturing and labor costs for these high-value treatments, as well as the costs associated with investigation, cleanup, corrective actions, and lost sales and additional risk for severely sick patients. This outcome is even more significant in certain applications, such as CAR-T cell therapies, which are generally the last treatment option for very sick patients for whom timing is critical, so there is only one chance to produce the product. It is absolutely essential to minimize the risk of contamination in order to ensure that a safe and effective cell therapy can be manufactured in such a limited timeframe.
Manual and Automated Disinfection
Destruction of contaminants in the biomanufacturing environment can be achieved using two main approaches: manual and automated. For manual approaches, a wide range of disinfectants can be used, namely alcohols and biocides, but it is critical that each agent is validated to ensure that it is fit for use in the specific context. A mix of spraying, mopping, and wiping are used, depending on the agent and the surface being treated.
Automated decontamination systems disinfect surfaces without human exposure. Vaporized hydrogen peroxide is the most common method of room and enclosure decontamination, although other methods, including UV irradiation, aerosolized hydrogen peroxide, and chlorine dioxide, can also be used for some specific applications.
For established biomanufacturing facilities and processes, routine daily cleaning is performed manually, while the more extensive automated decontamination is typically performed in between production campaigns and to remedy an unexpected contamination event. A notable exception is isolators, which generally use automated decontamination for routine disinfection of the enclosure surfaces and of load items entering the isolator.
The appetite for automated decontamination depends to some degree on the type of products being produced, however. Viral vaccine manufacturers and CDMOs (contract development and manufacturing organizations) that handle viral vectors lean toward automated decontamination, because they must achieve the removal of all traces of the virus used to make a particular product before switching to manufacturing another product or starting the next batch. It is necessary to kill any residual virus from the previous process, as well as any contamination resulting from the outside world.
In emerging fields, such as cell and gene therapy, where new production facilities must be constructed, biopharma manufacturers have many factors to consider. Manual processes require minimal capital investment and can be implemented quickly. Over the longer term, however, automated systems provide many benefits critical to cell therapy manufacturing: consistency and repeatability, reduced disinfection time and hence downtime, less operator resource required, ease of use, and reduced waste and health risk to operators. Many cell therapy developers adopt a timeline for moving toward automated decontamination, starting with manual decontamination processes first and working toward automated systems over time as products move closer to approval, while others elect to implement automated decontamination from the outset to minimize manual cleaning and disinfection requirements.
Another important consideration when deciding between manual and automated methods is the level of repeatability and traceability that can be achieved. Validating manual disinfection protocols involves proving that the selected disinfectant kills the microbes and other contaminants that are expected to be found on a particular surface after exposure for a specified period of time. Validation is ultimately difficult, because there is a human element to manual cleaning processes, which introduces variability and risk. Each person who mops a floor may get different coverage owing to variation from one person to another. In contrast, automated technology can be more easily validated, because the same conditions are replicated exactly in every cycle.
It is important to recognize that some extent of manual cleaning will always be required. But facilities that employ automated decontamination technology benefit from the ability to minimize the level of manual cleaning that is required.
Factors to Consider When Choosing an Automated Decontamination Strategy
Selection of an automated decontamination strategy is based on four main parameters:
Efficacy of the process: how well it kills the microbes, viruses, and other contaminants expected to be present in the environment
Cycle time: how quickly the decontamination process takes, which equates to production downtime and cost
Safety of the process, given that disinfectants are designed to kill microorganisms
Material compatibility, because many disinfectants can damage different types of surfaces through oxidation
Table. Comparison of leading automated decontamination methods for rooms and enclosures
| Contamination Control Method | Advantages | Disadvantages |
| UV Irradiation |
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| Chlorine dioxide |
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| Aerosolized hydrogen peroxide |
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| Hydrogen peroxide vapor |
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Evolving Regulatory Expectations
Regulatory standards and requirements for contamination control vary by region and evolve rapidly, particularly for newer modalities like cell therapy. At present, the most important requirements are presented in the following guidance documents.
Information covering regulatory concerns for production and quality control and preclinical testing for Advanced Therapy Medicinal Products (ATMPs) in the United States are presented in FDA Guidance for Human Somatic Cell Therapy and Gene Therapy.
In Europe, guidance for the manufacture of ATMPs is established in Annex 1 (Manufacture of Sterile Medicinal Products) of the EU Guidelines to Good Manufacturing Practice: Medicinal Products for Human and Veterinary Use,2 and guidance for nonclinical testing is presented in Part 4 of Annex 1 of Directive 2001/83/EC of the European Parliament.3
Requirements for the biocontamination control of cleanrooms are established in the ISO 14698 Standards, which include ISO 14698-1, Cleanrooms and associated controlled environments—Biocontamination control, Part 1: General principles and methods,4 and ISO 14698-2, Cleanrooms and associated controlled environments—Biocontamination control, Part 2: Evaluation and interpretation of biocontamination data.2
ISO 14698-1 describes the principles and basic methodology for a formal system to assess and control biocontamination in cleanrooms technology, including performing risk assessments and implementing suitable monitoring and control measures.4 ISO 14698-2 provides guidance on the analysis and trending of microbiological data generated from monitoring activities.5
In Europe only, EN 17141: 2020, Cleanrooms and associated controlled environments — Biocontamination control6 replaced the ISO 1469 standards in 2020.
In Europe only, EN 17272:2020, Chemical disinfectants and antiseptics: Methods of airborne room disinfection by automated process, provides a standard test that users of automated decontamination systems can compare the effectiveness of different decontamination technologies from different suppliers
Isolators Offer Real Advantages
For the manufacture of cell therapies, which currently involves processing multiple small-volume batches, isolators provide many advantages with respect to ensuring contamination control. For a relatively low cost compared with a conventional cleanroom, isolators provide manufacturers with a Grade A environment to perform aseptic processes. They are designed to allow all operations to take place within an enclosed environment, eliminating human intervention and exposure. Furthermore, when integrated with automated hydrogen peroxide decontamination technology, the risk of introducing contamination from the environment is reduced even further. The space is highly controllable and a very good alternative to expensive, large cleanrooms. Particularly for startups in the cell therapy area, isolators offer an effective solution with lower capital and operating costs.
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References
- FDA Guidance for Human Somatic Cell Therapy and Gene Therapy: Guidance for Industry. U.S. Food and Drug Administration. Mar. 1998
- EU Guidelines to Good Manufacturing Practice: Medicinal Products for Human and Veterinary Use. Annex 1: “Manufacture of Sterile Medicinal Products. European Medicines Agency.” 25 Nov. 2008.
- Directive 2001/83/EC. Annex 1: Analytical Pharmacotoxicological and Clinical Standards and Protocols in Respect of the Testing of Medicinal Products. European Parliament.. Nov. 2001
- ISO 14698-1:2003. “Cleanrooms and associated controlled environments — Biocontamination control — Part 1: General principles and methods.” Sep. 2003.
- ISO 14698-2:2003. “Cleanrooms and associated controlled environments — Biocontamination control — Part 2: Evaluation and interpretation of biocontamination data.” Sep. 2003.
- BS EN 17141:2020. “Cleanrooms and associated controlled environments. Biocontamination control.” 13 Aug. 2020.
- BS EN 17272:2020. “Chemical disinfectants and antiseptics. Methods of airborne room disinfection by automated process.” 7 Apr. 2020.
