Environmental Health and Biosafety Risk Assessment Guidance for Commercial-Scale Cell and Gene Therapy Manufacturing

Abstract

Introduction:

This article aims to identify best practices, improve risk controls, and aid regulatory agencies in developing guidance for environmental and biosafety risk assessment for commercial-scale cell and gene therapy manufacturing.

Methods:

A cross-functional team should start with hazard classification and testing requirements for materials used or generated by the process and process hazard characterization.

Results:

The team develops a safety profile of the process to mitigate risks, including:

• product biological contamination risk and process controls, including raw materials, facilities, operator and environmental controls, and method of detection;

• a technical review of the process to evaluate the operational and engineering controls;

• monitoring systems to mitigate the risk of failure and/or breach of the system, preventing the release of material to the facility or operator exposure;

• site sanitization strategy and facility containment measures, including engineering designs, air handling systems, spill containment measures, surface cleanability, waste flows, and decontamination practices;

• a review of site practices, including process, employee, material and waste flows, staff training, controlled access, operator gowning, and emergency response plans/measures.

Discussion:

The cross-functional team should regularly reconvene to provide solutions for enhanced process control, process life-cycle management, monitor assumptions, and track performance. The plan must be revised following any relevant failure event or process change.

Conclusion:

A risk assessment template is shared to bring to the reader’s attention the complexity of commercial-scale manufacturing, areas to assess, potential questions to ask, and other pertinent parties who may input to the risk assessment.

Introduction

The rapidly expanding field of cell and gene therapies (CGT) has the potential to revolutionize curative treatments. Alongside this scientific progress, regulatory guidance has developed to address manufacturing guidelines and chemistry, manufacturing, and controls (CMC) specifications for CGT drug products. Conversely, occupational health and safety guidelines for the manufacturing of CGT products have been lacking. There is no “universal” toxicological or potent compound classification document or risk assessment scheme for CGT manufacturing processes.

The BioPhorum CGT environmental health and safety (EHS) and biosafety team was developed to unite leaders in the biopharmaceutical industry to develop best practices for personal and environmental safety when conducting CGT research or clinical and commercial manufacturing.

The purpose of this article is to identify, share, and enhance best practices and standards; improve risk controls; increase the speed of learning; and ultimately influence and/or aid regulatory agencies in developing guidance or policies around CGT development and manufacturing. The scope of this guidance is strictly limited to the occupational health and safety risks associated with facilities and distribution sites in the clinical and commercial supply chain and does not attempt to address risks associated with clinical trials or patient safety. Although we recommend the use of medical surveillance by occupational health working alongside health care professionals, we do not attempt to define what should be included in the surveillance or any postexposure plans during this article. This is a key area for the industry to address in the future

For the purposes of this article, we selected the term commercial scale to cover not only the production of CGT products using processes described as large-scale (>10 L) but also where the commercial production is less than 10 L. The scale of production does not necessarily mean high risk. Frequent repetition of a process scale of less than 10 L in a commercial production environment may produce the same potential increase in possibility of exposure both in the number of humans and the amount of environmental exposure that might occur in the event of containment failure.

Current Industry Risk Assessment Methodologies

The CGT development and manufacturing community is increasingly challenged to keep employees, visitors (eg, scientists, clients, emergency responders), and the community at large safe from potential exposures. Some comprehensive guidance documents exist that provide risk-based analysis for working with infectious agents (eg, Biosafety in Microbiological and Biomedical Laboratories, or BMBL ¹ ; World Health Organization [WHO] Biosafety Manual ² ; and Bloodborne Pathogen Standards [Occupational Safety and Health Administration, or OSHA ³ ]) as well as recombinant or synthetic nucleic acid molecules, particularly in the laboratory environment (National Institutes of Health [NIH]). ⁴ However, there is a clear gap in guidance related to the CGT-associated risks and commercial-scale CGT manufacturing. ⁵ Commercial-scale EHS and biosafety guidance for safety professionals is limited to Appendix K of the NIH Guidelines ⁶ and the WHO Good Manufacturing Practices for Biological Products. ⁷

Several articles have been published addressing large-scale containment for recombinant technology. ^8,9 These guidelines were originally drawn up to address monoclonal antibody and biologic production and do not include specific considerations when it comes to assessing additional risks pertaining to CGT.

Workers performing small-scale laboratory support functions (eg, quality control or quality assurance analysis) that support the commercial scale manufacture are referred to BMBL ¹ and WHO Biosafety Manual. ²

Overview of Hazard and Risk Assessment for CGT Development and Manufacturing

The identification of hazards and assessment of risk are the initial steps in determining the safety practices necessary for the use of a hazardous agent. ¹⁰ A risk management system should ensure that there are adequate measures to assess safety performance and that the system is regularly audited by subject matter experts, including quality, engineering, process, occupational health, and EHS.

Risk assessments are a responsibility shared by multiple disciplines at all levels of an organization in addition to environmental health and biosafety professionals. A cross-site team must be assembled to execute the assessment and should include, at a minimum, representatives from manufacturing, quality control, raw materials management, quality assurance, facilities, health and safety, and process development/process sciences (including process, analytical, and formulation development). The aim of any risk assessment is to eliminate the risk of exposure by designing safe processes and procedures, and these individuals have a key role in the overall manufacturing process. When necessary, based on the risk assessment, occupational health should be consulted with to determine the appropriate medical treatment should an exposure occur and a postexposure plan should be put in place.

Conducting CGT risk assessments requires understanding of existing guidance for biosafety, recombinant DNA, chemical safety, and toxicology; it also requires a comprehensive understanding of the science of CGT as well as the development and manufacturing processes. Established risk groups and associated biosafety levels assigned to the agent during the risk assessment process provide guidance for an overall containment strategy; however, the risk assessment must consider the exposure of humans to CGT and its hazardous components (cells, vector, plasmids, etc) during both standard operations and during upset conditions that could result in an unintended release from containment. The risk assessment must include a traditional biosafety assessment of the infectivity of the agent, routes of exposure, severity of infection, availability of prophylaxis, level of containment afforded by the process and equipment used, volume of material being handled, and possible failure modes as well as the potential for target and off-target biological activity of the therapeutic molecule (drug substance, drug product, or synthetic nucleic acid constructs) in a healthy adult. With the advent of new technology, this should be expanded to cover the risk of exposure because not all biohazards will technically “infect,” such as plasmid products and other active synthetic acid constructs. A risk- and exposure-based approach should also be applied to the design of the manufacturing process because manufacturing risks may differ according to the type of product, nature/characteristics of the gene-editing materials, and level of complexity of the manufacturing process. The actual risks of exposure to some of these novel materials is unknown and in the case of some manufacturing components will not be evaluated in clinical trials (eg, the lentivectors for CAR-T production).

The risk assessment process should be an iterative one, changing and progressing as more experience and knowledge are gained. The cross-site team should reassess the plans and provide solutions for enhanced process control and for continued process life-cycle management.

Risk Assessment Processes, Including Facility Containment and Process Strategies

This section reviews elements of the process, facilities, and policies to consider when establishing a risk management system and control system. Appendix A includes a framework when conducting a risk assessment for CGT manufacturing and should take into consideration the following controls:

• process materials such as reagents, cell lines, vectors, helper viruses, and active synthetic nucleic acid materials;

• process operations and controls;

• other hazards (chemical, physical, and ergonomics);

• facilities, equipment, and utilities;

• employee gowning and personal protective equipment (PPE);

• employee and contractor training;

• site systems and policies;

• storage, shipping, and distribution;

• cleaning versus disinfection;

• waste management;

• emergency response (eg, spill, release, medical emergency in controlled areas, severe weather).

Biosafety risk profiles will differ according to the type of product being generated, any active or biohazard components used in development, manufacturing or testing, the risk classification of manufacturing materials used, the level of complexity of the manufacturing process, and the facility controls. Therefore, the risk assessment process must be tailored by an organization considering the unique biosafety profile relevant to its product, process, facility, and site practices. Risk assessments must include a competent and complete review of the manufacturing process, facilities, and site policies with the purpose of identifying failure modes and effective control measures. It applies equally to engineering modifications, which often require detailed drawings and approval of specifications and components, and to process changes, which involve alteration of chemical or biological constituents and operating conditions. ¹¹

Process Materials; Reagents, Cell Lines, Vectors, Helper Viruses, and Active Synthetic Nucleic Acid Constructs

Process materials, which may be of biological origin, chemical in nature, or synthetic nucleic acids, are relevant to employee and environmental safety, with implications on engineering controls and facility containment requirements. In the US, local institutional biosafety committees (IBC) may be required to define the handling and management requirements for biological agents, which in turn will define the biohazard risk profile of the process and the biosafety control requirements.

The NIH Guidelines ⁴ specify criteria that apply to the biological hazards associated with organisms containing recombinant or synthetic nucleic acid. However, other hazards accompanying the large-scale cultivation of such organisms, for example when generating cell banks, should be considered, such as the origin and traceability of the parental source cell line. Although cells may be tested for adventitious agents, the risk profile for potential endogenous virus contamination or introduction of human viruses from the staff, especially in human cell lines, should be considered in the safety process of the process feedstream.

The classification can impact the distribution and movement of materials between sites, potential site inspections, and across international borders (eg, permits may be required by government agencies for etiological agents, human- or animal-derived material, biological product, and drug intermediates and substances). Risk group classification levels for different CGT cells can be obtained from the ABSA International website at https://my.absa.org/tiki-index.php?page=Riskgroups.

Risk classification alone is a guideline, so hazards associated with project-specific materials and novel therapeutic molecules must also consider:

• the potency or ability of the engineered vector product to infect and transduce cells;

• the ability of product or vector to revert to replication competence;

• unintended effects (eg, expression in nontarget cells);

• the risk for vector shedding/mobilization if released ¹² ;

• density (concentration) of culture in both upstream processes (USP) and downstream processes (DSP);

• scale of production; although commercial scale does not necessarily mean high risk, increased scale may lead to an increased possibility of exposure in terms of the number of humans, cell-specific endogenous virus amplification, and the amount of environmental exposure that might occur in the event of containment failure. ¹³

Import and Export Permits

In the US, importation and exportation of biological materials is regulated under multiple US agencies. Materials that may require an import or export permit include but are not limited to:

• organisms and vectors that may cause disease in humans, animals, or plants;

• biological toxins;

• whole animals;

• human- and animal-derived materials (blood, blood products, tissues, cells, body fluids, etc);

• human and animal products;

• biological pharmaceuticals and vaccines;

• “Commerce Controlled” materials.

In the US, the following agencies have their own import and export rules for materials and products that are within their jurisdiction. Each regulatory authority broadly defines each of the categories of materials that require import and export permits through their agency. A material may require a permit from multiple agencies if it meets the definition and/or categorization of multiple agencies.

• Food and Drug Administration (FDA), US Department of Health and Human Services. The FDA has different import and export regulations based on the type of substance or product and it’s intended use.

• Centers for Disease Control (CDC) and Prevention, US Department of Health and Human Services. The CDC controls the import into the United States and interstate transportation of microorganisms that are or may be infectious to humans, including vectors, recombinant microorganisms, and nucleic acids with the potential to produce an infectious form of an agent.

• Animal Plant Health Inspection Service (APHIS), US Department of Agriculture. APHIS controls the import into the US, interstate transportation, and export outside of the US of animals, animal products, organisms (or their derivatives) that may introduce or disseminate any contagious or infectious disease in animals (including poultry) and vectors that have been treated or inoculated with organisms or are diseased or infected with any contagious, infectious, or communicable disease of animals or poultry or have been exposed to such disease. Veterinary services within APHIS has a separate import permitting process for animals and animal-derived products to ensure exotic and poultry diseases are not introduced into the US.

• Fish and Wildlife Service (FWS), US Department of the Interior. FWS issues permits under various wildlife laws and treaties to ensure that only legitimate wildlife-related activities are carried out and in a manner that safeguards wildlife.

In Europe, the type of product is considered; for any animal-derived products, there will almost certainly be a required inspection and import permit. For other products, an import permit may in certain circumstance not be required; an authorization such as an approved clinical trial may cover the import.

The first time a material is imported or exported by an organization and any time a requirement(s) is/are unclear, it is highly recommended that every agency who may have authority for that material is contacted for a requirement determination in writing.

Process Operations and Controls

Regardless of the manufacturing platform and unit operations employed, a well-established process definition and control strategy can mitigate the risk of failure and potential for exposure from both a product quality perspective but also from an employee safety and facility protection standpoint.

Process risk assessments consider the series of individual unit operations that comprise the manufacturing process. Occupational and environmental risks may differ during these individual unit operations; therefore, a brief description of each operation in the manufacturing process should be drafted to provide context and scope of the risk assessment, highlighting potential vulnerabilities and areas of increased risk. This must also be applied when engineering controls are modified and/or processes changed because new vulnerabilities may be identified. A typical, high-level CGT manufacturing process is shown in Figure 1. The user will need to go into more detailed examination of its own individual manufacturing process at each of the main stages shown in the following.

Figure 1.

Typical cell and gene therapy manufacturing processes.

The general pharmaceutical manufacturing process includes initial process steps, or USP, that include cell thaw, expansion, and production; the second stage process steps, or DSP, involve purification, formulation, and concentration; and the final stage, or fill finish, includes final formulation and fill operation.

Depending on the manufacturing platform and technologies utilized, USP can employ scale-up or scale-out strategies. Scale-up processes generally involve single-use vessels that are amenable for cells grown in suspension or in adherent mode as immobilized suspensions (eg, microcarriers), immobilized fixed bed (eg, Basket reactors), or multistack planar systems (eg, cell factories). Scale-out processes generally involve multiple, smaller volume units and can be for suspension or adherent cell cultures. The multiplication of complex setups can raise additional ergonomic concerns due to the increased number of unwieldy manipulations and manual agitation steps required by operators. These concerns should be addressed as part of basic manufacturing practice.

Downstream operations may be independent of scale-up or scale-out platforms but will likely utilize similar technologies operated under high pressure to concentrate and purify product (eg, cross-flow or dead filtration, chromatography). Fill finish can involve the handling of hundreds of units by either operator-dependent manual fills or semiautomated or fully automated fills. In the case of automated filling operations, consideration needs to be given to machine guarding of the equipment. Manufacturing process scales can routinely exceed 100 L, with typical commercial manufacturing volumes of 1000 L to 3000 L.

Ideally, all bioprocessing operations should take place in a closed system. A closed system, during normal operation when properly installed for the intended use, is defined as “a process step (or system) with equipment designed and operated such that the product or hazardous manufacturing components are not exposed to the immediate room environment.” ¹⁴ In practice, this is not possible for every individual operation, and expected failures of the closed system should be identified, addressed, or mitigated to eliminate the possibility of exposure. Occupational risks can be reduced and controlled by the correct application of internationally recognized procedures, such as good microbiological techniques, safety equipment, PPE, engineering controls, appropriate level of containment, and adequate facilities. This combination of mitigation factors is only effective when used consistently and correctly. Improper use, lack of validation, and neglecting regular reassessment may instill a false sense of security and increase the risk of exposure by decreasing the level of protection provided. ⁵ Substitution of PPE and procedures should not be a routine alternative.

Development of CGTs may encompass changes in the manufacturing process of the product itself and changes in the manufacturing of critical starting materials (eg, viral vector, cell source, modifying enzyme, synthetic nucleic acid molecules) that might impact the risks of the operators or the environment. It is important that all changes introduced during development are identified, assessed for potential impact on risk mitigation measures for the operators, and documented.

In all cases, viable organisms and recombinant DNA materials must be decontaminated. The identification of effective decontamination regimes ¹⁵ should be an integral part of the risk assessment. There is a need for solid, validated evidence for a range of disinfectants and procedures; however, in the absence of published data, the disinfection process should be selected based on product-specific data. ¹⁶

Other Hazards (Chemical, Physical, and Ergonomics)

The risk assessment must include a comprehensive review of unit operations that may involve other hazards, such as chemical, physical (eg, heat or pinch hazards, respirator evaluation, and fit), and ergonomics (eg, repetitive strain or heavy lifting), in accordance with other regulations governing the health and safety of employees. Consideration should be given for any potential conflicts for chemical and biohazard protection and control, for example, the use of safety showers and drains when responding to a spillage while needing to maintain containment of any biohazard or chemical compatibility for decontamination of a biological agent when in a specified buffer system.

Physical hazards that should be assessed from an ergonomics standpoint include lifting, hoisting, and transporting totes with large bags (>50 lb) of materials as well as standard workplace safety (slips, trips, falls; falling objects; hazard communication; and hazardous energy).

Facilities, Equipment, and Utilities

The type of manufacturing platform, process design, scale of operation, biosafety level, and material risk classification will determine the facility design and required floor plan. A manufacturability fit assessment should be performed to ensure the facility, equipment, and utilities can meet the desired process definition and that containment systems are adequate and compliant with the site sanitization and viral segregation plan and enough operating space is available to reduce trips and falls.

Facilities can be multiuse “ballroom” or, more likely for CGT products, batch-dedicated, with product changeover requirements. Processes can be segregated in space (eg, different suites) depending on the process step or the viral risk profile or executed in a single space with process operations separated in time.

Selection of the physical containment level required for recombinant or synthetic nucleic acid molecule research or production involving more than 10 L of culture is based on the containment guidelines established in Section III, Experiments Covered by the NIH Guidelines. ⁴ The NIH guideline sets 4 levels of containment based on the degree of the hazard to the health of the operators and the environment and are consistent with good manufacturing practice (GMP). Good large-scale practice (GLSP) is recommended for research/production involving viable, nonpathogenic/toxigenic recombinant or synthetic strains derived from host organisms that have an extended history of large-scale use, whereas BSL-3-LS is recommended for large-scale research or production of viable organisms containing recombinant or synthetic nucleic acid molecules that require biosafety level 3 (BSL-3) containment at the laboratory scale. For more detail, see Appendix K of the NIH Guidelines ⁶ and the Health and Safety Executive GMO regulations ¹³ with the Scientific Advisory Committee on Genetic Modification (SACGM) Guidance documents. ¹⁷

Employee Gowning and PPE

Employee gowning and PPE will be determined by the process requirements and biosafety risk profile. Engineering controls and monitoring systems may impact the level of gowning and required employee PPE needed. Generally, single-use, full-coverage, disposable gowning is preferred (eg, coverall). The risk assessment must consider both clean room requirements and features required to provide barrier protection (eg, chemical and moisture resistance).

Safety glasses and gloves are standard minimum PPE for skin and eye protection. However, the risk assessment must also consider chemical compatibility for gloves. For process operations involving chemical (eg, sodium hydroxide) or biological (eg, virus) splash hazards or risk of aerosols, additional protection is required (eg, aprons, face shields, and respirators), especially in the absence of splash guards or appropriate biosafety containment. Effective training supported by clearly written batch production records, with highlighted warnings for high-risk operations including safety limit values for monitoring control systems, are essential elements to ensure operator error is not a source of a hazard event.

Employee and Contractor Training

Employee and contractor training should be given before operations are initiated in a contained area and be documented in the individual employee records as well as signed off by both the trainer and the trainee. ¹¹ A rigorous and well-documented training plan includes a minimum number of supervised training events to ensure employees are adequately trained and aware of all operation risks prior to becoming the lead operator. Also, double employee coverage (eg, a buddy system) should be established to ensure adequate support for all operational needs as well as to support emergency response needs.

Additional training should include process flows, including materials, equipment, and waste management, so that all manufacturing employee and support staff are aware of the facility site segregation plan and any biohazard risk is managed and contained to the designated facility envelope, protecting the site and other employees. Regular refresher training about hazard communication, medical surveillance, use of technical containment, use of PPE (including respirator surveillance and fit test), and understanding of procedures and high-risk operations, with a thorough understanding of safety control measures and limits of operation, should be executed.

Site Systems and Policies

In addition to standard environmental monitoring and facility cleaning practices, a site sanitization and segregation strategy will also be necessary for CGT products. Such policies are necessary to provide containment and control of potentially biohazardous materials across the site and for the protection of nonessential employees. The site policies may include sanitization procedures, product, material and waste flows, containment systems, and emergency spill response plans. Site sanitization procedures, including liquid disinfectants, steam sterilization, and vapor phase hydrogen peroxide (VHP), need to be effective for the agents manufactured at the site and preferably validated to define effective decontamination/kill parameters. Due to limited viral models and effective analytics specific to the viral agents, identifying worse case viral models may be the preferred strategy using relevant viruses and process feed streams. In addition to efficacy, risks associated with employee exposure to chemical disinfectants must be considered and mitigated.

In addition to decontamination processes, the site containment and segregation policy needs to consider all process-supporting activities, including employee, material, equipment, and process waste flows; operator and support staff gowning requirements; employee training (eg, respirator training, air handling and room pressure designations, barrier systems, cleaning and changeover procedures); as well as packaging and transportation containment solutions. Furthermore, based on risk assessment and regional requirements, once commercial scale is reached and a routine process is in place, emergency response plan and/or exposure control plans should be in place and all staff adequately trained commensurate with their defined response capabilities. For first responder awareness or defensive measures, which are likely tasks under the manufacturing staff requirements, then annual training consistent with Code of Federal Regulations (CFR 29) for OSHA requirements may be applicable. ¹⁸ For full internal spill response plan management, the site may have its own HAZMAT team, trained at the technician response level up to incident commander status.

Together, the site sanitization and biohazard containment plans are necessary systems to establish and monitor to ensure adequate protection of the site, employee, and environments.

Transportation and Shipment

Certain biological and biologically derived materials must be shipped in accordance with international and country-specific regulations for the shipment of hazardous materials, referred to internationally as “dangerous goods.” The hazardous materials classifications 1 through 9 are also internationally accepted based on the Globally Harmonized System of Classification and Labeling of Chemicals (GHS). ¹⁹ Class 6 includes toxins and poisons (Division 6.1) and infectious agents (Division 6.2). Within Division 6.2, there are subclassifications of “infectious substances” based on the source of the material and the severity of the hazard to human and animal health. In addition, Hazard Class 9, Miscellaneous, includes “Genetically Modified Organisms, Genetically Modified Microorganisms” that do not meet the definition of a subclassification in Division 6.2.

All United Nations member states accept the International Air Transport Association (IATA) Dangerous Goods Regulations (DGR) ²⁰ as the international standard for the transportation of dangerous goods by air. Most countries base their own regulations on these model regulations. In addition, each individual country will have its own regulatory authority and rules for the interstate transportation of hazardous materials. For example, the US Department of Transportation (DOT) publishes and enforces Hazardous Materials Regulations (HMR) for the ground shipment and transportation of regulated materials on all public roads within the US (49 CFR Part 173). ²¹

Shipping regulations for biological and biologically derived materials are not based on risk groups of microorganisms or recommended biosafety levels. Professional judgment is required to properly identify and classify biological and biologically derived materials. A subject matter expert for the material needs to consider the source of the material and known hazardous characteristics of the material, derivative material, and/or its components as well as other materials to be included in the shipment (eg, fixatives, dry ice, liquid nitrogen, etc).

When hazardous characteristics are unknown but there is the potential for a biological or biologically derived material to contain an infectious agent or is in a form that may produce an infectious agent, it is advisable to ship in accordance with regulations for the infectious agent that may be present or produced in or by the material.

It is strongly recommended that biopharmaceutical manufacturer establishes in-house expertise on IATA and relevant member state hazardous materials regulations or work closely with a consultant to ensure compliance with state and international regulations.

Other Regulations Affecting International Transportation of Biological and Biologically Derived Materials

Geopolitical dynamics can change suddenly, and as a result, the designated status of countries and nongovernment organizations (NGOs), including private companies, can also change suddenly with respect to national security and trade. ⁵

Waste Management

Waste management can vary widely by organization and regulatory authorities so must be addressed as part of the risk assessment to ensure procedures that are implemented comply with applicable regulations. Decontamination strategies need to be established for liquid and solid waste management from lab-scale solutions (eg, bleach wipes, autoclave steam sterilization) to more scalable solutions (heat kill tanks, facility VHP). Care needs to be taken to ensure that decontamination strategies address both decontamination and minimum requirements established by regulatory agencies. It is important to note that disinfection (inactivation of the agent) is not the same as cleaning (physical removal of the agent).

Emergency Response

Upset conditions may not be predictable, and although not ideal, they cannot be avoided. Scenarios that should be considered include spills, uncontrolled releases, medical emergencies in controlled areas, and severe weather. To prepare and anticipate such events, emergency response plans and processes should be established commensurate with the risk profile associated with the manufacturing platform and aligned with the site EHS management policy. Such risks to the staff, facility, and the environment could be chemical, flammable, explosive, or biohazardous in nature, with the latter risk likely unique and specific to CGT processes due to the presence of human cells and virus. For commercial-scale manufacturing where closed-system practices are often employed in place of barrier systems, the failure of such systems, although infrequent and unexpected, can have a high impact on operator, facility, and the environment due to the use of biohazardous material, the high pressures associated with the processing of volumes, and possible lack of appropriate containment. The use of equipment with high-velocity moving parts increases the risk of physical injury to the operator. Therefore, it is necessary for the site to establish and institute emergency response plans under the guidance of the site biological biosafety officer and approval of the site IBC or equivalent site safety committee to account for all potential risk scenarios. Such guidance is defined in the Section III and Appendix K of the NIH guidelines for recombinant and synthetic nucleic acid molecules for different biosafety risk profiles. ⁶ For large-volume or commercial-scale biohazard response situations, where specialized training is required for cleanup and site decontamination, the site may want to consider outsourcing to a third-party contractor with HAZMAT capabilities. Alternatively, the site may consider establishing its own internal HAZMAT response team with members trained in Hazardous Waste Operations and Emergency Response (HAZWOPER) measures, as specified by OSHA regulations 1910.120 (29CFR 1910.120). ¹⁸ Such training may be offered to the manufacturing personnel as the first line of defense against the spill and initiators of the emergency response plan. In the case of physical injury or “person down” (eg, unconscious), the site may consider having Medical Emergency Response Trained (MERT) manufacturing staff or equivalent to provide appropriate decontamination and immediate medical attention to the injured person within the restricted access facility. It is advisable to work closely with local emergency services and authorities. In all cases, the emergency response plans must be adequately documented and maintained, supported by new employee and annual refresher training, and established as part of the site EHS management policy.

Risk Ranking

The combination of hazardous characteristics of the CGT, processes, facilities, and systems established to control and manage biohazard risk and containment need to be ranked for severity of impact and likelihood of occurrence to provide the overall risk score and to identify vulnerabilities that require risk mitigation.

Potent compound banding schemes are routinely used to categorize the hazards associated with drugs and therapeutic compounds. Most are based on traditional toxicology assessments of chemicals and small molecule drugs and are expressed as permissible exposure limit (PEL) or acceptable daily exposure (ADE) in units of milligram or microgram per kilogram of body weight. These evaluations do not easily apply to hazard assessments for CGT. First, there is no one-size-fits-all CGT banding or risk assessment scheme. The assessment process must be tailored to an organization and consider unique capabilities, processes, facilities, and engineering controls. Second, there is no permitted daily exposure limit for a biohazard.

Therefore, we recommend using the pathogen safety data sheets from Health Canada, ²² BMBL, ¹ agent summary statements, and current literature to help determine what anticipated exposure doses may be. Preclinical or clinical data may also be used, although these will not be available for the active or biohazard components of the process. We also recommend the inclusion of professional opinion based on the study design, indication of the drug, expected pharmacology, and spectrum of off-target effects. Effort must be made to account for the hazardous characteristics associated with routine exposure to CGT or its biohazardous components, such as protein/vector engineering, gene of interest, bioavailability of the therapeutic, occupational exposure risk versus clinical administration, individual human variability, and potential off-target effects. CGT risk assessments must consider the potential for expression in healthy human cells, potential to express in cell types other than those for which the therapeutic is designed, effect of expression in a healthy adult (and/or a specific risk population that could be represented in the workforce, eg, pregnant women or immunocompromised individuals), any special risk factors, and respiratory/aerosolization hazards.

Summary and Conclusions

Risks involved in CGT are not entirely novel in the biosafety community; however, clear guidance when considering commercial-scale CGT is lacking. Our intent of this article is to bring to the reader’s attention the complexity of commercial-scale manufacturing, areas to assess, potential questions to ask while assessing, and other parties who may have pertinent input to the overall risk assessment. We encourage groups who are just venturing into commercial-scale CGT manufacturing to involve a biosafety subject matter expert/biosafety officer in the planning phases and as part of the risk assessment process for the project. The risk assessment is a complex process that requires specific competencies that might vary from risk assessment to risk assessment. Unfortunately, there is not a one-size-fits-all approach when it comes to a risk assessment scheme because risk can vary based on infrastructure, scale, personal competencies, and so on. We hope this article offers a framework to the readers for assessing CGT commercial-scale projects.

Appendix

Appendix A. Risk Assessment Template

Due to variances both by industry and by country, this is only a general process designed to provide guidance. The template in Table A1 may form the basis for a starting discussion between a contract manufacturer and client or between production and development departments to ensure operators are not exposed to risky activities. The idea is to address the following questions when evaluating the process: What could go wrong? What controls are in place, and are they enough? What did we learn from this?

Table 1.

Risk Assessment Template

1.0 General Information
Date Conducted
Location and Description
Risk Assessment ID
Assessor (LEAD)
Other Participants
2.0 Background/Purpose
Description and summary of the equipment, task, process flow, room, scenario, in relation to the change that is occurring. Include details on Starting materials (cell banks, viral banks), Materials and Reagents, Process volumes, Operating monitoring systems and parameters (pressure, flow rates, temperature), Viral reduction steps or operations
3.0 Process Materials; Reagents, Cell lines, Vectors, Helper Viruses and Active Synthetic Nucleic Acid Constructs
Identify all the starting materials (i.e. cells, viral vectors, nucleic acid or synthetic DNA sequences, media) and reagents (i.e. serum, media, enzymes). Identify Drug Substance, Drug Product and all intermediate steps and hazardous components Consult local IBC or regional agencies to identify Risk Group designation
Volumes/ cell numbers reached, after how many passages?
Details of cells, vector (recombinant, defective or wildtype), serum, media and additives. Used separately or in combination? Any requirement for animal derived products?
Use of enzymes	□ Yes □ No
Use of nucleic acids/recombinant DNA	□ Yes □ No
Relevant import/export documentation obtained	□ Yes □ No
Impact upon Biosafety relevant to the scale of the process – vector copy number per transduced cell – vector integration profile; on and off-target modifications – vector replication competence; probability of reactivation – persistence of genome editing tools in the cells – expression controls (constitutive, cell specific promoters) – level of permissibility to human cells – natural mode of transmission
4.0 Process Operations and Control
Sketch out the process to be followed. Include details of individual unit operations, highlight potential vulnerabilities and areas of increased risk. Consider Up Stream Processes (USP), Down Stream Processes (DSP) and Fill Finish (FF). Consider the likely disinfection process requirements, and whether any steps are performed under pressure (flow chart could be substituted as well)
Upstream Processes
Downstream Processes
Fill Finish
Additional disinfection requirements
Steps performed under pressure
5.0 Other hazards
5.1 Chemical hazards (corrosive, flammable, toxic) Consider potential conflicts for chemical and biohazard protection and control. For example, the use of safety showers and drains when responding to a spillage whilst needed to maintain containment of any biohazard.
Corrosive
Flammable
Toxic
How/where will materials be stored
5.2 Physical hazards (e.g. heat, pinch) and Ergonomics (e.g. repetitive strain, heavy lifting) Physical hazards that should be assessed include lifting, hoisting, and transporting totes with large bags (>500 lbs.) of materials, as well as standard workplace safety (slips, trips, falls; falling objects, hazard communication, hazardous energy.
Physical (heat, pinch)
Ergonomics (repetitive strain, heavy lifting)
Slips, trips, falls, falling objects, hazard communication and hazardous energy
6.0 Facilities, Equipment and Utilities
Floor plan adequate for segregation, sanitization and operating space
Single Use Equipment– bioreactors, WAVE bags, filtration
Non-Single Use Equipment–Filtration columns, centrifuges
Break Points / Connections
Sharps
Restricted access necessary
7.0 Employee Gowning and PPE
The risk assessment must consider both clean room requirements and features required to provide barrier protection (e.g. chemical and moisture resistance)
PPE (skin and eye; safety glasses, disposable gowning, gloves, face shields, aprons, respirators)
8.0 Employee and Contractor Training
Adequate training plan in place (including correct use of Personal Protective Equipment, respirator fit and evaluation)
Refresher training scheduled
Medical surveillance in place
9.0 Site Systems and Policies Review all existing systems and policies for adequate coverage for the new process
Environmental monitoring
Facility cleaning
Site sanitization strategy (including liquid disinfectants and bleach wipes, steam sterilization, heat kill tanks and vapor phase hydrogen peroxide)
Segregation & Containment strategy (Consider flow of employee, materials, equipment and waste flow; PPE policy, air handling and room pressure designations, barrier systems and change over procedures)
Emergency spill response plan (Including first responder awareness and training)
Regular and Emergency Maintenance plans
10.0 Storage, Shipping and Distribution
Regulatory permits needed
How/where will materials be transported
How/where will materials be stored
Labelling and warnings
Temperature control
Containment solution
11.0 Waste Management
Adequate decontamination strategy
Strategy appropriate for scale of waste generated
12.0 Emergency Response
Accident and near miss reporting system adequate
Spillage kit available
Emergency contact information accessible for every employee
Relations established with local emergency responders and hospital
13.0 Controls Currently in Place
□ Elimination _______________________________________________________ □ Substitution _______________________________________________________ □ Engineering _______________________________________________________ □ Administrative _______________________________________________________ □ PPE _______________________________________________________
14.0 Data Collected In-House and/or Biological samples, electrical testing, space dimensions, tank volumes
15.0 Miscellaneous Information Follow-up required, comments, references
16.0 Corrective Action Items
Item	Corrective Action	Owner	Due Date	Tracking #
1
2
3

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.