Guest Column | March 14, 2022

The 13-Step Guide For Conducting Cell & Gene Therapy EHS Risk Assessments

By BioPhorum

Expert NetworkOne of the many hurdles for cell and gene therapies (CGT) is the lack of health and safety guidelines for manufacturing CGT products. There is no clear framework for considering risks for operators when working on a commercial scale. The result is that there is no “universal” biosafety risk assessment template for CGT manufacturing processes.

Industry needs best practices and improved risk controls when setting up a commercial-scale CGT manufacturing line — from an environmental health and safety (EHS) perspective.

This article summarizes the main steps in a suggested EHS risk assessment process. The template can form the basis for discussions between a contract manufacturer and client or between production and development departments. The aim is to address the following questions when evaluating the CGT manufacturing process: What could go wrong? What controls are in place and are they enough? What did we learn from this?

The following example shows how the template can be completed. It includes sample responses that focus on cell expansion operations working with the cell line only in research and development (R&D) labs to develop procedures and controls (it does not include production operations, such as transfection or viral infections).

Step 1 – General Information

Record general information, such as the date the risk assessment was conducted, the location, the risk assessment ID, the assessor, and other participants (e.g., quality assurance).

Step 2 – Background/Purpose

Describe and summarize the equipment, task, process flow, room, and scenario about the change that is occurring. The change could include adopting a new process or a new material. 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.

Sample Risk Assessment Response:

Background – A reasonably well-characterized but not-tested-to-full-compliance human cell line (e.g., HEK293) has just been delivered to your department in the U.S. The source of this cell line for this assessment could be an American Type Culture Collection (ATCC) repository, a third-party project collaborator, or different department of your company (an in-house cell line).

Materials and reagents; equipment – This cell line will be grown in serum-free, chemically defined media; no material or reagent contains animal-derived components.

Process development and process volumes – This will include operations ranging from 1 mL up to a final volume of 100 L, requiring up to four weeks of cell expansion and culturing process.

Operating monitoring systems and parameters (pressure, flow rates, temperature) – Process development will follow established techniques to mitigate contamination of viral and microbial agents from the environment. Operations will not be performed under GMP compliance, so they cannot be considered fully effective (processes are taking place in an R&D facility).

Viral reduction steps – These are yet to be determined for this new cell line and are not in the scope of this project.

Step 3 – Process Materials

Identify all the starting materials (e.g., cells) and reagents (e.g., serum). Next, identify drug substance, drug product, and all intermediate steps and hazardous components. Finally, consult local or regional agencies to identify the risk group designation.

Sample Risk Assessment Response:

  • 1 mL of cells (1e7 total viable count, TVC) up to 100 L (~1e11 TVC) over six passages of expansion.
  • Partially tested human cell line (e.g., HEK293).
  • Testing upon arrival – cells are placed into a -60ºC (or lower) freezer for storage. No additional testing will be performed before use in R&D.
  • Testing during and at the end of the process – following procedures performed in R&D; there will be no additional testing of the material.

Step 4 – Process Operations And Control

Sketch out the process to be followed. Include details of individual unit operations, highlighting potential vulnerabilities and areas of increased risk. Consider upstream processes, downstream processes, and fill/finish. Consider likely disinfection process requirements and whether any steps are performed under pressure (a flow chart could be substituted as well).

Sample Risk Assessment Response

Upstream And Downstream Processes

  1. Cell thaw and shake flask expansions in single-use, sterile disposable supplies (<5 L) – Example risk: Potential contamination of cells from operators during “open” liquid transfers. Example remediation: Gowning and aseptic technique training.
  2. Cell expansion, perfusion, and sampling of single-use, sterile disposable bioreactor systems (<100 L) – Example risk: Potential breach of containment for large-volume operations performed under pressure. Example remediation: Written process procedures with safety limits defined.


Vials will be stored in an ultra-low-temperature (<-60oC) freezer. Use appropriate personal protective equipment. When storing several different cell lines in the same location, the risk of cross-contamination is known due to vials leaking or breaking. Processes are in place to reduce this risk.

Steps Performed Under Pressure

Steps involving perfusion have been considered, including while the cells are being cultured. Integrity testers are available to check for bag integrity issues, either inherent or due to damage during bag installation.

Step 5 – Other Hazards

5.1 Chemical Hazards

Assess potential conflicts for chemical and biohazard protection and control, e.g., the use of safety showers when responding to a spillage. Refer to the safety datasheets of all chemicals used and the risk assessment provided by ATCC of the cells. Assess if materials are corrosive, flammable, toxic, irritant/other, and how/where materials will be stored.

Sample Risk Assessment Response:

  • Flammable – isopropanol IPA.
  • Toxic – bleach or other disinfectant.
  • Irritant/other – bleach or other disinfectant.

5.2 Physical Hazards And Ergonomics

Assess physical hazards, including lifting, hoisting, and transporting totes with large bags (&GT500 lbs.) of materials, as well as standard workplace safety (e.g., slips, trips, and falls).

Sample Risk Assessment Response:

  • Pinch hazards are present (e.g., with pumps).
  • There will be a need to lift large volumes.
  • Repetitive pipetting and liquid transfers may be required.

Step 6 – Facilities, Equipment, And Utilities

Assess if the floor plan is adequate for segregation, sanitization, and operating space; single-use equipment (e.g., bioreactors); non-single-use equipment (e.g., filtration columns); break points/connections; and sharps.

Sample Risk Assessment Response:

  • The suite is designated to support all operations exceeding 10 L and complies with NIH guidelines for large-scale BL2 (LS-BL2) as per Appendix K.
  • Single-use disposables will be used in an aseptic system and then a closed system.
  • Potential weld failures, connector problems, and tubing swelling under pressure are documented, and response plans are in place.
  • No needles will be used in the process.

Step 7 – Employee Gowning And Personal Protective Equipment (PPE)

Assess cleanroom requirements and features to provide barrier protection (e.g., chemical resistance). For PPE, this should consider protection for skin and eyes, safety glasses, etc.

Sample Risk Assessment Response:

Different PPE is required for different stages:

  • Small-scale work in laminar flow hood, e.g., disposable splash-resistant lab coat.
  • Large-scale operations in a reactor under pressure, e.g., face shield required during pump and high-pressure liquid transfers. Correct use of PPE is important but should not be relied upon to resolve this risk level.

Step 8 – Employee And Contractor Training

Assess whether an adequate training plan is in place (including the correct use of PPE, respirator fit, and evaluation), if refresher training is scheduled, and whether medical surveillance is in place.

Sample Risk Assessment Response:

  • Gowning procedures will be defined for large-scale operations, applied to all operators, and approved before use.
  • Training is reviewed annually and corrected as needed.
  • Any employee showing symptoms of illness will not be allowed into the facility.

Step 9 – Site Systems And Policies

Assess if all existing systems and policies are adequate for the new process. This should assess environmental monitoring, facility cleaning, site sanitization strategy, segregation and containment strategy, emergency spill response plan, and regular/emergency maintenance plans.

Sample Risk Assessment Response:

  • Routine cleaning – no special facility cleaning for development operations.
  • Deep cleaning – performed at the end of each campaign or batch.
  • Liquid waste – treatment with 1:10 bleach for a minimum of 30 minutes before disposal.
  • Solid waste – will be double bagged as biohazardous waste.
  • Access to the site will only be granted to trained operators as approved by the area supervisor.
  • Written procedures will be approved for the emergency response plans before manufacturing.
  • All equipment will be verified suitable for use following the preventive maintenance plan.

Step 10 – Storage, Shipping, And Distribution

Assess if regulatory permits are needed, how/where materials will be transported, how/where materials will be stored, labeling and warnings, temperature control, and the containment solution.

Sample Risk Assessment Response:

  • Shipping inside U.S. – samples will be shipped to partners and will not require special permits.
  • Shipping to EU – ensure receiving party has necessary permits/licenses per local requirements.
  • Materials will ship to EU and U.S.; 50 x 1 mL cryovials maximum.
  • Materials will be stored on-site in ultra-low temperature freezers, labeled as a biohazard.
  • The pack out, labeling, and transportation will be performed by trained shipping personnel.
  • Temperature loggers will be used for cells shipped on dry ice.
  • Triple containment is required (primary = cryovials, secondary = box or bag, tertiary = shipper).

Step 11 – Waste Management

Assess if there is an adequate decontamination strategy and if the strategy is appropriate for the scale of waste generated.

Sample Risk Assessment Response:

  • All disinfectant procedures have been demonstrated to be effective against the organism used.
  • R&D environment – disinfect in-vessel with 10% working volume of bleach.

Step 12 – Emergency Response

Assess if the accident and near-miss reporting system is adequate, if a spillage kit is available, if emergency contact information is accessible for every employee, and if relations have been established with local emergency responders and hospitals.

Sample Risk Assessment Response:

  • An emergency response plan is in place and approved by site EHS/biosafety officer and compliant with local authorities.
  • Spillages are managed by trained personnel.
  • Medical emergencies follow local site procedures.
  • Annual drills are conducted with the local fire department and emergency medical technicians.

Step 13 – Controls Currently In Place

These may include elimination, substitution, engineering, administrative, and other controls. The controls may already be noted earlier in the document – in which case, reference the appropriate section.

Additional Considerations

You should also record other data collected (e.g., in-house and/or biological samples, electrical testing, space dimensions, tank volumes), miscellaneous information (e.g., follow-up required, comments, and references), and corrective action items (these should be numbered, have an owner, have a due date, and use a tracking number).


The risks associated with commercial-scale CGT manufacture are not yet fully known and industry is building a body of knowledge of the hazards to operators. Understandably, industry lacks experience in these novel therapies and there is also no standardized CGT process, so this framework brings a more strategic than specific approach to assessing risks.

Completing the template will help share experiences and learning and build consensus across industry, while covering as many CGT-specific processes as possible. As with other areas of CGT, general biologics processes and controls cannot just be reused. The approach discussed above gives users a framework to develop their experience using a model that can be adapted for their own CGT EHS processes. The framework will help EHS and biosafety professionals use a best practice approach to assessing risks around these novel therapies.

This article summarizes two recent BioPhorum publications on this topic. To read more, check out the EHS risk assessment template and the completed template for the risk assessment of cells used in early development, which is the first in a series of follow-up articles considering real-world scenarios.