Designing Viral Vector Facilities: Key Issues to Consider

Unlike small-molecule or monoclonal antibody manufacturing, where decades of experience have produced well-established facility templates, gene therapy manufacturing continues to evolve. Processes change, as do manufacturers’ scaling requirements. New modalities—gene editing and combination therapies—add layers of infrastructure that other facility designs do not typically have to consider.

This article is a practical guide to the decisions that matter most when designing a viral vector manufacturing facility, based on some recent project experience across multiple products and scales.

Start with the Vector

The biggest decision in viral vector facility design is also the most basic: What type of viral vector will be manufactured? The answer to that question dictates crucial decisions, including space requirements and utility infrastructure.

By some estimates, AAV accounts for as much as 80 percent of viral vector applications and supports in vivo gene therapy at scales up to 2,000 liters.2 Lentiviral vectors (LVV) and retroviral vectors, used primarily in ex vivo cell therapy, represent roughly 10 percent of the market, and operate at a much smaller scale, typically topping out at 500-liter single-use bioreactors.

The design implications for these two market-leading applications are significant. A facility built around LVV manufacturing will have a fraction of the bioreactor footprint, utility load, and space requirements of one built around large-scale AAV production.

When AAV and LVV processes coexist in the same facility, designers have to address questions of scale, equipment compatibility, and process segregation at the same time, and before the facility footprint is committed.

Room Classification and Good Manufacturing Practice (GMP) Flows

Viral vector facilities typically operate at the Centers for Disease Control’s Biosafety Level 2 (BSL-2, indicating low mortality risk). While BSL-2 classifications may be unfamiliar, it’s important to keep in mind that in the context of biosafety, viral vectors are modified to retain only their delivery mechanism; pathogenic components are removed.

When designing a gene therapy facility, it is important to maintain strict cleanliness levels within suites. Upstream bioreactor and downstream purification spaces are typically classified as Grade C environments, with substantial gowning requirements and operator training. Fill-finish areas are subject to aseptic processing requirements.

Due to the risk of cross-contamination with viral vectors, gene therapy facilities are designed with unidirectional flows for all suites where product is processed. Suites where products are not processed (such as buffer prep, media, prep, etc.) can utilize bidirectional flows.

The Challenges of Scaling Up Production

One of the most common miscalculations in early facility planning lies in bioreactor scale-up. Passaging from a 50-liter seed bioreactor to a 2,000-liter production bioreactor typically requires a staged series of intermediate volumes. Several considerations need to be taken, including physical dimensions, high-hat needs, and utility requirements.

In viral vector manufacturing facilities with unidirectional flows, upstream and downstream suites may require operators to exit, de-gown, and re-gown when transitioning between upstream and downstream processing suites. This impact moves beyond manufacturing personnel, affecting maintenance personnel as well. As a result, facility design must carefully consider designated maintenance routes through corridors and access controls to clean suites.

The Truth about Flexible Suites—It’s a Utility Problem

A recurring request in viral vector projects is suite flexibility. Some manufacturers want the capability to run upstream processing in a suite that can also accommodate downstream equipment, or to interchange equipment between two suites depending on the product being manufactured. That type of requirement makes sense in the manufacturing world due to cadence differences between upstream and downstream manufacturing. After all, gene therapy programs and new business decisions can change direction faster than facilities can be rebuilt.

The challenge, however, is that flexibility is not generally a space problem. It is a utility problem. A suite configured for upstream bioreactors requires compressed air, oxygen, carbon dioxide, and nitrogen. Downstream chromatography and ultrafiltration equipment only require compressed air. If a facility is designed to allow upstream equipment to move into a downstream suite, that suite must be supplied with all utilities, and those utilities must be accessible at every point in the room where equipment might possibly be positioned.

Utility distribution and layout must be designed for the full range of intended configurations.

Biowaste Management: Common Challenges Facilities Face

Biowaste inactivation is one of the most critical infrastructure decisions in viral vector facility design. The fundamental requirement is clear: host cells and viral vectors cannot be released to municipal wastewater systems without inactivation by either thermal or chemical means.

Thermal inactivation uses elevated temperature to render any viable biological agents inactivated before discharge. Chemical inactivation, though less common, uses a validated biocidal agent, typically sodium hydroxide, to achieve the same result. Both require dedicated tanks, validated processes, and thorough documentation.

As an alternative, chemical inactivation can occur in suites, typically via bleach or sodium hydroxide, prior to sendoff to the pH neutralization system. With this approach, several considerations arise, including specifying waste piping to ensure compatibility with corrosive agents, operational complexity regarding added training for inactivating waste, and properly validated inactivation techniques to ensure regulatory compliance.

Municipal and state codes governing liquid waste discharge vary considerably across jurisdictions. Involving local authorities and building applicable state and municipal code requirements into the design is essential.

Releasing Potential: Modern Approaches to Cell Lysis

Cell lysis is a critical step in AAV gene therapy as it allows the viral vectors to be released from the cell prior to downstream processing. There are three main options for cell lysis:

• Freeze-thaw cycles
• Mechanical lysis via microfluidizer
• Chemical lysis via detergent

Freeze-thaw cycles are a valid option at research scales, but the technology hasn’t been scaled for successful implementation at production scales in cGMP environments.

Chemical lysis is the most prevalent route of cell lysis in the industry. The most commonly used detergent for cell lysis has historically been Triton X-100; however, the European Union has imposed a ban on its use, and its future usage in other countries is not certain.

Mechanical lysis passes cells through a microfluidizer at high pressures to disrupt the cell membrane. While a scalable option, they are often associated with higher levels of product loss due to shear-induced aggregation and precipitation.3

Though the ban of Triton-100 may make mechanical lysis seem a more attractive option, it is not without challenges. The inclusion of a microfluidizer requires clean-in-place or steam-in-place capabilities, which are not usually found in viral vector gene therapy facilities due to the primary usage of single-use equipment. Also, in a facility that produces multiple gene therapies via various viral vectors, ineffective cleaning of the microfluidizer can lead to cross-contamination of products. Alternatives to Triton X-100, such as Tween 20 and 80, NP-40, and sodium dodecyl sulfate, are being considered.

Water for Injection (WFI): Make or Buy?

WFI generation and distribution are commonplace in most pharmaceutical manufacturing facilities. However, if single-use equipment is being used throughout the process, which is common in gene therapy facilities, dedicated WFI generation and distribution equipment may not be a requirement based on economics and scale.

There is one caveat to this. If an in-house GMP autoclave is used, pure steam is required for effective sanitization. If the facility does not have pure steam, an onboard pure steam generator needs to be installed. Pure steam generators require WFI-quality water, and the lack of in-house WFI generation can prove challenging.

Conclusion

Designing a facility that safely accommodates biological classifications in a coherent operational flow is among the most demanding challenges in current gene therapy facility design.

Gene therapy manufacturing does not yet have the established design playbook of more mature modalities. The decisions made regarding vector type, scale requirements, suite flexibility, and waste management, dictate how subsequent decisions fit into the overall design. Changing those decisions late in the game can be an expensive endeavor.

Gene therapy programs are often scientifically ahead of their manufacturing planning, and the information needed to design a successful facility must sometimes be developed in parallel with a manufacturer’s business strategy.

About the Author

Kimberly Leyzerzon is a Process Engineer II at Arcadis. She is starting her career in the cell and gene therapy space. Kimberly holds a bachelor’s degree in chemical engineering and a master’s degree in biomedicine and cell and gene therapy.