The gene therapy sector is continuing to come into its own, and many developers are shifting their attention from nearly exclusively treating rare diseases to developing therapeutics for prevalent indications. An interesting dynamic of the gene therapy market is that gene therapies are generally designed to be curative. Therefore, the pressure to be the first to market is significant because, theoretically, even opportunities for fast-follower products might be limited.
However, to move effectively into higher-population disease states and expand access to a wide cross-section of patients, it is vital to meet volume demands and lower manufacturing costs while maintaining speed to market.
For gene therapies to reach their potential, drastically improving the scalability and cost-effectiveness of the adeno-associated virus (AAV) vector production process is paramount.
Why Are AAV Viral Vectors So Important?
Delivering a modified gene to a patient’s target cells requires passing numerous barriers within the extracellular and intracellular environments. However, nucleic acids are extremely vulnerable in their naked form. Specifically, other components within biological fluids can damage the integrity of naked genetic material.
Additionally, naked genetic material can cause undesirable immune responses, resulting in potential deactivation, elimination by the patient’s internal systems, and failure to perform intended therapeutic action. Therefore, gene therapies require a mechanism to deliver the modified genetic material safely and effectively within the target cells.
Nonviral delivery systems such as electroporation, magnetofection, and systems based on cationic polymers offer considerable promise and, if optimized, would allow developers to avoid the challenges of viral vector production. However, nearly all gene therapies today use one of three vector types: AAV, adenovirus, or lentivirus. Because AAV exhibits a strong safety profile, is capable of long gene expression windows, and can infect both dividing and nondividing cells, it has emerged as the leading vector in gene therapy delivery.1
The Criticality and Challenges of Improving AAV Viral Vector Production
AAV manufacturing, which is both cost-effective and capable of meeting volume demands, remains a considerable challenge. However, given its status as the vector of choice in today’s gene therapy market, drastically improving the availability and affordability of AAV vectors is paramount to extending gene therapies to more patients, increasing the range of conditions that can be addressed, and reducing the cost of goods to improve affordability.
Producing AAV vectors at scale presents numerous challenges,2 including the following:
- Nearly all AAV vectors are produced using transient transfection processes that offer the benefits of rapid program start-up and flexibility; however, they face the drawbacks of inherent scalability challenges and high cost of goods. AAV vector transient transfection requires the formation of transfection reagent–plasmid complexes that are unstable and shear-sensitive, creating mixing and transfer challenges—issues that are often central to hurdles presented in scaling processes.
- Standardizing manufacturing processes despite variability from one viral vector to
- Improving purification processes, including the particularly challenging empty/full capsid separation step.
However, significant progress can be made by focusing on the increased standardization and improvement of AAV vector chemistry, manufacturing, and control (CMC) methods.
Selecting the Right Production System
The choices among upstream production systems currently present a trade-off between the ability to rapidly start a program, flexibility, scalability, and quality. These criteria are all essential at the beginning of the development journey and are needed to achieve an approved, commercialized product as soon as possible.
Because no one AAV vector platform currently delivers on all criteria, developers need to make decisions early in the development process based on the size of the potential patient population (how much drug product will be required), their understanding of product characteristics, and market forces including the competitive landscape.
The four primary AAV vector manufacturing platform options gene therapy developers currently have at their disposal include the following:
- Transient transfection systems using HEK293 cells, which use plasmids in either adherent- or suspension-culture formats
- Producer cell lines involving stable transfection of HeLa cells with target and viral genes
- Baculovirus expression vector systems using Spodoptera frugiperda(Sf9) cells
- Herpes simplex virus (HSV) systems
As mentioned, there are pros and cons to each platform.3 For example, transient transfection systems using HEK293 cells offer the benefits of quick program start times and considerable flexibility. However, raw materials costs are high, and this production platform, particularly when adherent cell culture formats are used, presents decided scalability challenges.
A producer cell line manufacturing platform offers product quality, scalability, and cost efficiency at scale. However, program start-up takes considerably longer than transient transfection, which increases early-phase development costs and can present time-to-market concerns.
Baculovirus expression systems offer scalability and cost efficiencies at scale. However, baculovirus is generally characterized by lower infectivity, requiring higher dosing to deliver the needed efficacy.
HSV systems are newer possibilities and present considerable promise. They are natural residents of the peripheral nervous system and can be transformed into multiple transgenic cassettes, given their large genome. Interestingly, HSVs have mechanisms allowing them to remain in the latency phase for long periods without damaging host cells; therefore, they serve as excellent candidates for gene delivery to the nervous system.4
Each platform has advantages and disadvantages, but primary considerations weigh the trade-offs among the ability to rapidly and flexibly start programs, product volume needs, and the cost of goods.
Coordinate Analytical Method and Process Development
Optimizing upstream and downstream processes for producing AAV vectors with consistent quality relies on the development team’s ability to characterize accurately and quickly determine critical quality attributes (CQAs). Analytical method development and process development must proceed hand in hand to achieve this state.
For AAV vector production, virus titer, capsid content, and aggregation are generally identified as the CQAs most likely to affect a gene therapy’s potency, purity, and safety. However, analytical methods to assess these or other CQAs can challenge process development efforts if turnaround time frames are long or throughput is low.5
While rapid, high-throughput AAV vector analytical methods are beginning to be commercialized, turnaround times and throughput continue to pose challenges.
Ultimately, developing effective manufacturing platforms requires quick and accurate identification and understanding of CQAs and the functional relationship between CQAs and their process parameters.
Because a CQA is an attribute that corresponds to the product’s identity, potency, purity, or safety—with severe impacts on product quality and efficacy if outside its target range—it is crucial to understand efficiently and accurately these attributes and the resulting implications to changes in process parameters.
Optimizing Downstream Processes
As tends to be the case in biologics production, optimizing the control of AAV upstream processes sets the stage for increasing the productivity of downstream processes. For AAV vector processing, one of the crucial goals of upstream process development is achieving a high viral titer and a sufficient and predictable percentage of full capsids. However, the typical trade-off is that maximizing full-capsid purity often comes at the expense of yield and the possibility of risking volume objectives.
Another challenge is consistently removing cell debris in processes where different pretreatments can be utilized. Ultimately, the goal is to optimize chromatographic options to remove impurities efficiently while recovering as much AAV as possible. Leveraging well-designed experimentation and quality by design (QbD) methodology are the tools gene therapy development teams must utilize.
As an increasing number of gene therapy organizations shift focus from first launches in rare diseases to developing therapeutics for non-rare indications, making important manufacturing decisions early in the development lifecycle to support the goals of the commercial program becomes critical.
To this end, ongoing CMC methods and standardization work, including selecting optimal production systems, integrating process and analytical method development, and improving the efficiency of downstream processes, must be significant focuses of the gene therapy sector at large.
Additionally, while the FDA and other leading global regulatory bodies continue to develop new guidances to clarify expectations, collaboration with the industry must continue because a significant lack of standardization and clarity remains.
BioTechLogic has supported over 53 gene and 12 cell therapy programs, offering specific expertise guiding CMC programs. If we can help support your program, don’t hesitate to contact us.
- “Scaling AAV Production: Easing the Transition from Laboratory Scales to Commercial Manufacturing,” Bioprocessing International
- “Manufacturing an Abundance of AAV Vectors,” GEN
- “Viral-vector therapies at scale: Today’s challenges and future opportunities,” McKinsey & Company
- “Analytical Methods for Process and Product Characterization of Recombinant Adeno-Associated Virus-based Gene Therapies,” Molecular Therapy—Methods & Clinical Development
- “Recent advances in gene therapy: genetic bullets to the root of the problem,” Clinical and Experimental Medicine