In recent years, we have seen rapid growth in the development of adeno-associated virus (AAV)-mediated gene therapy products that target fatal and highly debilitating diseases. Growth is evident in the investments attracted by academic startups; in the mergers and acquisitions driven by large pharmaceutical companies; and in the number of products entering clinical trials.
According to a report issued by the Alliance for Regenerative Medicine in 2019, 320 gene therapy trials were in Phase I/II development and 32 were in Phase III. During 2019, the report noted, three gene therapies were approved. In the United States, Zolgensma was approved for spinal muscular atrophy; in Europe, Zynteglo was approved for β-thalassemia; and in Japan, Collategene was approved for critical limb ischemia. Also, chimeric antigen receptor (CAR) T-cell therapies that had already been approved in the United States received additional approvals in Canada and Japan.
Many gene therapy products have been fast tracked through clinical development. Now what is apparent is the need to address manufacturing challenges such as speed and scale of operations.
Establishing manufacturing capabilities
We have moved beyond establishing “proof of principle” for gene therapy products. The current challenge is establishing the manufacturing capabilities to support development pipelines and scale production. These capabilities include the platforms that are used to produce gene therapy vectors.
While some large pharmaceutical companies are establishing their own production capabilities, many development companies are relying on contract development and manufacturing organizations (CDMOs) to provide both development and manufacturing services to generate material for clinical trials and potentially commercial activities. According to Masri et al. (Cell & Gene Therapy Insights 2009; 5(Suppl. 5): 949–970), annual vector requirements for AAV2 to treat Leber’s congenital amaurosis, AAV1 to treat lipoprotein lipase deficiency, AAV9 to treat spinal muscular atrophy, and AAV9 to treat Duchenne muscular dystrophy are estimated to be 1.20E13, 1.5E5, 6.00E17, and 5.00E20 genomic particles, respectively.
To date, AAV therapies have been targeted most often at diseases with relatively small patient populations, that is, populations smaller than 1,000. In the near future, it is likely that developers will start to target much larger patient populations, which in turn may require the support of larger clinical trials as well as larger in-market production.
AAV production approaches are still evolving (Figure). Although some operations are not ideal, such as 2D cell culture and ultracentrifugation, they have provided routes to supply vectors for both clinical and commercial production for early products.
The development of manufacturing processes for AAV vectors poses several difficulties. For example, AAV vectors are generated by cells that predominantly rely on transient production routes and offer relatively low yields (1E5 vectors/cell). These cells also produce many nonfunctional empty capsids that need to be separated from functional full capsids.
Another difficulty in AAV vector manufacturing, besides low productivity, is variable productivity. It can vary depending on the AAV serotypes or cargos that are needed for different clinical applications. Accommodating different serotypes or cargos can lead to >10-fold variations in productivity, wide ranges in the levels of empty capsids generated, and large differences in the amounts of product that accumulate in cells or in cell culture media.
More basic platforms, such as 2D cell culture and ultracentrifugation, can to a large degree accommodate processes that may result in different AAV vector types, but are unsuited to producing large quantities of vectors. Increasingly, drug developers/customers are counting on CDMOs to offer more scalable production approaches, such as suspension-based culture processes and chromatography-based purification processes. By retaining CDMOs, drug developers/customers hope to avoid significant process changes during clinical development, as well as potentially costly delays and risks.
This switch from basic to advanced manufacturing requires more development and optimization work if processes are to accommodate variable productivity levels and different AAV vector types.
The challenge of scalable operations
When AAV vector preparations are purified chromatographically, the development of scalable downstream processing operations can become difficult because product concentrations are low and contaminant levels are high, especially if high-dose therapies are being produced, or if full (infective) capsids need to be separated from empty (noninfective) capsids.
There is an increasing recognition that downstream processing operations can be simplified, and made more robust, by using approaches that focus on the recovery of AAV vectors from culture supernatant, rather than cell lysate. Using these approaches means accepting some loss in yield as well as the need to develop a more detailed knowledgebase of upstream processing operations. Drawing on such a knowledgebase can help manufacturers increase vector titers while controlling the quality of vectors produced in the cell culture process.
The potential range of options poses a challenge for CDMOs seeking to balance available resources while trying to meet customer needs and expectations about production platforms and scales.
Additionally, as with any biological production process, there is a need to recognize potential risks—specifically, risks of delays and failures related to process scale-up and manufacturing operations—and to adopt approaches that mitigate those risks. With batch costs (including consumables) potentially exceeding $500,000, risk mitigation is a significant issue for all customers, especially for small and mid-sized enterprises.
Learning from mAb development
In many ways, the challenges posed by AAV vector manufacturing resemble those posed by the production of monoclonal antibodies (mAbs). For mAb production, development groups have been able to establish generic process development, analytical, and manufacturing platforms that apply to multiple drug candidates. Stages are optimized using scale-down models of key process steps, both in upstream and downstream operations, and high-throughput assay systems are designed to accommodate combined access, allowing detailed studies to be performed. Consequently, critical process parameters can be defined early in process development, helping to de-risk the transition from laboratories to production facilities.
If these process improvements are to be emulated by CDMOs that produce AAV vectors, the CDMOs must begin by splitting AAV vector manufacturing’s key steps into modular activities. CDMOs need to identify which procedures can be applied to multiple vectors—including vectors of the same serotype, vectors with differing transgenes, and vectors of different serotypes. Also, CDMOs need to determine the level of optimization they will apply within each process stage. Finally, CDMOs need to develop robust high-throughput assays that are suitable for use with multiple vectors, and that are accurate enough to support these types of development approaches.
Looking to the future
The next few years will bring significant opportunities to engage in the production of AAV vectors and to participate in a rapidly expanding marketplace. However, to compete, manufacturers need to exercise wisdom when investing in processing capabilities and selecting production platforms. The relevant production processes include cell build, transfection, vector production, vector recovery, capture chromatography, purification of full vectors, and formulation.
The development of large-scale AAV production platforms presents many challenges to in-house manufacturing operations and CDMOs alike. It is clear, however, that manufacturers of AAV vectors can emulate the work achieved by manufacturers of mainstream biopharmaceutical products. Specifically, manufacturers of AAV vectors can, like manufacturers of mainstream biopharmaceutical products, establish the platforms they need—platforms that will allow AAV-mediated gene therapies to fulfill their promise as next-generation therapies that will meet the needs of patients.
Tony Hitchcock is technical director at Cobra Biologics.