AAV vectors are widely used in gene therapy, largely because they do not become integrated into the patient’s DNA and are safe from an immunogenicity perspective. AAVs also offer great versatility, with different targets possible using different serotypes. Relative to other vectors, AAVs are also generally produced at higher titers, which reduces costs. In addition, they present long-lasting effects and tend to be more stable than other vectors.
The market for AAV vectors is predicted to expand at approximately 20% annually over the next five years. In addition to more approvals of AAV-based gene therapies, this growth rate will likely only be maintained if innovations leading to improved titers continue. More efficient and effective production processes are needed to reduce the costs of gene therapy. For instance, a ten-fold improvement in titers could cut costs by at least 80%. Some advances have been made already, and we know from our experience with monoclonal antibodies (mAbs) that titers will likely continue to improve.
When gene therapies first emerged, each process was by necessity customized for each product and customer. Working in this manner is highly inefficient, because additional time is required to develop each process, obtain or manufacture the myriad unique raw materials and get them approved, train the operators, design and install the equipment, and every other step in the program. Lead times can be extensive, and present a risk of derailing the program.
By taking a platform approach, common sources of problems can be minimized, with the process development strategy, raw materials, and equipment all standardized. Raw materials other than the plasmid for the gene of interest are maintained in stock, the batch record has been drafted, and the manufacturing team is already familiar with the process when the program begins. Because design-of-experiment studies were completed and a wide range of process parameters and conditions have been evaluated in advance, it is possible to troubleshoot novel issues that arise faster, so process optimization and scale-up proceed smoothly and rapidly.
In order for CDMOs to successfully demonstrate their ability to execute AAV vector programs at the quality and efficiency levels required, they must have established AAV manufacturing platforms. Only with a platform approach is it possible to demonstrate to potential customers that the CDMO has the knowledge, and expertise required for efficient process development and manufacturing, as well as the flexibility and agility needed to minimize disruptions and reach the clinic efficiently.
Production of AAV vectors in adherent cell lines is difficult to scale — particularly in plasticware, but even in bioreactors — because adherent surfaces are needed to support the cells. It is easier to scale suspension cell culture processes, but suspension cell lines have been less productive.
At WuXi Advanced Therapies, we established an AAV adherent platform but over time realized that this technology was not sufficiently scalable to support our anticipated production levels/needs. The development of gene therapies for indications that are systemic or involve large parts of the body and/or the use of gene therapies in much larger patient populations, such as heart failure, driving the need to comprehensively scale AAV manufacturing.
We invested in the development of a suspension cell line and our AAV suspension platform was launched in January 2020. AAV 1.0 is a robust producer cell line with productivity comparable to conventional adherent cells. With the ability to readily scale processing using this suspension cell line, we will be better positioned to meet future customer needs.
Once we had a platform for manufacturing AAV, we knew there were opportunities to improve the new suspension cell line, the upstream process, and the downstream process, with the objective of further improving the titer and the infectivity rate.
By the end of 2020, WuXi Advanced Therapies will be launching AAV 2.0, an improved suspension cell line for AAV manufacture. The AAV 1.0 cell line was cloned, and the top clones presenting 2–3-fold improvements in titer were selected. In addition, we have further refined our upstream process. As a result, depending on the relevant serotype, we have been able to improve the titer from 3× to 20×.
There are some manufacturing solutions for improving the full-to-empty capsid ratio. Using ultracentrifugation rather than ion-exchange chromatography during down-stream processing generally provides a better ratio, but it is currently only feasible at smaller volumes. However, downstream purification only removes empty capsids, but what is really needed is a means to increase the number of full capsids produced upstream, optimizing the true yield rather than improving the separation of the empties.
Plasmids and HEK293 are not the only approach to AAV vector production. The use of insect (baculovirus) rather than human cells is attracting attention. The titers are better, the manufacturing process is simpler, and the times are shorter, which all lead to lower costs. However, the potency is often viewed to be lower, and thus support for this technology is split in the gene therapy community.
Completely nonviral methods for the introduction of genetic material into patient cells are also being explored, including electroporation and pressure. There is interest in moving away from viruses for both safety reasons and potential cost advantages. We are actively working with several different approaches, often in collaboration with customers.
We have found so far that these methods are less efficient, and most are not yet sufficiently advanced to displace viral vectors. It will be interesting to see, as AAV, lentivirus, and other vector technologies improve, whether these alternatives can advance as quickly.
In addition to reliably manufacturing AAVs — or any pharma product — CDMOs must also have the ability to develop process- and product-specific assays within the established timelines and perform those tests to support process development and product release. For AAVs, many CDMOs outsource analytical work to third parties, which can impact their responsiveness.
At WuXi Advanced Therapies, we conduct our testing in-house and have this capability integrated into our service offering. We have coined a new term for CDMOs that offer testing in conjunction with development and manufacturing support — CTDMO (contract testing, development, and manufacturing organization).
The testing capabilities are important, because it doesn’t benefit a customer to have a CDMO manufacture a product that they are ultimately unable to release in a timely manner. In an emerging market like gene therapy, with so many candidates in clinical trials, access to third-party testing services can be challenging. Lack of analytical development resources is a key constraint in this industry. CTDMOs with the ability to offer integrated analytical support will have a distinct advantage. However, there will be a limited number of organizations that can claim to be true CTDMOs, because it is very difficult and time-consuming to set up broad testing capabilities that include analytical, molecular biology, virology, and other laboratories staffed by experienced technicians.
Felix Hsu is Senior Vice President and Global Head at WuXi Advanced Therapies located in Philadelphia, PA. He has nearly 31 years of experience in the life sciences industry and serves as an Advisory Board Member for the Jefferson Institute for Bioprocessing. He has mainly held executive and senior positions at the following companies: WuXi AppTec, Medtronic and Abbott Laboratories. He has studied at the University of Michigan – Stephen M. Ross School of Business with a Masters in Business Administration and Management.