(93b) Continuous Processing of Viral Gene Therapy Vectors

As gene therapy clinical trials and FDA approvals accelerate at a breath-taking speed, the ability to manufacture these lifesaving drugs is lagging. The cost to manufacture AAV is high due to large amount of GMP DNA that is needed in the bioreactor. But continuous manufacturing can also reduce the cost at process scales above 0.5 x 10¹⁹ vg/year [1]. As more and more viral gene therapies continue to come to market, a more scalable process is needed. And economically, it likely needs to be continuous.

Upstream production of viral gene therapy products can be done with a perfusion bioreactor. We are developing a multi-reactor system to continuously produce and harvest AAV from HEK cells. The focus of the work has been on the downstream purification. Our process involves a single-pass tangential flow filtration (SPTFF) for concentration, two aqueous two-phase system (ATPS) units, a flow-through polishing filter, and a final SPTFF. We have focused heavily on the ATPS units, which use high PEG and salt concentrations to create two phases for purification and extraction. The first ATPS is a purification step and the second is to recover the viral product from the viscous polymer phase. The mixing and settling of these systems have been challenging for our continuous runs. Methods to improve and monitor each unit, including optimizing mixing, tubing for settling, and turbidity to measure mixing are some of our system improvements. We can recover 66% of the infectious titer of a model non-enveloped porcine parvovirus with 91% removal of protein impurities and 94% host cell DNA removal with only the ATPE steps. Additional protein and DNA removal occurs with the flow through AEX filter. We have preliminary data that the ATPE steps can recover 68% of AAV viral genomes, 74% of herpes simplex virus infectious titer, and 100% of lentivirus viral genomes while maintaining transduction ability. Economically, we have determined that the downstream purification system will save 50% on production costs and up to 90% on capital costs.

This continuous process would increase the equity and availability of viral gene therapy vectors by lowering production costs. There is not currently a continuous process for viral vectors and this process has the potential to not only continuous produce viral vectors, but to also be a platform for viral gene therapy and vaccine vectors.