A scalable adeno associated virus production process: From cell culture to purified bulk

Adeno-associated virus (AAV) vectors are increasingly being used in clinical trials for gene therapy applications. Manufacturing of safe and efficacious clinical-grade virus relies on a scalable and cost-effective production process. In this study we have combined experimental work and process economy calculations to develop an efficient and scalable process for AAV production. Each process step, from AAV production in suspension cell culture to purified bulk product up to the 10L scale, was evaluated and optimized. We used AAV2 for process development, since this is the most used AAV vector in addition to serotype 5, 8, 9, and various synthetic capsid variants. AAV2 expressing green fluorescent protein (GFP) was used for process development to facilitate analysis.

First, suspension culture of HEK 293T cells adapted to serum-free medium and triple plasmid transfection were optimized using a DoE strategy for transfection efficiency and AAV2 production. The conditions were further developed for production in a single use stirred tank bioreactor system. Novel affinity and ion exchange based purification step alternatives were compared, including different chromatography resins and membrane formats. Finally, ion exchange chromatography and core bead technology were evaluated as an alternative to size exclusion chromatography for the polishing step before the formulation into storage buffer. Analytical methods for virus titer are challenging and depend on purity and quality of the sample. For viral genome titer qPCR was used and for total virus titer ELISA and HPLC methods were used. For infectious virus titer, we used a flow cytometry-based assay counting GFP producing cells. Based on analytical data different downstream process alternatives were compared regarding load capacity, recovery, reduction of empty capsids, and purity. We propose a robust and scalable process with a favorable process economy.

Speaker:
Åsa Hagner Mcwhirter
Senior Scientist, Cytiva

Åsa is a Senior Scientist at GE Healthcare’s Life Sciences business, now Cytiva, in Uppsala, Sweden. She has been 17 years in R&D focusing on applications and product development within downstream purification and analytics. During the last 4 years she has been working with viral vectors. In her current role, she is a team leader for several research projects and customer collaborations. Previously, she worked with proteomics and fluorescent based protein analysis. Prior to joining GE Healthcare’s Life Sciences business, now Cytiva, Åsa did a PhD and a post-doc in Medical Biochemistry at Uppsala University within the field of Glycobiology.