Adenovirus associated virus (AAV) vectors are increasingly used in clinical trials, evaluated 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, from AAV production in suspension cell culture to purified bulk product up to 10L scale. An efficient and scalable process for AAV production was developed by evaluation and optimization of each process step. 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 the 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 vial genome titer qPCR was used and for total virus titer ELISA and HPLC methods were used. For infectious virus titer, we have 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 and we propose a robust and scalable process with a favorable process economy.<br/>

Scalable adeno associated virus production process, from cell culture to purified bulk

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.