Adeno-associated virus (AAV) vectors are increasingly used in clinical trials for the evaluation of gene therapy applications. The manufacture of safe and efficacious clinical-grade viruses relies on a scalable and cost-effective production process. In this study, we developed a method for AAV production in suspension cell culture to purified bulk products up to the 10 L scale. Each process step — from harvest, clarification, concentration and buffer exchange, affinity capture, and polishing — was evaluated and optimized. We used the rAAV serotype 5 expressing the green fluorescent protein (GFP) as our model for process development since this serotype is one of the most used. The purification process included an affinity capture step with Capto™ AVB chromatography resin and further anion exchange-based polishing to enrich full capsids. Finally, concentration and buffer exchange into storage buffer and sterile filtration was performed.<br/><br/>Analytical methods for determining virus titer are challenging and depend on the purity and quality of the sample. For viral genome titer, we used qPCR. For total virus titer, ELISA and HPLC were used to characterize the purified AAV. We also developed a reproducible and easy-to-use SPR assay for total virus titer. For infectious virus titer, we used a flow cytometry-based assay for counting GFP-producing cells.<br/><br/>Based on the data, different downstream step conditions were compared to give load capacity, recovery, reduction of empty capsids, and purity. From these findings, we propose a scalable purification process, which is GMP-compliant.

Development of a scalable Adeno-associated virus purification process

ACS Spring 2022

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