Production and purification of adeno-associated virus (AAV) at the 50 L scale

To build upon previous work demonstrating the scalability of the Gibco™ CTS™ AAV-MAX Helper-Free AAV Production System to produce AAV6-GFP in the Thermo Scientific™ DynaDrive™ Single-Use Bioreactor (S.U.B.)[1], here we provide data on the production, purification, and characterization of an AAV6 anti-CD19 chimeric antigen receptor (AAV6-CD19CAR) in the DynaDrive S.U.B. at the 50 L scale. Two DynaDrive S.U.B.s were run in parallel with shake flask controls to demonstrate scalability of the process using a non-GFP gene of interest. Furthermore, we provide guidance for a scalable downstream process for purification of the 50 L AAV6-CD19CAR. This purification strategy utilizes depth filters for supernatant clarification and tangential flow filtration (TFF) for concentration and buffer exchange prior to loading of the AAV onto the Thermo Scientific™ POROS™ CaptureSelect™ AAVX Affinity Resin. Utilizing this process, we obtained and purified a biologically active AAV6-CD19CAR, demonstrating a feasible and further scalable end-to-end workflow.