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.
In all steps of the process, Gibco™ CTS™ Viral Production Cells
2.0 (VPC 2.0) were maintained in Gibco™ CTS™ Viral Production
Medium containing 4 mM Gibco™ GlutaMAX™ Supplement
(Table 1). The cells were thawed and maintained in shake flasks
as recommended in the AAV-MAX system user guide [2] before
inoculating each of the bioreactors at a low turndown ratio to
further expand the cells prior to each production run.
