Author: F. Michael Haller, PhD, Principal Scientist, R&D
Abstract:
One major obstacle to achieving shortened development times and reduced costs of adeno-associated gene therapy products is the lack of standardized and cGMP-compliant downstream purification platforms. Besides providing high yields and purities, such platforms should be applicable to a wide variety of AAV serotypes as well as being customizable with minimal process modifications to allow rapid development of scalable downstream purification processes for clinical testing. Previously, we developed a comprehensive AAV production platform consisting of a proprietary high-producer suspension HEK293 cell line, a high-performing packaging plasmid system and an optimized triple transfection-based upstream process. In this study, we developed a streamlined downstream purification process using a GFP-encoding AAV9 construct (AAV9-GFP) as model. The applicability of the process was subsequently verified for several other AAV serotype/GOI combinations. For AAV9-GFP, the total process recovery was up to 38 %, starting from unclarified cell lysate at 3 L bioreactor scale, with post lysis titers of ~1.0 E+15 vg per liter cell culture by ddPCR titer assay. After affinity capture, circa 60 % full capsid content were obtained, which could be further enriched to up to 74-90% full, based on AUC capsid only quantification and/or SEC-MALS or mass photometric analyses. For an AAV9 model construct encoding a therapeutically relevant protein (AAV9-GOI1), a post-lysis intact genomic titer by 2D ddPCR of 4.1E+14 vg per liter production scale was achieved. After affinity capture, a Full:Empty ratio of 1.1 was obtained which increased to 11.5 for the BDS, with the final product containing 86 % full and less than 8 % empty capsid (AUC capsid only quantification). Starting from crude cell lysate, an overall downstream process yield of 33 % was obtained, while residual host cell DNA was reduced to below 2.5 ng/E+12 vg and HCP levels below the assay’s lower limit of quantification. In conclusion, we developed a streamlined AAV downstream purification process with high total recovery and full% capsid content of the final product at high levels of purity.