Improved adeno‐associated virus empty and full capsid separation using weak partitioning multi‐column AEX chromatography

Abstract

Recombinant adeno‐associated virus (rAAV) empty and full capsid separation has been a topic of interest in the rAAV gene therapy community for many years and the anion exchange chromatography (AEX) step has undergone various process optimizations to improve rAAV empty capsid separation, including AEX stationary phase, mobile phase, and process parameters. Here, we present a new AEX method that employs both weak partitioning chromatography (WPC) and multi‐column chromatography (MCC) to achieve improved full rAAV percentage in the AEX pool. The WPC technology allows empty rAAV to be displaced by full rAAV during loading, while the MCC technology enables parallel column processing which further increases AEX step productivity. Our results show that, compared to baseline AEX batch chromatography, the AEX‐WPC‐MCC method demonstrated improvements in both AEX pool full rAAV percentage (∼ 20% increase) and rAAV genome recovery (∼ 20% increase). As a result, the productivity (full capsid generated per liter of AEX column per hour of processing time) of the AEX step increased by ∼34‐fold from the baseline AEX batch run to the AEX‐WPC‐MCC run. It is foreseeable that this AEX‐WPC‐MCC method could find applications in large‐scale rAAV manufacturing processes to improve AEX yield and reduce the cost of goods of rAAV manufacturing.

Removal of empty capsids from high‐dose adeno‐associated virus 9 gene therapies

“…Di et al recently reported weak partition chromatography of three parallel AEX columns in which rAAV8 full capsids displaced ECs (Di et al, 2023). Di et al's approach increased yield by 20% and increased full capsid levels by 20%, compared to baseline batch chromatography (Di et al, 2023). Our partition approach also leverages the tighter binding of full rAAV9…”

“…For instance, Joshi et al loaded rAAV9 onto POROS 50 HQ with no apparent flow-through, indicating complete binding of all capsid species (Joshi et al, 2021). Di et al recently reported weak partition chromatography of three parallel AEX columns in which rAAV8 full capsids displaced ECs (Di et al, 2023). Di et al's approach increased yield by 20% and increased full capsid levels by 20%, compared to baseline batch chromatography (Di et al, 2023).…”

Tuning capsid formation dynamics in recombinant adeno‐associated virus producing synthetic cell lines to enhance full particle productivity

“…[22] In laboratory scales, empty particles can be separated from full particles by ultracentrifugation exploiting their slightly different buoyant densities, but this method is not scalable. [23] Anion exchange chromatography has been successfully employed for largescale separation of empty particles from full particles, relying on the difference in isoelectric points between empty and full particles, [24,25] and recent studies have demonstrated improvement in separation of full and empty particles by using weak partitioning chromatography [26] and adjusting mobile phase properties. [27] An ideal new system for rAAV production should be robust, free of virus infection or plasmid transfection, with a high productivity and high full particle content.…”