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.