Displacement chromatography for enrichment of rAAV genome-containing capsids using weak organic acid

Separation of empty and genome-containing particles is a challenging requirement for recombinant adeno-associated virus gene therapy vectors. Strong anion exchange chromatography operated with a linear salt concentration gradient to achieve a high degree of resolution has emerged as the replacement for traditional density-gradient ultracentrifugation to improve efficiency, consistency, and scalability. Herein, an innovative mechanism of anion exchange chromatography using weak organic acid present in the load is shown to selectively displace empty capsids into the flowthrough, enabling an isocratic elution and eliminating operational challenges associated with linear gradients. This separation technique was applied to AAV2 and AAV8 serotypes using three chromatographic media (monoliths, resin, and membranes) and showed comparable genome-containing capsid enrichment levels to that of density gradient ultracentrifugation. Processes for both serotypes were successfully transferred from lab to production scale.