High-throughput development and scale-up of recombinant adeno-associated virus 5 anion-exchange chromatography for...

Recombinant adeno-associated virus (rAAV) production commonly yields a high proportion of empty and partially filled capsids, necessitating downstream purification to enrich full capsids. Among purification approaches, anion-exchange chromatography (AEX) has emerged as a scalable alternative to ultracentrifugation. In this study, a structured and scalable development strategy for AAV5 AEX purification is presented. A high-throughput, ultra-scale-down platform using 200 μL RoboColumns™ was established to enable rapid screening of salt systems, additives, and operating conditions with minimal material requirements. Dual-salt systems were evaluated to decouple ionic strength from ion-specific effects, leading to the identification of a magnesium-only system (MgCl₂/MgSO₄) that improved both enrichment and recovery. Optimized conditions achieved >85% full capsids with robust recovery and >90% full capsids in peak fractions. Scalability was demonstrated through strong agreement between ultra scale-down (200 μL) and scale-down (1 mL) formats. A QbD-aligned framework was applied to define the purity-recovery design space and guide selection of operating conditions based on target product profile. The optimized gradient process was translated into a manufacturing-relevant isocratic step format, with systematic evaluation of buffer conductivity sensitivity and load robustness to address large-scale operational constraints.