High‐throughput development and scale‐up of recombinant adeno‐associated virus 5 anion‐exchange chromatography for high‐purity capsid separation

Abstract 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 (MgCl 2 /MgSO 4 ) 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.