Purification of AAV8 through a scalable two-step monolithic chromatography approach.

Adeno-associated viruses (AAV) are becoming increasingly popular as a powerful tool for gene delivery therapy applications. Although processes to produce AAV are established, future demand for this type of viral vector requires further development of manufacturing processes to make them more robust, scalable, and flexible to accommodate the rise of engineered capsids. This study focuses on designing and evaluating a two-step chromatography process for capturing and polishing AAV8 using monolith chromatography media. A cation-exchange-based capture step was established, using CIMmultus® SO3, resulting in a virus recovery of approximately 70 % of total capsids. This step also achieved high protein removal (>95 %) and considerable DNA clearance (>80 %). For the polishing step, three different CIMmultus® monoliths were evaluated: anion-exchange-based QA (strong anion exchange), multimodal-based PrimaS (weak anion exchange and hydrogen bonding) and multimodal-based PrimaT (weak anion exchange, hydrogen bonding and metal affinity) ligands. High viral vector genome recoveries, DNA and protein clearance and a three-fold full capsid enrichment were observed at a 1 mL column scale. Similar results were obtained during a scale-up to 8 mL and 4 mL monolith volumes for capture and polishing, respectively. These results provide a strong and robust alternative to conventional AAV purification methods, such as resin-based affinity chromatography and density gradient ultracentrifugation.