Downstream processing of AAV based gene therapy vectors
Alois Jungbauer, Scott M. Wheelwright
Abstract
Adeno-Associated Virus (AAV) purification remains a major challenge due to the presence of empty, overfilled, aberrantly assembled, and aggregated virions. The unfavorable empty-to-full ratio and the low viral titers necessitate purification methods with high resolution and capacity. AAV’s relatively large size (25 nm) requires chromatography materials with large pores to mitigate diffusion limitations, yet larger pores reduce binding capacity. Additionally, virus clearance strategies must be carefully designed when mammalian cells are used for virus production. Continuous ultracentrifugation presents a scalable solution for virus capture, concentration, and separation of different capsid populations, but its high infrastructure costs and limited availability hinder widespread adoption. Affinity chromatography using serotype-specific or agnostic ligands offers an alternative platform approach, enabling direct loading of clarified lysates with elution at low pH, leveraging AAV’s stability under acidic conditions. Ion exchange chromatography, while highly selective, remains difficult to standardize due to AAV’s sensitivity to electrolyte concentration, salt type, and pH, though its high resolution facilitates full-particle enrichment. However, the separation of partially filled and aberrantly filled particles remains an unmet challenge, underscoring the need for new high-capacity, high-resolution chromatography materials. To enhance AAV production efficiency and reduce costs, advancements in both upstream and downstream processing are necessary. Improved expression technologies should aim to increase titers and optimize the full-to-empty ratio, reducing purification burdens. Novel chromatography solutions, including high-resolution continuous separation techniques like Multi-Column Solvent Gradient Purification (MCSGP) and Simulated Moving Bed (SMB) chromatography, hold promise for industrial-scale purification. Optimized feedstock clarification strategies will also be critical in preventing column clogging and ensuring scalability. By addressing these challenges, the field can improve the cost-effectiveness, scalability, and efficiency of AAV production, ultimately enhancing access to gene therapies.