Downstream processing of AAV based gene therapy vectors

作者：Alois Jungbauer, Scott M. Wheelwright · 发表于：Separation and Purification Technology · 年份：2025 · DOI：10.1016/j.seppur.2025.133051 · 被引用次数：22 · 研究领域：Virus-based gene therapy research、Viral Infectious Diseases and Gene Expression in Insects、RNA Interference and Gene Delivery

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, a...