Impact of Adeno-Associated Virus Production on Capsid Heterogeneity and Product Quality.

Adeno-associated virus (AAV) capsids are inherently heterogeneous, arising from viral protein stoichiometric assembly, post-translational modifications, genome packaging, and manufacturing process conditions. Understanding how AAV manufacturing processes affect capsid heterogeneity and corresponding drug product quality impact is essential for AAV-based gene therapies. We investigated the effects of extended production duration and capsid localization at harvest on capsid heterogeneity. Analytical characterization revealed that prolonged production increased extracellular AAV concentrations but also elevated the proportion of empty capsids, overall hydrophobicity, and deamidation of viral protein (VP). These changes altered charge-dependent separation during anion exchange chromatography (AEX). Capsids derived from intracellular fractions showed even higher levels of deamidation and empty particles compared to extracellular populations, further impacting AEX purification performance. Collectively, these findings demonstrate that both production time and capsid localization significantly influence AAV purification process performance and critical quality attributes. Process control strategies to minimize capsid heterogeneity are consequently critical to ensure a consistent quality profile in AAV manufacturing.