Efficient and High-Resolution Separation of Partial, Empty, and Full AAV Capsids by Optimized Iodixanol Density Gradient Ultracentrifugation.

Recombinant adeno-associated virus (rAAV) vectors are essential for gene therapy, but their production generates empty and partial capsids as product-related impurities that can compromise efficacy and safety. While empty capsids have been partially characterized, the isolation and study of partial capsids remain hindered by a lack of robust separation methods. Here, we present an optimized iodixanol density gradient ultracentrifugation strategy enabling high-resolution separation and enrichment of empty, partial, and full AAV capsids. Key innovations include expanding the 40% iodixanol layer to create a broader intermediate-density zone and implementing controlled bottom-puncture fractionation. Applied to a rAAV2 preparation, this method enriched partial capsids from 18.9% in the starting material to approximately 94.9% in the target fraction (determined by AUC integration under the equal response assumption). For a rAAV2 variant with a low baseline of partial capsids (1.3%), over 20-fold enrichment was achieved, reaching 30.1% purity as determined by AUC integration under the equal response assumption. This approach provides partial-capsid-enriched fractions, enabling detailed characterization of partial capsids, supporting critical quality attribute assessment, and facilitating the development of safer, more effective rAAV-based therapeutics.