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

Authors

Shan Fang, Jun Wu, Yanfang Shen, Dandan Qi, Zhuoyu Ni, Fenghua Wang

Published in

Biotechnology journal. Volume 21. Issue 10. Pages e70319.

Abstract

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.

PMID:
42875965
Bibliographic data and abstract were imported from PubMed on 15 Oct 2026.

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