Development of a scalable and robust AEX method for enriched rAAV preparations in genome-containing VCs of serotypes 5, 6, 8, and 9

Pranav R H Joshi; Alice Bernier; Pablo D Moço; Joseph Schrag; Parminder S Chahal; Amine Kamen

doi:10.1016/j.omtm.2021.03.016

Development of a scalable and robust AEX method for enriched rAAV preparations in genome-containing VCs of serotypes 5, 6, 8, and 9

Mol Ther Methods Clin Dev. 2021 Mar 23:21:341-356. doi: 10.1016/j.omtm.2021.03.016. eCollection 2021 Jun 11.

Authors

Pranav R H Joshi¹, Alice Bernier¹, Pablo D Moço¹, Joseph Schrag², Parminder S Chahal², Amine Kamen¹

Affiliations

¹ Viral Vectors and Vaccine Bioprocessing Group, Department of Bioengineering, McGill University, Montreal, QC, Canada.
² Human Health Therapeutics, National Research Council of Canada, Montreal, QC, Canada.

Abstract

Removal of empty capsids from adeno-associated virus (AAV) manufacturing lots remains a critical step in the downstream processing of AAV clinical-grade batches. Because of similar physico-chemical characteristics, the AAV capsid populations totally lacking or containing partial viral DNA are difficult to separate from the desired vector capsid populations. Based on minute differences in density, ultracentrifugation remains the most effective separation method and has been extensively used at small scale but has limitations associated with availabilities and operational complexities in large-scale processing. In this paper, we report a scalable, robust, and versatile anion-exchange chromatography (AEX) method for removing empty capsids and subsequent enrichment of vectors of AAV serotypes 5, 6, 8, and 9. On average, AEX resulted in about 9-fold enrichment of AAV5 in a single step containing 80% ± 5% genome-containing vector capsids, as verified and quantified by analytical ultracentrifugation. The optimized process was further validated using AAV6, AAV8, and AAV9, resulting in over 90% vector enrichment. The AEX process showed comparable results not only for vectors with different transgenes of different sizes but also for AEX runs under different geometries of chromatographic media. The herein-reported sulfate-salt-based AEX process can be adapted to different AAV serotypes by appropriately adjusting elution conditions to achieve enriched vector preparations.

Keywords: AAV serotype-5, 6, 8, 9; AAV vectors; anion-exchange chromatography; downstream processing; gene therapy; scale-up; separation of genome containing/empty AAV capsids.