A critical step in manufacturing of adeno-associated viral vectors (AAVs) is separation of full and empty capsids. The current consensus in the industry is that it is critical to enrich for full capsids to deliver safer and more efficacious drug products to patients. Anion-exchange chromatography is well established as a scalable method for full/empty separations. However, due to the poor peak resolution, especially at process-scale column loadings, anion exchange gradient methods are unlikely to be able to achieve >70% full capsids in cases of 5%-20% full capsids in the load material, as the low starting percent full truncates the yield and purity of the elution pools. To overcome this challenge, we propose a "two-pass" anion exchange method which is effective in maximally enriching for full capsids. The method consists of a "first-pass" elution using a series of microstep increases in conductivity to remove the bulk of the empty capsids, followed by a "second-pass" elution in which selected enriched fractions from the first pass are reloaded onto the chromatography column for a final, targeted enrichment. The two-pass AEX strategy has been tested on four serotypes (AAV1, AAV2, AAV8, and AAV9) and demonstrated at pilot and production scale.
Keywords: adeno-associated viral vector; anion-exchange chromatography; empty capsids; full capsids; manufacturing; purification.
© 2025 The Authors.