Enrichment of full AAV capsids by preparative strong anion exchange chromatography.

Abstract

Recombinant adeno-associated virus (rAAV) vectors are the leading in vivo gene delivery platform for the treatment of various human diseases. Scalable manufacturing of rAAV has been successfully demonstrated; however, the presence of non-genome containing empty AAV capsids still remains a significant downstream bottleneck. Separation of empty and full rAAV vectors with linear gradient anion exchange chromatography is challenging to implement at large scale and often achieves only a low recovery of full rAAV capsids. Here we present a workflow to separate empty from full rAAV capsids using Capto Q™ resin with isocratic elution as an alternative. The workflow is based on a preliminary conductivity screening that identifies an optimal empty capsid removal salt concentration, followed by an isocratic two-step elution method. This approach was successfully demonstrated with rAAV serotypes 8 and 9. Approximately 65% of full rAAV8 and rAAV9 capsids were recovered with an enrichment to greater than 80% and 90% full capsids, respectively. Process development using the same approach for rAAV6.2 proved to be more challenging and required a switch in elution salt and an increased concentration of MgCl₂. The optimized two-step purification protocol for AAV6.2 achieved the recovery of 68% of full capsids with a purity of greater than 80% full capsids.