Analyzing full & empty AAV capsid ratio in less than 5 minutes

Cell & Gene Therapy Insights 2023; 9(5), 549–559


Published: 6 July 2023
Innovator Insight

In gene therapy, there is a need for quick, robust, and cost-efficient full and empty capsid analytical methods during adeno-associated virus (AAV) process development. The gold standard for full and empty capsid ratio is analytical ultracentrifugation (AUC). However, AUC is costly, time-intensive, and poorly suited for high-throughput screening of conditions. Several other methods are used with varying performance and cost; most require relatively pure and concentrated samples in large quantities to give accurate results. Separation of full and empty capsids can be achieved with ion exchange by using a small difference in charge, as full capsids have a lower pI compared to empty capsids, on average. This article will explore the critical parameters for high-performance separation, and how Capto™ Q (HiTrap™ column, 1 mL) run on an ÄKTA pure™ 25 chromatography system can be used to determine percent full capsids of AAV8 and AAV9 in less than 5 minutes with low sample consumption. 

The top AAV processing challenges in the polishing stage include incomplete separation of full and empty capsids with overlapping peaks and the need to optimize to maximize separation for each serotype, often leading to poor enrichment of full capsids and low viral genome (VG) recoveries. Cytiva has sought to overcome these challenges with a one-resin, one-protocol method for all serotypes tested: AAV2, AAV5, AAV8, and AAV9. In the analytical stage, challenges remain around confirming the full and empty capsid ratio in the separated peaks, which is required to optimize the polishing step. During process development, there is a need for quick analysis to access the proportion of full capsids, and for the optimized process several analytic methods are required to confirm the percent full capsids using qPCR:ELISA ratio, analytical ultracentrifugation, transmission electron cryomicroscopy (CryoTEM), or other often time-consuming and costly analytics.