Analyzing full and empty AAV capsid ratio - in less than 5 minutes

Adeno-associated virus (AAV) is the main vector for gene therapy, and there is need for a quick, robust, and cost-efficient full and empty capsid analytic method during 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 (full capsids have lower pI compared to empty capsids, on average). We have previously presented preparative full and empty capsid separation for AAV2, AAV5, AAV8, and AAV9 with anion exchange using Capto™ Q chromatography resin. Here we show how Capto™ Q (HiTrap™ column, 1 mL) run on an ÄKTA pure™ 25 chromatography system can be used to determine % full capsids of AAV8 and AAV9 in less than 5 minutes with low sample consumption. We also expect this analytical tool to work for the other serotypes and capsid variants.

In this webinar:
• Dive into details of the quick analytical protocol that uses one buffer system and only requires 1010 viral particles per run.
• Understand that only one pre-screening procedure is needed per AAV capsid serotype/variant to determine the conductivity for eluting the empty capsids.
• See a comparison of full capsid/full + empty capsid peak areas and qPCR/ELISA (viral genomes/viral total capsids) for determining % full capsids.
• Discover hints and tips to avoid common pitfalls.