Automated zonal-rotor CsCl ultracentrifugation with ÄKTA-based fractionation removes empty and intermediate AAV...

Recombinant adeno-associated virus (AAV) preparations contain heterogeneous full, empty, and intermediate capsids. Although anion-exchange chromatography is scalable, it does not always resolve these populations, particularly intermediate capsids. A workflow integrated zonal-rotor cesium chloride (CsCl) density-gradient ultracentrifugation with ÄKTA-based loading, inline monitoring, unloading, and fraction collection. SW41Ti swing-bucket experiments defined the empirical 1,240-1,360 mg/cm³ AAV density range and identified a two-layer CsCl configuration for transfer to a 1.65-L Ti-15 zonal rotor. The same process was demonstrated with AAV8-X and AAV9-Y. The AAV8-X full fraction contained 95.5% full and 0.4% empty capsids; the AAV9-Y full fraction contained 91.8% full capsids, with no empty capsids detected by sedimentation velocity analytical ultracentrifugation (SV-AUC). Vector-genome-based full-fraction recoveries were 78.1% and 93.4%, respectively. On an equal-capsid basis, the AAV8-X intermediate fraction showed an SV40-targeted quantitative polymerase chain reaction (qPCR) signal equivalent to 48.2% of the full fraction. Separately, a Huh-7 assay tested the intermediate fraction at a higher capsid input than the full fraction, yielding approximately 4% of full-fraction X-gene mRNA expression. Thus, the intermediate fraction retained genome-targeted qPCR signal but showed limited expression under the tested conditions. This automated ÄKTA-coupled zonal CsCl workflow provides a scalable, high-resolution polishing operation for separating AAV capsid populations.