Assessing AAV stability on affinity resins and the effects of long contact times through extensive washing

Affinity chromatography represents the standard platform for the capture of recombinant adeno-associated viral vectors (rAAVs). However, low upstream titers frequently necessitate long processing time exceeding multiple hours, the biophysical and functional consequences of which remain largely uncharacterized. Here, we present an extensive dataset characterizing the effects of long capsid-ligand interaction times on rAAV critical quality attributes. We examined the impact of binding durations (0–24 h) at room temperature versus 10 °C, using three different serotypes (rAAV2, AAV8 wild-type (WT), AAV9WT) and several affinity ligands, all based on single-domain V H H-fragments (POROS™ CaptureSelect™ AAVX, POROS™ CaptureSelect™ AAV9, Capto™ AVB). Using CaptureSelect AAVX, extended hold times ( ≥ 8 h) resulted in serotype-specific yield losses correlated with shifts in capsid viral protein (VP)-ratios, alongside enhanced host-cell impurity clearance, without compromising transgene integrity. Washing for 8 h or longer reduced elution yields by 28% for AAV9 and 35% for rAAV2, whereas AAV8 yield remained unaffected. This yield loss coincided with an increased relative abundance of VP3 for rAAV2 (+1.5% at 8 h), while AAV8 exhibited no changes in VP-ratio. Crucially, host-cell protein clearance was substantially improved across all serotypes, with log removal factor increasing from 3.34 to 4.71 for AAV8, 3.34 to 4.87 for rAAV2, and 2.77 to 4.91 for AAV9. LC-MS-based proteomics revealed that DNA-interacting proteins, known to co-elute with rAAVs during affinity chromatography, were cleared more than 10-fold during several hour-long binding times compared to standard processes.