Improving AAV Production Yield and Quality for Different Serotypes Using Distinct Processing Methods

Adeno-associated viruses (AAVs) have emerged as a promising tool for gene therapy due to their excellent safety profile and efficient transduction in multiple target tissues. Currently generated AAV yields at lab scale are in the range of 10¹²-10¹⁴ vgc/L or between 10⁴ and 10⁵ vg/cell. Maximizing yields would significantly impact the overall production cost and accessibility. However, existing challenges to improving the overall yield from upstream and downstream processes persist. Using an adherent cell manufacturing process, we compared two different purification approaches to optimize the recovery from cell lysate (CsCl ultracentrifugation) and media supernatant (affinity column chromatography). We achieved combined purified yields of 2.25 × 10¹³ for AAV6 (1.00 × 10¹³ (44.44%) from ultracentrifugation and 1.25 × 10¹³ (55.56%) from affinity chromatography), 1.11 × 10¹⁴ for AAV8 (2.37 × 10¹³ (21.39%) from ultracentrifugation and 8.70 × 10¹³ (78.61%) from affinity chromatography), and 7.32 × 10¹³ for AAV9 (1.10 × 10¹³ (15.03%) from ultracentrifugation and 6.22 × 10¹³ (84.97%) from affinity chromatography), with AAV9 showing a ∼1.5-fold increase compared to previous reports. Interestingly, we found each serotype to elute at different pH values which may influence yields. The chromatography approach obtained higher percentage recoveries for AAV6 (88.35 ± 12.87%), AAV8 (96.67 ± 0.60%), and AAV9 (93.54 ± 1.08%) from the media supernatant and also demonstrated significantly higher transduction rates. Thus, we demonstrate the potential for further expanding the AAV production capacity while also improving the final product quality.