Automated plasmid purification system enables increased AAV production

Gene therapies are transforming modern medicine, with adeno-associated virus (AAV) being the most used vector for delivery. High manufacturing costs however continue to limit drug discovery, clinical development, and patient access. Plasmid DNA (pDNA) has been identified as a critical bottleneck in AAV development and small-scale production. In this study, we compared conventional manual plasmid purification kits with a scalable, automated purification system across Maxi, Mega, and Giga scales. Time savings increased with purification scale, e.g., when purifying 4 plasmid samples at Giga scale (10 mg plasmid from ~7 g of bacterial pellet), 8 hours of active time was saved using the automated system and total time was cut from almost 10 hours to 5 hours, although with generally lower yields (3–67% lower) and higher endotoxin levels. Both purification methods produced comparable AAV9 titres using standard PEI-based transfection of HEK293F cells. Surprisingly the AAV-MAX production system, plasmids purified by the automated method generated 14-fold higher titres than manually purified plasmids (3.2×10¹¹ vg/ml vs. 2.3×10¹⁰ vg/ml, respectively). Further analyses, including single and triple plasmid transfections and dynamic light scattering, revealed that the elution buffer used in the automated system formed larger pDNA transfection complexes in the AAV-MAX system and resulted in increased transfection efficiency. These findings provide insights that extend beyond AAV manufacturing, highlighting opportunities to optimise the transient production of other biologics and further improvement of plasmid preparation methods. • Automated plasmid purification reduced manual labour by up to 95% • AAV yield could be increased 14-fold using the automatically purified plasmids • Plasmid resuspension buffer can have a large impact on transfection efficiency and polyplex formation