Monitoring full-length recombinant adeno-associated virus genome DNA in large-scale production using automated electrophoresis

Abstract

Large-scale production of recombinant adeno-associated viruses (rAAV) for clinical use involves bioreactors and complex purification processes. Monitoring genomic DNA during manufacturing procedures is crucial to ensure the quality of the final product, as production can vary from batch to batch. We conducted a 50-L bioreactor production, with sampling conducted at each processing step, from transfection to anion-exchange chromatography purification. The rAAV genome can be monitored in transfected cells via a concise DNA extraction method, even in the presence of cellular impurities, using automated electrophoretic quantification with a TapeStation. The rAAV genomic DNA recovery rate was 64% for the affinity column and 41% for anion exchange, as measured using the TapeStation, whereas droplet digital polymerase chain reaction yielded recovery rates of 51% and 45% for the affinity and anion-exchange columns, respectively. Therefore, automated electrophoresis is a reliable and quantitative method for monitoring rAAV genome integrity during production.