Production of Recombinant Adeno-Associated Virus Through High-Cell-Density Transfection of HEK293 Cells Based on...

Adeno-associated virus (AAV)-associated gene therapy has been increasingly promising, in light of the drugs progressed to clinical trials or approved for medications internationally. Therefore, scalable and efficient production of recombinant AAV is pivotal for advancing gene therapy. Traditional methods, such as the triple-plasmid transfection of human embryonic kidney 293 cells in suspension culture, have been widely employed but often hampered by low unit yield. In this study, we optimized the cell culture process with high cell density up to 2 × 10⁷ cells/mL by employing a perfusion culture system with centrifugation and medium exchange in shake flasks and perfusion device in bioreactor. Furthermore, we utilized a design of experiments strategy to systematically modulate a series of transfection-related variables including the quantity of plasmid DNA, the DNA-to-polyethylenimine ratio, incubation duration, and the impact of post-transfection feeding strategies on the yield of recombinant AAV (rAAV). Our comprehensive analysis and subsequent optimizations actualized a remarkable unit yield reaching nearly 2 × 10¹² vector genomes (vg)/mL. Importantly, the resulting single-cell yield and biological activity were found to be comparable with those obtained from fed-batch cultures, underscoring the efficacy of our approach. Based on these findings, we investigated rAAV yield via high-density suspend culture in bioreactor, particularly focusing on cell aggregation and the use of perfusion technology. Intriguingly, we attempted to elevate the yield of an oversized recombinant coagulation factor VIII AAV843 vector by 3.5-fold, reaching a yield of 1 × 10¹² vg/mL. Concurrently, the medium usage rate was only double that of batch feeding, thereby significantly shrinking the upstream cost of rAAV manufacture. In summary, this strategy significantly benefits large-scale AAV production for both commercial and clinical applications.