Scalable process development for rAAV transient transfection production using computational fluid dynamics modeling

Abstract Recombinant adeno‐associated virus (rAAV) is a promising delivery vehicle for cell and gene therapies. Upstream development faces challenges like low productivity and inconsistent performance despite advancements. This study presents a scale‐up design for robust rAAV production at 250 L scale using a transfection system. Initial process development in shake flasks optimized plasmid ratio to improve rAAV production. However, genome titer decreased by up to 50% in stirred‐tank bioreactors, likely due to mechanical shear forces. Stirred‐tank bioreactors were modeled with computational fluid dynamics (CFD) by M‐STAR (250 mL, 5 L, 50 L) and with empirical correlations by Dynochem (250 L). Hydrodynamics were characterized to provide normalized shear stress across different geometries. The power per unit volume (P/V) of 71 W/m 3 was optimal for the 250 mL bioreactor, focusing on cell growth, rAAV genome titer, capsid titer, and full capsid ratio. Based on CFD modeling, a P/V of 20 W/m 3 was projected to perform best at 5 and 50 L scales during development, confirmed by comparable genome titer to low shear shake flask culture. A P/V of 15 W/m 3 was subsequently projected for final production at the 250 L scale. The negative impact of shear stress could be further mitigated by adding extra Poloxamer‐188 as a shear protectant. Additionally, pre‐transfection viable cell density (VCD) was identified as a critical attribute. The final process included a 30% fixed‐volume dilution of the cell culture along with controlled DNA complexation conditions to improve process robustness. Sequential production at the 250 L scale demonstrated consistent cell growth and rAAV production.