P06

Maximizing Upstream rAAV Yield: A Design of Experiments Approach to Plasmid Ratio Optimization

I A Mancilla(1) J Challita(1) I RS Ferreira(1) K Strasser(1)

1:Revvity Gene Delivery GmbH

Plasmids, essential for rAAV production via transient transfection, represent a significant expense and optimization target in early-stage development. This study aimed to identify optimal plasmid ratios for maximizing functional rAAV yield in preclinical settings using Design of Experiments (DoE), with potential applications in both discovery and early preclinical development stages.

We employed an R-based open-source DoE pipeline to reduce the number of 3-plasmid ratio combinations from 14,553 to 28, maintaining statistical power while minimizing experimental load. Using a HEK293F-derived clonal suspension cell line, we conducted transfections with the 3-plasmid system in 50 mL tubes containing 10 mL of cell culture. After 70 hours, cells were harvested, lysed, and clarified. Vector genome titers were quantified by ITR2-qPCR. Our analysis revealed optimal conditions for total plasmid DNA and ratios of pHelper, pRepCap, and pTransgene, yielding up to 4-fold higher productivity compared to other combinations. A Response Surface Model (RSM) was generated and validated through ridge analysis and comparison with previously established ratios. The optimized plasmid ratios resulted in up to 3-fold increase in vector genome titers across four common serotypes and two transgenes of varying lengths, with some serotypes achieving productivity exceeding 7E+14 VG/L of clarified cell culture. This method demonstrates potential for application in discovery stages to identify high-yield capsids and transgenes, as well as in preclinical stages to fine-tune plasmid ratios for improved empty:full capsid ratios. By optimizing plasmid usage, this approach contributes to enhancing rAAV manufacturing efficiency and potentially reducing development costs.