Buffer Valency Engineering Enables High-concentration and Shelf-stable DNA Transfection Particles for Viral Vector Production

Cost-effective and scalable production is critical for advancing the clinical translation of viral vector-mediated gene therapy. The widely used transient transfection method for the production of adeno-associated virus (AAVs) and lentivirus utilizes polyethyleneimine (PEI)/DNA particles loaded with multiple plasmids; however, the current pDNA/PEI particles must be prepared under dilute concentrations (< 20 µg mL⁻¹) and used immediately, hampering scale-up and reproducibility. Here, we introduce a kinetic-gating strategy in which transient binding of trivalent citrate ions slows complexation, enabling a streamlined and scalable strategy to generate shelf-stable, highly concentrated pDNA/poly(ethylenimine) (PEI) transfection particles. By incorporating trivalent citrate ions in the assembly buffers, we kinetically modulate electrostatic complexation to achieve uniform particle assembly and prevent aggregation at high concentrations. This enables a tenfold increase in pDNA concentration in stabilized transfection particles from a typical range of 10–20 µg/mL to 200 µg/mL, while reducing the required dosing volume from 5–10% to 0.5% of the cell culture medium. The particle assembly approach is mixer-agnostic, GMP-compatible, and compatible with standard workflows. We demonstrate equivalent AAV production efficiencies to standard methods and consistent performance in various production scales, which confirms the practical utility of this assembly method in developing robust, scalable, and cost-effective AAV manufacturing processes.