Sodium Chloride Enhances Recombinant Adeno-Associated Virus Production in a Serum-Free Suspension Manufacturing Platform Using the Herpes Simplex Virus System.

The increase in effective treatments using recombinant adeno-associated viral (rAAV) vectors has underscored the importance of scalable, high-yield manufacturing methods. Previous work from this group reported the use of recombinant herpes simplex virus type 1 (rHSV) vectors to produce rAAV in adherent HEK293 cells, demonstrating the capacity of this system and quality of the product generated. Here we report production and optimization of rAAV using the rHSV system in suspension HEK293 cells (Expi293F) grown in serum and animal component-free medium. Through adjustment of salt concentration in the medium and optimization of infection conditions, titers greater than 1 × 10 ¹⁴ vector genomes per liter (VG/liter) were observed in purified rAAV stocks produced in Expi293F cells. Furthermore, this system allowed for high-titer production of multiple rAAV serotypes (2, 5, and 9) as well as multiple transgenes (green fluorescent protein and acid α-glucosidase). A proportional increase in vector production was observed as this method was scaled, with a final 3-liter shaker flask production yielding an excess of 1 × 10 ¹⁵ VG in crude cell harvests and an average of 3.5 × 10 ¹⁴ total VG of purified rAAV9 material, resulting in greater than 1 × 10 ⁵ VG/cell. These results support the use of this rHSV-based rAAV production method for large-scale preclinical and clinical vector production.