292. Towards Large-Scale Manufacturing of Adeno-Associated Virus by Transient Transfection of HEK293 Suspension Cells in a Stirred Tank Bioreactor Using Serum-Free Medium
Authors: Parminder Chahal, Érica Alessandra Schulze, Alice Bernier, Stephane Lanthier, Nathalie Coulombe, Amine Kamen, Renald Gilbert
Journal: Molecular Therapy (2016)

Adeno-Associated Virus (AAV) vectors showing safety profile in phase I clinical trials and its ability to transduce gene expression in various tissues have made it a vector of choice for gene delivery. There are different modes of AAV vector production and each has advantages and disadvantages. Here we demonstrated that the production of AAV by transient transfection in a serum-free medium using NRC's patented cGMP compliant human embryonic kidney HEK293 cell line (clone HEK293SF-3F6) adapted for growth in suspension can be readily scaled-up in stirred tank bioreactors. We employed triple-plasmid / polyethylenimine (PEI) based transient transfection technique. As a proof of concept, we demonstrated that nine serotypes of AAV (AAV-1 to AAV-9) encoding GFP can be produced by our cell line HEK293SF with yields of about 1E+13 genome-containing particles per liter (Vg/L). Depending on the serotypes 4-30% of AAV is present in the supernatant of the cell culture at 48hpt. The presence of plasmids and plasmid polyplexes that were not taken up by the cells or were not brought into the cell nucleus were removed by Iodixanol-ultracentrifugation method and Benzonase treatment before analyzing by real-time PCR. About 25% loss in genome containing viral particle counts were observed by Iodixanol purification method based on infectivity assay. Productions of AAV2 and AAV6 encoding GFP were demonstrated in 3L stirred tank bioreactors. Purification scheme was based on column chromatography - a scalable process. Different chromatography media, such as cation exchanger, anion exchanger and hydrophobic interaction chromatography, were tested with each AAV serotypes for their ability to adsorb and elute efficiently. The purification scheme was then adopted by integrating best chromatography medium and sequence dependent upon the AAV serotype in use. We demonstrated the purification scheme for AAV2 based on ion-exchange and hydrophobic interaction chromatography steps. The SDS-PAGE showed the purity of the final product and the presence of three capsid proteins VP1, VP2 and VP3 on Western blot corresponding to the only three bands present in the final product on SDS-PAGE. To extend the storage life of AAV we explored lyophilization technique to study the stability of AAV2 and AAV6 under lyophilized conditions. The AAV2 and AAV6 were stable for over 40 weeks based on infectivity assay. We demonstrated the scalability of the process up to 45L. Productions tested in 20 and 500 mL cultures in shake flasks were scaled up in 2 and 45L cultures (in 3- and 60-L stirred tank bioreactors, respectively). The volumetric yields and purification recoveries were comparable at all of these production scale levels demonstrating scalability of transient transfection at even larger scale is possible to generate material necessary for dosages required for gene therapy application.