308. Ultracentrifugation-Free Chromatography-Mediated Large-Scale Purification of Recombinant Adeno-Associated Virus Serotype 1 (rAAV1) and rAAV9 from the Serum-Free Culture Supernatant

[Background]The current production of rAAV from the transfected cell lysate and purification based on CsCl or iodixanol density ultracentrifugation are not suitable for large-scale processing. Although rAAV1 and rAAV9 are promising therapeutic vectors for genetic neuromuscular disorders, the large-scale purification method for those vectors has not yet been established. In this study, we elaborate the novel chromatography-mediated methods for purification of rAAV1 and rAAV9 from the serum-free culture supernatant with ultracentrifugation-free technique towards large-scale and GMP production.[Methods]rAAV1 (scAAV1-CBA-EGFP) and rAAV9 (scAAV9-CBA-EGFP) were produced by the triple-transfection to HEK293 or HEK293EB cells in serum-free medium with polyethyleneimine (PEI). Five days later, the culture supernatant was tangential flow-filtrated (TFF) and concentrated by the Hollow Fiber system. After reducing protein debris by 1/3-saturated ammonium sulfate (1/3 AS) precipitation, rAAV1 or rAAV9 was precipitated in 1/2 AS solution.Subsequently, the precipitated rAAV1 was dissolved in 10 ml of PBS containing 3 mM MgCl2. After the sample was dialyzed against a buffer containing 5 mM NaCl, the dialysate was diluted with dH2O and loaded to tandem quintet cation-exchange membranes (Mustang SXT) and quintet anion-exchange membranes (Mustang QXT) with a column volume of 0.86 ml. After rAAV1 was eluted from Mustang QXT, it was finally purified by gel-filtrated chromatography (Superdex 200 10/300 GL).The precipitated rAAV9 was dissolved in 22 ml of 3.3 mM MES, 3.3 mM HEPES, 3.3 mM sodium acetate (MHN) buffer (pH8.0) containing 50 mM NaCl and 0.01% Pluronic F-68. After the resultant sample was diluted with MHN buffer, it was loaded to tertiary amine charged anion-exchange column. The passed through fraction containing rAAV9 was finally purified by gel-filtrated chromatography.The physiological and biological properties of the purified rAAV1 and rAAV9 were characterized by qPCR, electron micrograph, FACS, western blot and SDS-PAGE.[Results]The purified rAAV1 and rAAV9 displayed three major bands (VP1, VP2, and VP3) on SDS-PAGE and more than 90% of rAAV1 particles or 95% of rAAV9 particles was contained fully packaged viral genomes according to electron micrographic analysis. We confirmed increasing infectivity of rAAV1 products depending on chromatographic purification step and that of rAAV9 is under evaluation. The resultant genomic titer of the purified rAAV1 was 4.17 × 1013 v.g. (3.63 × 1013 v.g./ml) from the 4 × 109 HEK293 cells and the purified rAAV9 was 1.49 × 1015 v.g. (1.14 × 1014 v.g./ml) from the 5.1 × 109 HEK293EB cells.[Conclusion]Chromatography-mediated purification from the culture supernatant is a major breakthrough, especially for the production of rAAV9. We obtained rAAV1 and rAAV9 by this protocol with high titer, high purity and minimum contamination of empty particles. This novel chromatography-based method would be consistent with GMP production and facilitate clinical studies in the future.