554. Efficient Scalable Purification of rAAV1 Using Ion-Exchange and Gel-Filtration Chromatography To Avoid Ultracentrifugation Procedure

[Background]: Recombinant adeno-associated virus vectors (rAAVs) are attractive tools for gene transfer and are evaluated for use in human gene therapies. However, the current production and purification of rAAVs based on CsCl or iodixanol density ultracentrifugation are not suitable for large-scale production in GMP (Good Manufacturing Practice) and inadequate to meet SOP (Standard Operating Procedure). In this study, we elaborate the novel methods for production and purification of type1 AAV vector (rAAV1) using chromatography steps (ion-exchange and gel-filtration chromatography). We study here for the chromatography-based purification of rAAV1, because Glybera, the first rAAV-based drug with the official approval of the European Medicines Agency (EMA), was produced as the rAAV1.[Methods]: rAAV1 (scAAV1-CBA-EGFP) was produced by triple-transfection of HEK293 cells in serum free medium with PEI (polyethyleneimine). The transfected cells were cultured for five days without the medium replacement. The cultured medium was subjected to ultrafiltration and concentration by the Hollow Fiber (exclusion limit is 750 kDa). After reducing protein debris by 1/3-saturated ammonium sulfate precipitation, rAAV1 was precipitated in 1/2-saturated ammonium sulfate solution. Subsequently, rAAV1 was dissolved in PBS (with 3mM MgCl2) and dialyzed against 20mM Tris-HCl (pH8.0) buffer containing 50mM NaCl and 3mM MgCl2 for 3-4 hours. Next, the sample was dialyzed against 20mM Tris-HCl (pH8.0) buffer containing 5mM NaCl and 3mM MgCl2 for 20 hours with a buffer replacement. After the sample was diluted with dH2O to adjust conductivity around 1.0mSV/cm, it was loaded at a rate of 1ml/min to tandem dual cation-exchange (Mustang SXT) and anion-exchange (Mustang QXT) high-capacity membranes. After removing the Mustang SXT, rAAV1 was eluted from the Mustang QXT by stepwise NaCl gradient and the vector-containing fraction was eluted by a buffer containing 140mM NaCl. After ultrafiltration with Ultracel 30K, rAAV1 was finally purified by gel-filtration chromatography with Superdex 200 HR using AKTA Explorer 100 HPLC system. The physiological and biological properties of the resultant rAAV samples were characterized by qPCR, electron micrograph, fluorescent micrograph and SDS-PAGE.[Results]: The purified rAAV1 represented three major bands (VP1, VP2, VP3) on SDS-PAGE and more than 90% of rAAV1 particles were fully packaged virions by electron micrographic analysis. Consequently, the resultant genomic titer of the purified rAAV1 was 4.6×1013 vg/ml from the 2×109 of HEK293 cells. This system would be scalable up to 1015 with our improved culture system and membrane capsule.[Conclusion]: We refined rAAV1-production protocol with high purity with minimum empty particles. This steamlined method with high performance scalable ion-exchange membrane would be useful for the large scale production of rAAV1 to facilitate clinical studies.