title: Ultracentrifugation-free chromatography-mediated large-scale purification of recombinant adeno-associated virus serotype 1 (rAAV1).
abstract: Recombinant adeno-associated virus (rAAV) is an attractive tool for gene transfer and shows potential for use in human gene therapies. The current methods for the production and purification of rAAV from the transfected cell lysate are mainly based on cesium chloride and iodixanol density ultracentrifugation, although those are not scalable. Meanwhile, chromatography-based systems are more scalable. Therefore, in this study, we developed a novel method for the production and purification of rAAV serotype 1 (rAAV1) from serum-free culture supernatant based on ion-exchange and gel-filtration chromatography to obtain highly purified products with an ultracentrifugation-free technique towards Good Manufacturing Practice (GMP) production. The purified rAAV1 displayed three clear and sharp bands (VP1, VP2, and VP3) following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and more than 90% of rAAV1 particles contained fully packaged viral genomes according to negative-stain electron micrographic analysis. Consequently, the resultant genomic titer of the purified rAAV1 was 3.63 × 10(13) v.g./ml (the total titer was 4.17 × 10(13) v.g.) from the 4 × 10(9) HEK293 cells. This novel chromatography-based method will facilitate scale-up of manufacturing for clinical applications in gene therapy.
authors.affiliation: Department of Biochemistry and Molecular Biology, Nippon Medical School, Tokyo, Japan; Department of Human Genetics, National Center for Child Health and Development, Tokyo, Japan; Graduate School of Comprehensive Human Sciences, Master's Program in Medical Sciences, University of Tsukuba, Ibaraki, Japan.
authors.forename: Taro
authors.initials: T
authors.lastname: Tomono
authors.affiliation: Department of Biochemistry and Molecular Biology, Nippon Medical School , Tokyo, Japan.
authors.forename: Yukihiko
authors.initials: Y
authors.lastname: Hirai
authors.affiliation: Department of Biochemistry and Molecular Biology, Nippon Medical School , Tokyo, Japan.
authors.forename: Hironori
authors.initials: H
authors.lastname: Okada
authors.affiliation: Department of Biochemistry and Molecular Biology, Nippon Medical School , Tokyo, Japan.
authors.forename: Kumi
authors.initials: K
authors.lastname: Adachi
authors.affiliation: Department of Neurology, Faculty of Medicine, University of Tsukuba , Ibaraki, Japan.
authors.forename: Akiko
authors.initials: A
authors.lastname: Ishii
authors.affiliation: Department of Biochemistry and Molecular Biology, Nippon Medical School , Tokyo, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Shimada
authors.affiliation: Department of Human Genetics, National Center for Child Health and Development , Tokyo, Japan.
authors.forename: Masafumi
authors.initials: M
authors.lastname: Onodera
authors.affiliation: Department of Neurology, Faculty of Medicine, University of Tsukuba , Ibaraki, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Tamaoka
authors.affiliation: Department of Biochemistry and Molecular Biology, Nippon Medical School , Tokyo, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Okada