Format

Send to

Choose Destination
Mol Ther Methods Clin Dev. 2016 Feb 10;3:15058. doi: 10.1038/mtm.2015.58. eCollection 2016.

Ultracentrifugation-free chromatography-mediated large-scale purification of recombinant adeno-associated virus serotype 1 (rAAV1).

Author information

1
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.
2
Department of Biochemistry and Molecular Biology, Nippon Medical School , Tokyo, Japan.
3
Department of Neurology, Faculty of Medicine, University of Tsukuba , Ibaraki, Japan.
4
Department of Human Genetics, National Center for Child Health and Development , Tokyo, Japan.

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.

Supplemental Content

Full text links

Icon for Elsevier Science Icon for PubMed Central
Loading ...
Support Center