Ultracentrifugation-free chromatography-mediated large-scale purification of recombinant adeno-associated virus serotype 1 (rAAV1)
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 ultracentr...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2016-01-01
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| Series: | Molecular Therapy: Methods & Clinical Development |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2329050116301486 |
| _version_ | 1818040369530535936 |
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| author | Taro Tomono Yukihiko Hirai Hironori Okada Kumi Adachi Akiko Ishii Takashi Shimada Masafumi Onodera Akira Tamaoka Takashi Okada |
| author_facet | Taro Tomono Yukihiko Hirai Hironori Okada Kumi Adachi Akiko Ishii Takashi Shimada Masafumi Onodera Akira Tamaoka Takashi Okada |
| author_sort | Taro Tomono |
| collection | DOAJ |
| description | 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 × 1013 v.g./ml (the total titer was 4.17 × 1013 v.g.) from the 4 × 109 HEK293 cells. This novel chromatography-based method will facilitate scale-up of manufacturing for clinical applications in gene therapy. |
| first_indexed | 2024-12-10T08:13:26Z |
| format | Article |
| id | doaj.art-68e4a33f357e47d58c5eb4c031780ed7 |
| institution | Directory Open Access Journal |
| issn | 2329-0501 |
| language | English |
| last_indexed | 2024-12-10T08:13:26Z |
| publishDate | 2016-01-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Molecular Therapy: Methods & Clinical Development |
| spelling | doaj.art-68e4a33f357e47d58c5eb4c031780ed72022-12-22T01:56:31ZengElsevierMolecular Therapy: Methods & Clinical Development2329-05012016-01-013C10.1038/mtm.2015.58Ultracentrifugation-free chromatography-mediated large-scale purification of recombinant adeno-associated virus serotype 1 (rAAV1)Taro Tomono0Yukihiko Hirai1Hironori Okada2Kumi Adachi3Akiko Ishii4Takashi Shimada5Masafumi Onodera6Akira Tamaoka7Takashi Okada8Department of Biochemistry and Molecular Biology, Nippon Medical School, Tokyo, JapanDepartment of Biochemistry and Molecular Biology, Nippon Medical School, Tokyo, JapanDepartment of Biochemistry and Molecular Biology, Nippon Medical School, Tokyo, JapanDepartment of Biochemistry and Molecular Biology, Nippon Medical School, Tokyo, JapanDepartment of Neurology, Faculty of Medicine, University of Tsukuba, Ibaraki, JapanDepartment of Biochemistry and Molecular Biology, Nippon Medical School, Tokyo, JapanDepartment of Human Genetics, National Center for Child Health and Development, Tokyo, JapanDepartment of Neurology, Faculty of Medicine, University of Tsukuba, Ibaraki, JapanDepartment of Biochemistry and Molecular Biology, Nippon Medical School, Tokyo, JapanRecombinant 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 × 1013 v.g./ml (the total titer was 4.17 × 1013 v.g.) from the 4 × 109 HEK293 cells. This novel chromatography-based method will facilitate scale-up of manufacturing for clinical applications in gene therapy.http://www.sciencedirect.com/science/article/pii/S2329050116301486 |
| spellingShingle | Taro Tomono Yukihiko Hirai Hironori Okada Kumi Adachi Akiko Ishii Takashi Shimada Masafumi Onodera Akira Tamaoka Takashi Okada Ultracentrifugation-free chromatography-mediated large-scale purification of recombinant adeno-associated virus serotype 1 (rAAV1) Molecular Therapy: Methods & Clinical Development |
| title | Ultracentrifugation-free chromatography-mediated large-scale purification of recombinant adeno-associated virus serotype 1 (rAAV1) |
| title_full | Ultracentrifugation-free chromatography-mediated large-scale purification of recombinant adeno-associated virus serotype 1 (rAAV1) |
| title_fullStr | Ultracentrifugation-free chromatography-mediated large-scale purification of recombinant adeno-associated virus serotype 1 (rAAV1) |
| title_full_unstemmed | Ultracentrifugation-free chromatography-mediated large-scale purification of recombinant adeno-associated virus serotype 1 (rAAV1) |
| title_short | Ultracentrifugation-free chromatography-mediated large-scale purification of recombinant adeno-associated virus serotype 1 (rAAV1) |
| title_sort | ultracentrifugation free chromatography mediated large scale purification of recombinant adeno associated virus serotype 1 raav1 |
| url | http://www.sciencedirect.com/science/article/pii/S2329050116301486 |
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