[rAAV-Edu] rAAV9の精製方法 – 特許,2018 – △ID2559 [2019/10/05]

rAAV9 vectorの精製方法

培養液のバッファ組成をTFF処理で調整、硫安(AmSo4)沈殿処理と第4級アンモニウム陰イオン交換クロマトグラフィー(HiPrep Q XL)によるFlow through mode、最後にSECによる精製でrAAV9を得る

rAAV9 vectorの調製方法

1. Transfection by 3 plasmid (with PEI, FBS-free)
2. Post culture
3. Harvest supernatant
4. TFF process
5. 1/3 sut. concentration AmSO4 precipitation
6. centrifuge
   • to take the sup.
7. 1/2 sut. concentration AmSO4 precipitation
8. centrifuge
   • to take the sup.
9. Dilution to 7.3 mS/cm
10. AEX
    • HiPress Q XL 16/10 column chromatograph with flow through mode
11. TFF process
    • 30kDa
12. SEC column chromatography
    • HiLoad 16/60 Superdex 200, MNH pH6.5, 300mM NaCl, 0.01% F-68

Highly Efficient Ultracentrifugation-free
Chromatographic Purification of Recombinant
AAV Serotype 9

https://www.cell.com/molecular-therapy-family/methods/pdfExtended/S2329-0501(18)30111-6

Improved methods for purification of recombinant aav vectors, EP2443233A1, 2009

https://patents.google.com/patent/EP2443233A1/en

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[rAAV] PEG沈殿法によるrAAVの精製手順, 2012 – △ID2429 [2019/09/26] [rAAV] rAAVのUSP/DSP – 製造方法 – emptyとfullの比重, 2015 – SM-ID2585 [2019/10/05] [Data Link] Purification of rAAV-1 and -9 with ultracentrifugation-free technique towards GMP production.(2013-2015, 科研費研究) – △□ID2225 [2019/09/15] [rAAV] rAAVの従来からの精製方法 (PEG沈殿、超遠心), △2010 – ID2417 [2019/09/26] [rAAV] 遺伝子治療薬としての遺伝子組換えAAV(rAAV)の沈殿法による精製のいろいろ – △ID2452 [2019/09/27] [rAAV-Edu] rAAV9の精製方法 – 特許, 2017 – ID2566 [2019/10/05]

AAV