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[rAAV] PEG沈殿法によるrAAVの精製手順, 2012 – △ID2429 [2019/09/26]

[rAAV] PEG沈殿法によるrAAVの精製手順, 2012 – △ID2429 [2019/09/26]
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rAAV vectorの精製方法

以下のReviewにおけるOptimized purificationの精製方法は、Ultracentrifugationを用いずにAAVを精製している文献である。

[rAAV] rAAVの従来からの精製方法 (PEG沈殿、超遠心), 2010 – ID2417 [2019/09/26]

Transfection of HEK293, and post culture

20 Flasks of T-150, 3 Plasmid PEI

Centrifugation / cell pellet

3,000 xg 10min

(A) Lysis by Freeze-thaw, and (B) Nuclease, (C) sodium deoxycholate

(A) 50mM Tris-HCl, 0.15M NaCl, 2mM NgCl2, pH8, Dry ice-ethanol ↔︎ 37℃ water
(B) Benzonase: 50u/mL, RNase: 10U/mL, 37℃, 30min
(C) 0.5% sodium deoxycholate, 37℃, 30min

cfg (2,500 g x 10min)/sup

Pooling with cell culture supernatant

PEG 8000, NaCl Treatment / ppt

1. 8% PEG 8000, 0.5M NaCl by 40% PEG 8000, 2.5M NaCld, incubation 1h, RT.
2. centrifuge 2,000g x 30min
3. Re-suspend with HEPES buffer

Chloroform Extraction / cfg-sup /Evaporate

疎水性タンパクとPEGを除去
1. Add equal volume of chloroform
2. Vortex going , 2min , RT.
3. cfg (370g x 5min) (おそらく水層にAAV)
4.. Evaporate 30min , RT

PEG 8000, AmSO4 Treatment / AAV in sup

1. 10% PEG 8000, 13.2% AmSO4 pH8.0 adjusted by stocked solution
2., Incubation at RT.
(Impurity salt out in ppt), HEPES buffer pH has to be kept at pH8.0, AAV stable is Alkaline that acid.

Dialysis using 50kDa

diligent: PBS or MEMEM media with 0.001% puluronic F68 for preventing the aggregation.

関連記事:

[rAAV-Edu] rAAV9の精製方法 – 特許,2018 – △ID2559 [2019/10/05] AEX purification[rAAV-Edu] rAAV9の精製方法 – 特許, 2017 – ID2566 [2019/10/05] [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] [Data Link] Pharmacology of Recombinant Adeno-associated Virus Production – ID2415 [2019/09/26] [rAAV] rAAVの従来からの精製方法 (PEG沈殿、超遠心), △2010 – ID2417 [2019/09/26]

用語の解説リンク、関連投稿および広告

AAV

[用語] rAAV [Biotech] [2022/09/03]

…end

DSP

[用語] DSP; Down Stream Process [2022/11/14] ID24806

…end

purification

…end

rAAV-production

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