Process Development for the Production and Purification of Adeno-Associated Virus (AAV)2 Vector using Baculovirus-Insect Cell Culture System.
J Vis Exp
; (179)2022 01 13.
Article
en En
| MEDLINE
| ID: mdl-35098946
ABSTRACT
Adeno-associated viruses (AAV) are promising vectors for gene therapy applications. Here, the AAV2 vector is produced by co-culture of Spodoptera frugiperda (Sf9) cells with Sf9 cells infected with baculovirus (BV)-AAV2-GFP (or therapeutic gene) and BV-AAV2-rep-cap in serum-free suspension culture. Cells are cultured in a flask in an orbital shaker or Wave bioreactor. To release the AAV particles, producer cells are lysed in buffer containing detergent, which is subsequently clarified by low-speed centrifugation and filtration. AAV particles are purified from the cell lysate using AVB Sepharose column chromatography, which binds AAV particles. Bound particles are washed with PBS to remove contaminants and eluted from the resin using sodium citrate buffer at pH 3.0. The acidic eluate is neutralized with alkaline Tris-HCl buffer (pH 8.0), diluted with phosphate-buffered saline (PBS), and further concentrated with tangential flow filtration (TFF). The protocol describes small-scale pre-clinical vector production compatible with scale-up to large-scale clinical-grade AAV manufacturing for human gene therapy applications.
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Baculoviridae
/
Dependovirus
Tipo de estudio:
Guideline
/
Risk_factors_studies
Límite:
Animals
/
Humans
Idioma:
En
Revista:
J Vis Exp
Año:
2022
Tipo del documento:
Article