Scalable Production and Purification of Adeno-Associated Viral Vectors (AAV).

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Version: Author's accepted manuscript
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Serval ID
serval:BIB_B1349188A5CB
Type
Article: article from journal or magazin.
Publication sub-type
Review (review): journal as complete as possible of one specific subject, written based on exhaustive analyses from published work.
Collection
Publications
Institution
Title
Scalable Production and Purification of Adeno-Associated Viral Vectors (AAV).
Journal
Methods in molecular biology
Author(s)
Blessing D., Déglon N., Schneider B.L.
ISSN
1940-6029 (Electronic)
ISSN-L
1064-3745
Publication state
Published
Issued date
2018
Peer-reviewed
Oui
Volume
1850
Pages
259-274
Language
english
Notes
Publication types: Journal Article ; Research Support, Non-U.S. Gov't
Publication Status: ppublish
Abstract
Here we describe methods for the production of adeno-associated viral (AAV) vectors by transient transfection of HEK293 cells grown in serum-free medium in orbital shaken bioreactors and the subsequent purification of vector particles. The protocol for expression of AAV components is based on polyethyleneimine (PEI) mediated transfection of a 2-plasmid system and is specified for production in milliliter to liter scales. After PEI and plasmid DNA (pDNA) complex formation the diluted cell culture is transfected without a prior concentration step or medium exchange. Following a 3-day batch process, cell cultures are further processed using different methods for lysis and recovery. Methods for the purification of viral particles are described, including iodixanol gradient purification, immunoaffinity chromatography, and ultrafiltration, as well as quantitative PCR to quantify vector titer.
Keywords
Chromatography, Affinity, Dependovirus/genetics, Dependovirus/isolation & purification, Genetic Therapy, Genetic Vectors/genetics, Genetic Vectors/isolation & purification, HEK293 Cells, Humans, Adeno-associated viral vector, Affinity chromatography purification, HEK293, Orbital shaken bioreactors, Suspension cell culture, Transient transfection
Pubmed
Create date
29/10/2018 9:56
Last modification date
20/08/2019 15:20
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