Scalable Production and Purification of Adeno-Associated Viral Vectors (AAV)
| Autor: | Blessing, D., Déglon, N., Schneider, B.L. |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2018 |
| Předmět: |
viruses
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 |
| Zdroj: | Methods in molecular biology, vol. 1850, pp. 259-274 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
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