Scalable Production and Purification of Adeno-Associated Viral Vectors (AAV).
- Blessing D Laboratory of Neurotherapies and Neuromodulation, Department of Clinical Neurosciences, Lausanne University Hospital, Lausanne, Switzerland. daniel.blessing@epfl.ch.
- Déglon N Laboratory of Neurotherapies and Neuromodulation, Department of Clinical Neurosciences, Lausanne University Hospital, Lausanne, Switzerland.
- Schneider BL Brain Mind Institute, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland. bernard.schneider@epfl.ch.
- 2018-09-23
Published in:
- Methods in molecular biology (Clifton, N.J.). - 2018
Adeno-associated viral vector
Affinity chromatography purification
HEK293
Orbital shaken bioreactors
Suspension cell culture
Transient transfection
Chromatography, Affinity
Dependovirus
Genetic Therapy
Genetic Vectors
HEK293 Cells
Humans
English
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.
- Language
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- English
- Open access status
- green
- Identifiers
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- DOI 10.1007/978-1-4939-8730-6_17
- PMID 30242692
- Persistent URL
- https://sonar.rero.ch/global/documents/20512
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