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Recombinant Protein Expression in Mammalian Cells pp 259–274Cite as

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

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1850))

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.

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Acknowledgments

This work was supported by the SwissTransMed Platforms for Translational Research in Medicine. This study was conducted at the EPFL platform for gene therapy, which is supported by the Bertarelli Foundation. The authors would like to thank Vivianne Padrun, Fabienne Pidoux, and Aline Aebi for expert technical assistance in this project.

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Authors and Affiliations

  1. Laboratory of Neurotherapies and Neuromodulation, Department of Clinical Neurosciences, Lausanne University Hospital, Lausanne, Switzerland

    Daniel Blessing & Nicole Déglon

  2. Neurosciences Research Center (CRN), LNTM, Lausanne University Hospital, Lausanne, Switzerland

    Daniel Blessing & Nicole Déglon

  3. Brain Mind Institute, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland

    Daniel Blessing & Bernard L. Schneider

Authors
  1. Daniel Blessing
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  2. Nicole Déglon
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  3. Bernard L. Schneider
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Corresponding authors

Correspondence to Daniel Blessing or Bernard L. Schneider .

Editor information

Editors and Affiliations

  1. Protein Production and Structure Core Facility (PPSCF), Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland

    David L. Hacker

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Blessing, D., Déglon, N., Schneider, B.L. (2018). Scalable Production and Purification of Adeno-Associated Viral Vectors (AAV). In: Hacker, D. (eds) Recombinant Protein Expression in Mammalian Cells. Methods in Molecular Biology, vol 1850. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8730-6_17

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  • DOI: https://doi.org/10.1007/978-1-4939-8730-6_17

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8729-0

  • Online ISBN: 978-1-4939-8730-6

  • eBook Packages: Springer Protocols

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