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. 2024:2810:55-74.
doi: 10.1007/978-1-0716-3878-1_4.

Production and Purification of Adeno-Associated Viral Vectors (AAVs) Using Orbitally Shaken HEK293 Cells

Jean-Philippe Gaudry  1 , Aline Aebi  1 , Pamela Valdés  1 , Bernard L Schneider  2   3   4
Affiliations

Production and Purification of Adeno-Associated Viral Vectors (AAVs) Using Orbitally Shaken HEK293 Cells

Jean-Philippe Gaudry et al. Methods Mol Biol. 2024.

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 using 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 three-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 7-day batch process, cell cultures are further processed using a set of methods for cell lysis and vector recovery. Methods for the purification of viral particles are described, including immunoaffinity and anion-exchange chromatography, ultrafiltration, as well as digital PCR to quantify the concentration of vector particles.

Keywords: Adeno-associated viral vector; Affinity chromatography; Anion-exchange purification; HEK293; Orbital shaken bioreactors; Suspension cell culture; Transient transfection.

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