Biotechnology Letters

, Volume 29, Issue 11, pp 1713–1721 | Cite as

High-titer, serum-free production of adeno-associated virus vectors by polyethyleneimine-mediated plasmid transfection in mammalian suspension cells

  • Markus Hildinger
  • Lucia Baldi
  • Matthieu Stettler
  • Florian M. Wurm
Original Research Paper


Adeno-associated virus (AAV)-based vectors belong to the most promising gene transfer vectors in clinical studies. To provide vector for late-stage clinical trials as well as for a potential commercial phase, a scalable, cGMP-compliant process is required. Nearly all vector production protocols currently approved in Phase I clinical trials rely on AAV production in adherent HEK 293 cells in the presence of serum. In this study, we present a helper- and serum-free production method of AAV vectors in suspension-adapted HEK 293 cells. The method is based on plasmid transfection with 25 kDa linear polyethyleneimine. Compared to existing methods, our system is highly scalable as cells grow in suspension, does not require animal-derived products or the use of an exogenous virus (adenovirus or baculovirus) and yields genomic titers equal to those obtained in adherent HEK 293 cells in the presence of serum. Most importantly, work load and cost could be dramatically reduced in comparison to earlier methods, when comparing the production of equivalent volumes of cell culture media. Thus, our protocol should appeal to both basic research laboratories and cGMP manufacturing units.


Adeno-associated virus Gene therapy GMP Production Serum-free Suspension 



This work was financially supported by a KTI-grant obtained from the Swiss government.


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Copyright information

© Springer Science+Business Media B.V. 2007

Authors and Affiliations

  • Markus Hildinger
    • 1
  • Lucia Baldi
    • 2
  • Matthieu Stettler
    • 2
  • Florian M. Wurm
    • 1
    • 2
  1. 1.ExcellGene S.AMontheySwitzerland
  2. 2.Institute of Biological Engineering and Biotechnology, Laboratory of Cellular Biotechnology Swiss Federal Institute of Technology (EPFL)LausanneSwitzerland

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