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Quantification of Polyethylenimine in Transient Gene Expression: On the Way to GMP Compliance

  • Sophie Nallet
  • Zuzana Kadlecova
  • Lucia Baldi
  • Harm-Anton Klok
  • Florian M. Wurm
Conference paper
Part of the ESACT Proceedings book series (ESACT, volume 5)

Abstract

Transient gene expression (TGE) allows production of virtually any recombinant protein (r-protein) in mammalian cells. Its flexibility, speed, scalability, and cost-effectiveness have been widely demonstrated. However, good manufacturing practices (GMP) have not been established for the production of r-proteins by TGE. In this study, a method was developed for the detection and quantification of polyethylenimine (PEI), the DNA delivery agent in TGE. Currently, there are no established methods to track this polymer during r-protein production and purification. Linear 25 kDa PEI was labelled with fluorescein, and the modified PEI was characterized by NMR and UV/VIS spectroscopy. The optimal conditions for an accurate measurement of PEI by fluorescence were defined, and the limit of detection and quantification were determined. Importantly, the labeling of PEI did not alter its capacity to form polyplexes with plasmid DNA and to efficiently transfect HEK-293 cells in suspension. The assay we developed is expected to be an essential tool for the establishment of GMP protocols for the production of r-proteins by TGE.

Keywords

Fluorescence Signal Good Manufacturing Practice Transient Gene Expression Buffer Composition Linear Standard Curve 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  • Sophie Nallet
    • 1
  • Zuzana Kadlecova
    • 2
  • Lucia Baldi
    • 1
  • Harm-Anton Klok
    • 2
  • Florian M. Wurm
    • 1
  1. 1.Laboratory of Cellular Biotechnology, School of Life SciencesÉcole Polytechnique Fédérale de LausanneLausanneSwitzerland
  2. 2.Laboratory of PolymersÉcole Polytechnique Fédérale de Lausanne, Institute of MaterialsLausanneSwitzerland

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