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Glyco-engineered HEK 293-F cell lines for the production of therapeutic glycoproteins with human N-glycosylation and improved pharmacokinetics.

  • Uhler, Rico1, 2
  • Popa-Wagner, Ruth2
  • Kröning, Mario3
  • Brehm, Anja3
  • Rennert, Paul3
  • Seifried, Annegrit2
  • Peschke, Madeleine2
  • Krieger, Markus2
  • Kohla, Guido3
  • Kannicht, Christoph2, 3
  • Wiedemann, Philipp1
  • Hafner, Mathias1, 4
  • Rosenlöcher, Julia3
  • 1 Institute of Molecular and Cell Biology, Mannheim University of Applied Sciences, 68163 Mannheim, Germany. , (Germany)
  • 2 Octapharma Biopharmaceuticals GmbH, 69120 Heidelberg, Germany. , (Germany)
  • 3 Octapharma Biopharmaceuticals GmbH, 12489 Berlin, Germany. , (Germany)
  • 4 Institute for Medical Technology, University Heidelberg and the Mannheim University of Applied Sciences, 68163 Mannheim, Germany. , (Germany)
Published Article
Oxford University Press
Publication Date
Jan 05, 2021
DOI: 10.1093/glycob/cwaa119
PMID: 33403396


N-glycosylated proteins produced in human embryonic kidney 293 (HEK 293) cells often carry terminal N-acetylgalactosamine (GalNAc) and only low levels of sialylation. On therapeutic proteins, such N-glycans often trigger rapid clearance from the patient bloodstream via efficient binding to asialoglycoprotein receptor (ASGP-R) and mannose receptor (MR). This currently limits the use of HEK 293 cells for therapeutic protein production. To eliminate terminal GalNAc, we knocked-out GalNAc transferases B4GALNT3 and B4GALNT4 by CRISPR/Cas9 in FreeStyle 293-F cells. The resulting cell line produced a coagulation factor VII-albumin fusion protein without GalNAc but with increased sialylation. This glyco-engineered protein bound less efficiently to both the ASGP-R and MR in vitro and it showed improved recovery, terminal half-life and area under the curve in pharmacokinetic rat experiments. By overexpressing sialyltransferases ST6GAL1 and ST3GAL6 in B4GALNT3 and B4GALNT4 knock-out cells, we further increased factor VII-albumin sialylation; for ST6GAL1 even to the level of human plasma-derived factor VII. Simultaneous knock-out of B4GALNT3 and B4GALNT4, and overexpression of ST6GAL1 further lowered factor VII-albumin binding to ASGP-R and MR. This novel glyco-engineered cell line is well-suited for the production of factor VII-albumin and presumably other therapeutic proteins with fully human N-glycosylation and superior pharmacokinetic properties. © The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: [email protected]

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