CRISPR/Cas9-multiplexed editing of Chinese hamster ovary B4Gal-T1, 2, 3 and 4 Tailors N-Glycan Profiles of Therapeutics and Secreted Host Cell Proteins

Thomas Amann, Anders Holmgaard Hansen*, Stefan Kol, Gyun Min Lee, Mikael Rørdam Andersen, Helene Faustrup Kildegaard

*Corresponding author for this work

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In production of recombinant proteins for biopharmaceuticals, N-glycosylation is often important for protein efficacy and patient safety. IgG with agalactosylated (G0)-N-glycans can improve the activation of the lectin-binding complement system and be advantageous in the therapy of lupus and virus diseases. In this study, the authors aimed to engineer CHO-S cells for the production of proteins with G0-N-glycans by targeting B4Gal-T isoform genes with CRISPR/Cas9. Indel mutations in genes encoding B4Gal-T1, -T2, and -T3 with and without a disrupted B4Gal-T4 sequence resulted in only ≈1% galactosylated N-glycans on total secreted proteins of 3-4 clones per genotype. The authors revealed that B4Gal-T4 is not active in N-glycan galactosylation in CHO-S cells. In the triple-KO clones, transiently expressed erythropoietin (EPO) and rituximab harbored only ≈6% and ≈3% galactosylated N-glycans, respectively. However, simultaneous disruption of B4Gal-T1 and -T3 may decrease cell growth. Altogether, the authors present the advantage of analyzing total secreted protein N-glycans after disrupting galactosyltransferases, followed by expressing recombinant proteins in selected clones with desired N-glycan profiles at a later stage. Furthermore, the authors provide a cell platform that prevalently glycosylates proteins with G0-N-glycans to further study the impact of agalactosylation on different in vitro and in vivo functions of recombinant proteins.
Original languageEnglish
Article number1800111
JournalBiotechnology Journal
Issue number10
Number of pages9
Publication statusPublished - 2018


  • Chinese hamster ovary cells
  • CRISP/Cas9
  • Erythropoietin
  • Glycoengineering
  • Multiplexing
  • N‐glycosylation
  • Rituximab

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