title: Multi-level glyco-engineering techniques to generate IgG with defined Fc-glycans.
abstract: Immunoglobulin G (IgG) mediates its immune functions through complement and cellular IgG-Fc receptors (FcγR). IgG contains an evolutionary conserved N-linked glycan at position Asn297 in the Fc-domain. This glycan consists of variable levels of fucose, galactose, sialic acid, and bisecting N-acetylglucosamine (bisection). Of these variations, the lack of fucose strongly enhances binding to the human FcγRIII, a finding which is currently used to improve the efficacy of therapeutic monoclonal antibodies. The influence of the other glycan traits is largely unknown, mostly due to lack of glyco-engineering tools. We describe general methods to produce recombinant proteins of any desired glycoform in eukaryotic cells. Decoy substrates were used to decrease the level of fucosylation or galactosylation, glycosyltransferases were transiently overexpressed to enhance bisection, galactosylation and sialylation and in vitro sialylation was applied for enhanced sialylation. Combination of these techniques enable to systematically explore the biological effect of these glycosylation traits for IgG and other glycoproteins.
authors.affiliation: Sanquin Research, Department Experimental Immunohematology, Amsterdam, The Netherlands, and Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Gillian
authors.initials: G
authors.lastname: Dekkers
authors.affiliation: Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Rosina
authors.initials: R
authors.lastname: Plomp
authors.affiliation: Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Carolien A M
authors.initials: CA
authors.lastname: Koeleman
authors.affiliation: Sanquin Research, Department Experimental Immunohematology, Amsterdam, The Netherlands, and Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Remco
authors.initials: R
authors.lastname: Visser
authors.affiliation: ProBioGen AG, Berlin, Germany.|HTW-Berlin University of Applied Sciences, Life Science Engineering, Berlin, Germany.
authors.forename: Hans H
authors.initials: HH
authors.lastname: von Horsten
authors.affiliation: ProBioGen AG, Berlin, Germany.
authors.forename: Volker
authors.initials: V
authors.lastname: Sandig
authors.affiliation: Sanquin Research, Department Immunopathology, Amsterdam, The Netherlands, and Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Theo
authors.initials: T
authors.lastname: Rispens
authors.affiliation: Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Manfred
authors.initials: M
authors.lastname: Wuhrer
authors.affiliation: Sanquin Research, Department Experimental Immunohematology, Amsterdam, The Netherlands, and Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Gestur
authors.initials: G
authors.lastname: Vidarsson