title: Glycoengineering of Chinese hamster ovary cells for enhanced erythropoietin N-glycan branching and sialylation.
abstract: Sialic acid, a terminal residue on complex N-glycans, and branching or antennarity can play key roles in both the biological activity and circulatory lifetime of recombinant glycoproteins of therapeutic interest. In order to examine the impact of glycosyltransferase expression on the N-glycosylation of recombinant erythropoietin (rEPO), a human α2,6-sialyltransferase (ST6Gal1) was expressed in Chinese hamster ovary (CHO-K1) cells. Sialylation increased on both EPO and CHO cellular proteins as observed by SNA lectin analysis, and HPLC profiling revealed that the sialic acid content of total glycans on EPO increased by 26%. The increase in sialic acid content was further verified by detailed profiling of the N-glycan structures using mass spectra (MS) analysis. In order to enhance antennarity/branching, UDP-N-acetylglucosamine: α-1,3-D-mannoside β1,4-N-acetylglucosaminyltransferase (GnTIV/Mgat4) and UDP-N-acetylglucosamine:α-1,6-D-mannoside β1,6-N-acetylglucosaminyltransferase (GnTV/Mgat5), was incorporated into CHO-K1 together with ST6Gal1. Tri- and tetraantennary N-glycans represented approximately 92% of the total N-glycans on the resulting EPO as measured using MS analysis. Furthermore, sialic acid content of rEPO from these engineered cells was increased ∼45% higher with tetra-sialylation accounting for ∼10% of total sugar chains compared to ∼3% for the wild-type parental CHO-K1. In this way, coordinated overexpression of these three glycosyltransferases for the first time in model CHO-K1 cell lines provides a mean for enhancing both N-glycan branching complexity and sialylation with opportunities to generate tailored complex N-glycan structures on therapeutic glycoproteins in the future.
authors.affiliation: Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland.
authors.forename: Bojiao
authors.initials: B
authors.lastname: Yin
authors.affiliation: Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Gao
authors.affiliation: Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland.
authors.forename: Cheng-Yu
authors.initials: CY
authors.lastname: Chung
authors.affiliation: Department of Pathology, Johns Hopkins University, Baltimore, Maryland.
authors.forename: Shuang
authors.initials: S
authors.lastname: Yang
authors.affiliation: Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland.
authors.forename: Emily
authors.initials: E
authors.lastname: Blake
authors.affiliation: Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland.
authors.forename: Mark C
authors.initials: MC
authors.lastname: Stuczynski
authors.affiliation: Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland.
authors.forename: Juechun
authors.initials: J
authors.lastname: Tang
authors.affiliation: Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hoersholm, Denmark.
authors.forename: Helene F
authors.initials: HF
authors.lastname: Kildegaard
authors.affiliation: Department of Systems Biology, Technical University of Denmark, Kgs. Lyngby, Denmark.
authors.forename: Mikael R
authors.initials: MR
authors.lastname: Andersen
authors.affiliation: Department of Pathology, Johns Hopkins University, Baltimore, Maryland.
authors.forename: Hui
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland. beten@jhu.edu.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Betenbaugh
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003412:Cricetulus; D004921:Erythropoietin / Q000378:metabolism*; D005260:Female; D006031:Glycosylation; D016695:Glycosyltransferases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D037102:Lectins / Q000378:metabolism; D013058:Mass Spectrometry; D060847:Metabolic Engineering; D011134:Polysaccharides / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism*
chemical_list: D037102:Lectins; D011134:Polysaccharides; D011994:Recombinant Proteins; D004921:Erythropoietin; D016695:Glycosyltransferases
keywords: CHO; GNTIV/Mgat4; GNTV/Mgat5; N-glycosylation; erythropoietin; sialic acid