title: A novel sugar analog enhances sialic acid production and biotherapeutic sialylation in CHO cells.
abstract: A desirable feature of many therapeutic glycoprotein production processes is to maximize the final sialic acid content. In this study, the effect of applying a novel chemical analog of the sialic acid precursor N-acetylmannosamine (ManNAc) on the sialic acid content of cellular proteins and a model recombinant glycoprotein, erythropoietin (EPO), was investigated in CHO-K1 cells. By introducing the 1,3,4-O-Bu3ManNAc analog at 200-300 µM into cell culture media, the intracellular sialic acid content of EPO-expressing cells increased ∼8-fold over untreated controls while the level of cellular sialylated glycoconjugates increased significantly as well. For example, addition of 200-300 µM 1,3,4-O-Bu3ManNAc resulted in >40% increase in final sialic acid content of recombinant EPO, while natural ManNAc at ∼100 times higher concentration of 20 mM produced a less profound change in EPO sialylation. Collectively, these results indicate that butyrate-derivatization of ManNAc improves the capacity of cells to incorporate exogenous ManNAc into the sialic acid biosynthetic pathway and thereby increase sialylation of recombinant EPO and other glycoproteins. This study establishes 1,3,4-O-Bu3ManNAc as a novel chemical supplement to improve glycoprotein quality and sialylation levels at concentrations orders of magnitude lower than alternative approaches. Biotechnol. Bioeng. 2017;114: 1899-1902. © 2017 Wiley Periodicals, Inc.
authors.affiliation: Department of Chemical and Biomolecular Engineering, Johns Hopkins University, 221 Maryland Hall, 3400 N. Charles St., Baltimore, Maryland 21218.
authors.forename: Bojiao
authors.initials: B
authors.lastname: Yin
authors.affiliation: Department of Chemical and Biomolecular Engineering, Johns Hopkins University, 221 Maryland Hall, 3400 N. Charles St., Baltimore, Maryland 21218.
authors.forename: Qiong
authors.initials: Q
authors.lastname: Wang
authors.affiliation: Department of Chemical and Biomolecular Engineering, Johns Hopkins University, 221 Maryland Hall, 3400 N. Charles St., Baltimore, Maryland 21218.
authors.forename: Cheng-Yu
authors.initials: CY
authors.lastname: Chung
authors.affiliation: Department of Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland.
authors.forename: Rahul
authors.initials: R
authors.lastname: Bhattacharya
authors.affiliation: Department of Chemical and Biomolecular Engineering, Johns Hopkins University, 221 Maryland Hall, 3400 N. Charles St., Baltimore, Maryland 21218.
authors.forename: Xiaozhi
authors.initials: X
authors.lastname: Ren
authors.affiliation: Department of Chemical and Biomolecular Engineering, Johns Hopkins University, 221 Maryland Hall, 3400 N. Charles St., Baltimore, Maryland 21218.
authors.forename: Juechun
authors.initials: J
authors.lastname: Tang
authors.affiliation: Department of Chemical and Biomolecular Engineering, Johns Hopkins University, 221 Maryland Hall, 3400 N. Charles St., Baltimore, Maryland 21218.|Department of Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland.
authors.forename: Kevin J
authors.initials: KJ
authors.lastname: Yarema
authors.affiliation: Department of Chemical and Biomolecular Engineering, Johns Hopkins University, 221 Maryland Hall, 3400 N. Charles St., Baltimore, Maryland 21218.
authors.forename: Michael J
authors.identifier: 0000-0002-6336-4659
authors.initials: MJ
authors.lastname: Betenbaugh
mesh_terms: D000818:Animals; D016466:CHO Cells; D003412:Cricetulus; D003470:Culture Media / Q000737:chemistry / Q000378:metabolism; D004921:Erythropoietin / Q000096:biosynthesis* / Q000235:genetics*; D006595:Hexosamines / Q000378:metabolism*; D019158:N-Acetylneuraminic Acid / Q000096:biosynthesis* / Q000302:isolation & purification; D009844:Oligosaccharides; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D003470:Culture Media; D006595:Hexosamines; D009844:Oligosaccharides; D011994:Recombinant Proteins; D004921:Erythropoietin; D019158:N-Acetylneuraminic Acid; C002022:N-acetylmannosamine
keywords: Chinese hamster ovary (CHO) media; N-acetylmannosamine (ManNAc) analog; glycosylation; metabolic oligosaccharide engineering; sialylation