title: Chinese hamster ovary (CHO) host cell engineering to increase sialylation of recombinant therapeutic proteins by modulating sialyltransferase expression.
abstract: N-Glycans of human proteins possess both α2,6- and α2,3-linked terminal sialic acid (SA). Recombinant glycoproteins produced in Chinese hamster overy (CHO) only have α2,3-linkage due to the absence of α2,6-sialyltransferase (St6gal1) expression. The Chinese hamster ST6GAL1 was successfully overexpressed using a plasmid expression vector in three recombinant immunoglobulin G (IgG)-producing CHO cell lines. The stably transfected cell lines were enriched for ST6GAL1 overexpression using FITC-Sambucus nigra (SNA) lectin that preferentially binds α2,6-linked SA. The presence of α2,6-linked SA was confirmed using a novel LTQ Linear Ion Trap Mass Spectrometry (LTQ MS) method including MSn fragmentation in the enriched ST6GAL1 Clone 27. Furthermore, the total SA (mol/mol) in IgG produced by the enriched ST6GAL1 Clone 27 increased by 2-fold compared to the control. For host cell engineering, the CHOZN(®) GS host cell line was transfected and enriched for ST6GAL1 overexpression. Single-cell clones were derived from the enriched population and selected based on FITC-SNA staining and St6gal1 expression. Two clones ("ST6GAL1 OE Clone 31 and 32") were confirmed for the presence of α2,6-linked SA in total host cell protein extracts. ST6GAL1 OE Clone 32 was subsequently used to express SAFC human IgG1. The recombinant IgG expressed in this host cell line was confirmed to have α2,6-linked SA and increased total SA content. In conclusion, overexpression of St6gal1 is sufficient to produce recombinant proteins with increased sialylation and more human-like glycoprofiles without combinatorial engineering of other sialylation pathway genes. This work represents our ongoing effort of glycoengineering in CHO host cell lines for the development of "bio-better" protein therapeutics and cell culture vaccine production.
authors: Cell Sciences and Development, SAFC/Sigma-Aldrich, 2909 Laclede Avenue, Saint Louis, MO, 63103.
authors: Nan
authors: N
authors: Lin
authors: Joaquina
authors: J
authors: Mascarenhas
authors: Natalie R
authors: NR
authors: Sealover
authors: Henry J
authors: HJ
authors: George
authors: Jeanne
authors: J
authors: Brooks
authors: Kevin J
authors: KJ
authors: Kayser
authors: Brian
authors: B
authors: Gau
authors: Isil
authors: I
authors: Yasa
authors: Parastoo
authors: P
authors: Azadi
authors: Stephanie
authors: S
authors: Archer-Hartmann
mesh_terms: D000818:Animals; D016466:CHO Cells; D060846:Cell Engineering / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D060847:Metabolic Engineering; D019158:N-Acetylneuraminic Acid / Q000032:analysis / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism*; D012799:Sialyltransferases / Q000378:metabolism*
chemical_list: D011134:Polysaccharides; D011994:Recombinant Proteins; D012799:Sialyltransferases; D019158:N-Acetylneuraminic Acid
keywords: Chinese hamster ovary cells; St6gal1; glycoengineering; host cell engineering; α2,6-linked sialic acid
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