title: Understanding of decreased sialylation of Fc-fusion protein in hyperosmotic recombinant Chinese hamster ovary cell culture: N-glycosylation gene expression and N-linked glycan antennary profile.
abstract: To understand the effects of hyperosmolality on protein glycosylation, recombinant Chinese hamster ovary (rCHO) cells producing the Fc-fusion protein were cultivated in hyperosmolar medium resulting from adding NaCl (415 mOsm/kg). The hyperosmotic culture showed increased specific Fc-fusion protein productivity (qFc) but a decreased proportion of acidic isoforms and sialic acid content of the Fc-fusion protein. The intracellular and extracellular sialidase activities in the hyperosmotic cultures were similar to those in the control culture (314 mOsm/kg), indicating that reduced sialylation of Fc-fusion protein at hyperosmolality was not due to elevated sialidase activity. Expression of 52 N-glycosylation-related genes was assessed by the NanoString nCounter system, which provides a direct digital readout using custom-designed color-coded probes. After 3 days of hyperosmotic culture, nine genes (ugp, slc35a3, slc35d2, gcs1, manea, mgat2, mgat5b, b4galt3, and b4galt4) were differentially expressed over 1.5-fold of the control, and all these genes were down-regulated. N-linked glycan analysis by anion exchange and hydrophilic interaction HPLC showed that the proportion of highly sialylated (di-, tri-, tetra-) and tetra-antennary N-linked glycans was significantly decreased upon hyperosmotic culture. Addition of betaine, an osmoprotectant, to the hyperosmotic culture significantly increased the proportion of highly sialylated and tetra-antennary N-linked glycans (P ≤ 0.05), while it increased the expression of the N-glycan branching/antennary genes (mgat2 and mgat4b). Thus, decreased expression of the genes with roles in the N-glycan biosynthesis pathway correlated with reduced sialic acid content of Fc-fusion protein caused by hyperosmolar conditions. Taken together, the results obtained in this study provide a better understanding of the detrimental effects of hyperosmolality on N-glycosylation, especially sialylation, in rCHO cells. Biotechnol. Bioeng. 2017;114: 1721-1732. © 2017 Wiley Periodicals, Inc.
authors.affiliation: Department of Biological Sciences, KAIST, 335 Gwahak-ro, Yuseong-gu, Daejeon 305-701, Republic of Korea.
authors.forename: Jong Hyun
authors.initials: JH
authors.lastname: Lee
authors.affiliation: Department of Biological Sciences, KAIST, 335 Gwahak-ro, Yuseong-gu, Daejeon 305-701, Republic of Korea.
authors.forename: Yeong Ran
authors.initials: YR
authors.lastname: Jeong
authors.affiliation: Biotechnology Process Engineering Center, KRIBB, Ochang-eup, Cheongwon-gu, Cheongju, Republic of Korea.|Department of Bioprocess Engineering, Korea University of Science and Technology (UST), Gajeong-ro, Yuseong-gu, Daejeon, Republic of Korea.
authors.forename: Yeon-Gu
authors.initials: YG
authors.lastname: Kim
authors.affiliation: Department of Biological Sciences, KAIST, 335 Gwahak-ro, Yuseong-gu, Daejeon 305-701, Republic of Korea.
authors.forename: Gyun Min
authors.identifier: 0000-0002-9876-7378
authors.initials: GM
authors.lastname: Lee
mesh_terms: D000818:Animals; D016466:CHO Cells; D003412:Cricetulus; D006031:Glycosylation; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry* / Q000378:metabolism*; D009997:Osmotic Pressure / Q000502:physiology*; D011134:Polysaccharides / Q000737:chemistry* / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D007141:Immunoglobulin Fc Fragments; D011134:Polysaccharides; D011993:Recombinant Fusion Proteins
keywords: CHO cells; Fc-fusion protein; N-glycosylation; betaine; hyperosmolality; nanostring nCounter system