Metabolic control of recombinant protein N‐glycan processing in NS0 and CHO cells
Abstract
Chinese hamster ovary and murine myeloma NS0 cells are currently favored host cell types for the production of therapeutic recombinant proteins. In this study, we compared N‐glycan processing in GS‐NS0 and GS‐CHO cells producing the same model recombinant glycoprotein, tissue inhibitor of metalloproteinases 1. By manipulation of intracellular nucleotide‐sugar content, we examined the feasibility of implementing metabolic control strategies aimed at reducing the occurrence of murine‐specific glycan motifs on NS0‐derived recombinant proteins, such as Galα1,3Galβ1,4GlcNAc. Although both CHO and NS0‐derived oligosaccharides were predominantly of the standard complex type with variable sialylation, 30% of N‐glycan antennae associated with NS0‐derived TIMP‐1 terminated in α1,3‐linked galactose residues. Furthermore, NS0 cells conferred a greater proportion of terminal N‐glycolylneuraminic (sialic) acid residues as compared with the N‐acetylneuraminic acid variant. Inclusion of the nucleotide‐sugar precursors, glucosamine (10 m M, plus 2 m M uridine) and N‐acetylmannosamine (20 m M), in culture media were shown to significantly increase the intracellular pools of UDP‐N‐acetylhexosamine and CMP‐sialic acid, respectively, in both NS0 and CHO cells. The elevated UDP‐N‐acetylhexosamine content induced by the glucosamine/uridine treatment was associated with an increase in the antennarity of N‐glycans associated with TIMP‐1 produced in CHO cells but not N‐glycans associated with TIMP‐1 from NS0 cells. In addition, elevated UDP‐N‐acetylhexosamine content was associated with a slight decrease in sialylation in both cell lines. The elevated CMP‐sialic acid content induced by N‐acetylmannosamine had no effect on the overall level of sialylation of TIMP‐1 produced by both CHO and NS0 cells, although the ratio of N‐glycolylneuraminic acid:N‐acetylneuraminic acid associated with NS0‐derived TIMP‐1 changed from 1:1 to 1:2. These data suggest that manipulation of nucleotide‐sugar metabolism can promote changes in N‐glycan processing that are either conserved between NS0 and CHO cells or specific to either NS0 cells or CHO cells. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 73: 188–202, 2001
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Approaches based on supplementation of cell culture media with precursors of the sialic acid pathway such as CMP-sialic acid, ManNAc and NeuNAc have limited and still debated effects.
“Some of these approaches are based on the supplementation of cell culture media with different precursors of the sialic acid pathway, such as CMP-sialic acid, ManNAc and NeuNAc, but their effects are limited and still debated (Gu and Wang, 1998;Baker et al., 2001;Hills et al., 2001;Bork et al., 2009;Hossler et al., 2009;Wong et al., 2010;Kildegaard et al., 2016).”
From Therapeutic glycoprotein production in mammalian cells (2017) · cites this paper as Baker et al., 2001
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Umaña, P., Jean–Mairet, J., Moudry, R., Amstutz, H., & Bailey, J. E. (1999) · Nature Biotechnology · doi:10.1038/6179 · Cited by 764 · see citing works
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Therapeutic glycoprotein production in mammalian cells
Lalonde, M.-E., & Durocher, Y. (2017) · Journal of Biotechnology · doi:10.1016/j.jbiotec.2017.04.028 · Cited by 296 · see citing works
Glycoengineering in CHO cells: advances in systems biology
Tejwani, V., Andersen, M. R., Nam, J. H., & Sharfstein, S. T. (2018) · Biotechnology Journal · doi:10.1002/biot.201700234 · Cited by 70 · see citing works
Lee, J. H., Jeong, Y. R., Kim, Y., & Lee, G. M. (2017) · Biotechnology and Bioengineering · doi:10.1002/bit.26284 · Cited by 34 · see citing works
Fan, L., Frye, C. C., & Racher, A. J. (2013) · Pharmaceutical Bioprocessing · doi:10.4155/pbp.13.56 · Cited by 35 · see citing works
Kim, S.-M., Chang, K.-H., & Oh, D. J. (2018) · Biotechnology and Bioprocess Engineering · doi:10.1007/s12257-018-0109-8 · Cited by 11 · see citing works
- Authors
- Baker, K. N., Rendall, M. H., Hills, A. E., Hoare, M., Freedman, R. B., & James, D. C.
- Affiliations
- University of Kent (GB)
- University College London (GB)
- Corresponding author
- James, David C.
- ORCID
- James, David C.
- Venue
- Biotechnology and Bioengineering
- Publisher
- Wiley
- Published
- 2001-03-02
- Volume
- 73
- Issue
- 3
- Pages
- 188-202
- ISSN
- 0006-3592 (ISSN-L)
- 1097-0290
- OpenAlex
- W1886156337
- Cited by
- 184
- References
- 1
- Citations by year
- 2026: 3 · 2025: 2 · 2024: 1 · 2023: 2 · 2022: 2 · 2021: 7 · 2020: 6 · 2019: 7 · 2018: 13 · 2017: 16 · 2016: 4 · 2015: 16 · 2014: 12 · 2013: 11 · 2012: 7
- FWCI
- 1.27
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Biotechnology and Bioengineering (publishedVersion)
- PubMed (publishedVersion)
- UCL Discovery (University College London) (submittedVersion)
- Kent Academic Repository (University of Kent) (submittedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Viral Infectious Diseases and Gene Expression in Insects
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Glycosylation and Glycoproteins Research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein purification and stability
- Keywords
- N-glycan processing, recombinant protein glycosylation, NS0 cells, CHO cells, TIMP-1, nucleotide sugar metabolism, UDP-N-acetylhexosamine, CMP-sialic acid, sialylation, N-glycolylneuraminic acid, N-acetylneuraminic acid, N-acetylmannosamine
- MeSH terms
- Animals, Cell Division, Chromatography, High Pressure Liquid, Galactose, Glucosamine, Glycosylation, Cricetinae, Hexosamines, Nucleotides, Polysaccharides, Recombinant Proteins, Sialyltransferases, Tumor Cells, Cultured, CHO Cells, Bioreactors, N-Acetylneuraminic Acid, Tissue Inhibitor of Metalloproteinase-1, Mice
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