Enhanced sialylation of recombinant human erythropoietin in Chinese hamster ovary cells by combinatorial engineering of selected genes
Abstract
Therapeutic glycoproteins with exposed galactose (Gal) residues are cleared rapidly from the bloodstream by asialoglycoprotein receptors in hepatocytes. Various approaches have been used to increase the content of sialic acid, which occupies terminal sites of N- or O-linked glycans and thereby increases the half-life of therapeutic glycoproteins. We enhanced sialylation of human erythropoietin (EPO) by genetic engineering of the sialylation pathway in Chinese hamster ovary (CHO) cells. The enzyme GNE (uridine diphosphate-N-acetyl glucosamine 2-epimerase)/MNK (N-acetyl mannosamine kinase), which plays a key role in the initial two steps of sialic acid biosynthesis, is regulated by cytidine monophosphate (CMP)-sialic acid through a feedback mechanism. Since sialuria patient cells fail in regulating sialic acid biosynthesis by feedback mechanism, various sialuria-like mutated rat GNEs were established and subjected to in vitro activity assay. GNE/MNK-R263L-R266Q mutant showed 93.6% relative activity compared with wild type and did not display feedback inhibition. Genes for sialuria-mutated rat GNE/MNK, Chinese hamster CMP-sialic acid transporter and human α2,3-sialyltransferase (α2,3-ST) were transfected simultaneously into recombinant human (rh) EPO-producing CHO cells. CMP-sialic acid concentration of engineered cells was significantly (>10-fold) increased by sialuria-mutated GNE/MNK (R263L-R266Q) expression. The sialic acid content of rhEPO produced from engineered cells was…
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An engineered CHO cell line stably expressing the human α2,3-sialyltransferase ST3Gal3, the rat mutated GNE/MNK-R263L-R266Q glycosylation enzymes and the Chinese hamster CMP-sialic acid transporter was successful in increasing tetra-sialylation of recombinant human EPO.
“An engineered CHO cell line stably expressing the human α2,3-sialyltransferase ST3Gal3, the rat mutated GNE/MNK-R263L-R266Q glycosylation enzymes and the Chinese hamster CMP-sialic acid transporter was successful in increasing tetra-sialylation of recombinant human EPO (Son et al., 2011).”
From Therapeutic glycoprotein production in mammalian cells (2017) · cites this paper as Son et al., 2011
Cites
RNA interference of sialidase improves glycoprotein sialic acid content consistency
Ngantung, F. A., Miller, P. G., Brushett, F. R., Tang, G. L., & Wang, D. I. C. (2006) · Biotechnology and Bioengineering · doi:10.1002/bit.20997 · Cited by 97 · 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
Glycoengineering Chinese hamster ovary cells: a short history
Donini, R., Haslam, S. M., & Kontoravdi, C. (2021) · Biochemical Society Transactions · doi:10.1042/bst20200840 · Cited by 37 · see citing works
Improving sialylation of recombinant biologics for enhanced therapeutic efficacy
Chan, K. F., Goh, J. S., & Song, Z. (2014) · Pharmaceutical Bioprocessing · doi:10.4155/pbp.14.44 · Cited by 4 · see citing works
- Authors
- Son, Y.-D., Jeong, Y. T., Park, S.-Y., & Kim, J. H.
- Affiliations
- Korea Advanced Institute of Science and Technology (KR)
- Venue
- Glycobiology
- Publisher
- Oxford University Press (OUP)
- Published
- 2011-03-24
- Volume
- 21
- Issue
- 8
- Pages
- 1019-1028
- ISSN
- 0959-6658 (ISSN-L)
- 1460-2423
- OpenAlex
- W2126371691
- Cited by
- 73
- References
- 1
- Citations by year
- 2026: 3 · 2025: 2 · 2024: 2 · 2023: 3 · 2022: 3 · 2021: 5 · 2020: 3 · 2019: 5 · 2018: 9 · 2017: 5 · 2016: 4 · 2015: 6 · 2014: 5 · 2013: 7 · 2012: 9
- FWCI
- 2.85
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Glycobiology (publishedVersion)
- PubMed (publishedVersion)
- KAIST Institutional Repository (KAIST) (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
- Physical Sciences › Chemistry › Organic Chemistry › Carbohydrate Chemistry and Synthesis
- Keywords
- sialylation, recombinant human erythropoietin, sialic acid biosynthesis, CMP-sialic acid, CMP-sialic acid transporter, alpha2,3-sialyltransferase, asialoglycoprotein receptor, CHO cell engineering, product inhibition
- MeSH terms
- Animals, Carbohydrate Epimerases, Cricetulus, Cytidine Monophosphate, Erythropoietin, Genetic Engineering, Cricetinae, Humans, Sialyltransferases, CHO Cells, Phosphotransferases (Alcohol Group Acceptor), N-Acetylneuraminic Acid, Rats
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