Choice of host cell line is essential for the functional glycosylation of the Fc region of human IgG1 inhibitors of influenza B viruses
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Abstract
Abs are glycoproteins that carry a conserved N-linked carbohydrate attached to the Fc whose presence and fine structure profoundly impacts on their in vivo immunogenicity, pharmacokinetics, and functional attributes. The host cell line used to produce IgG plays a major role in this glycosylation, as different systems express different glycosylation enzymes and transporters that contribute to the specificity and heterogeneity of the final IgG-Fc glycosylation profile. In this study, we compare two panels of glycan-adapted IgG1-Fc mutants expressed in either the human endothelial kidney 293-F or Chinese hamster ovary–K1 systems. We show that the types of N-linked glycans between matched pairs of Fc mutants vary greatly and in particular, with respect, to sialylation. These cell line effects on glycosylation profoundly influence the ability of the engineered Fcs to interact with either human or pathogen receptors. For example, we describe Fc mutants that potently disrupted influenza B–mediated agglutination of human erythrocytes when expressed in Chinese hamster ovary–K1, but not in human endothelial kidney 293-F cells.
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Some hypersialylated mutants bind influenza viruses with high affinity and inhibit influenza-mediated hemagglutination.
“Some of these hypersialylated mutants were also found to bind influenza viruses with high-affinity and inhibit influenza-mediated hemagglutination [121,122].”
From Glycoengineering Chinese hamster ovary cells: a short history (2021) · cites this paper as [122]
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BackgroundAuthor response: Influenza A virus surface proteins are organized to help penetrate host mucus
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Li, S., McCraw, A. J., Gardner, R. A., Spencer, D. I. R., Karagiannis, S. N., & Wagner, G. K. (2021) · Antibodies · doi:10.3390/antib10040044 · Cited by 27 · see citing works
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“For practical utility as a vaccine or research reagent, a spike immunogen must be able to be efficiently produced, properly folded, and natively glycosylated in cell lines appropriate for industrial production (Blundell et al. 2020; Raska et al. 2010).”
BackgroundQuantitative profiling of N-glycosylation of SARS-CoV-2 spike protein variants
Xie, Y., & Butler, M. (2023) · Glycobiology · doi:10.1093/glycob/cwad007 · Cited by 23 · see citing works
“…been well established that the glycosylation profiles of wild type or recombinant glycoproteins produced in different cellular expression systems may differ (Yu et al. 2011; Croset et al. 2012; Bohm et al. 2015; Goh and Ng 2018; Blundell et al. 2020), and SARS-CoV-2 spike protein is no exception.”
The therapeutic potential of sialylated Fc domains of human IgG
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“that are not attached to any of the inhibitory Fcs expressed in either CHO or HEK cells.(51) Because this specificity is also shared with the 2009 pandemic H1N1, enhanced Fc blockers may potentially be created by manufacturing these molecules”
BackgroundInfluentialSelf-citationThe MUC2 Gene Product: Polymerisation and Post-Secretory Organisation—Current Models
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“Moreover, humans cannot attach N-glycolylneuraminic acid, whereas many non-mammalian cells like CHO cells can [103].”
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“The degree of glycosylation can be influenced by cell lines,[7,24] cell culture conditions,[21,25] andmedia composition.”
BackgroundHuman IgG Subclasses Differ in the Structural Elements of Their N -Glycosylation
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Serum immunoglobulin and the threshold of Fc receptor-mediated immune activation
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“F cells compared to CHO-K1 cells, which was speculated to be due increased sialyation at this site [380].”
Glycoprofile Comparison of the SARS-CoV-2 Spike Proteins Expressed in CHO and HEK Cell Lines
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“However, differences in the glycosylation machinery exhibited by different cell types can produce vastly altered glycoforms of the spike protein, which may impact properties such as circulating half-life, binding affinity, thermal stability and potency for immune response [27–30].”
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Genetic Divergence and Antibody Expression Influence the N‐Glycomes of CHO‐K1 and CHO‐S Cells
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Self-citationSynthetic RNA–protein decoy granules prevent SARS-CoV-2 infection
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Vrettou, C., Blundell, P. A., Gaunt, E. R., & Pleass, R. J. (2022) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2022.10.06.511107 · Cited by 1 · see citing works
“Although HIA is a good correlate for the potential in vivo efficacy of these reagents, the findings need to be supported with more demanding virus neutralisation experiments with permissive cell lines that better mimic the human lung infected with live virus.”
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Willinger, O., Granik, N., Goldberg, S., & Amit, R. (2024) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2024.08.28.610020
Modulation of N-glycosylation in bispecific antibody biosimilars through combined modulators
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“Contrastingly, the N-glycans of an Asn297Ala/Cys575Ala mutant exhibit a considerably more glycan heterogeneity, including: antennary fucosylation, sialylation, LacdiNAc, tri-and tetra-antennary complex structures [72].”
InfluentialPairing of high-mannose glycans in human IgE-Fc: implications for secretion and stability
Nomura, S., Iwabuchi, T., Sawataishi, H., Kamo, K., Manabe, N., Uematsu, R., Sakamoto, I., Hirose, K., Kwon, E., & Yamaguchi, Y. (2026) · Clinical Proteomics · doi:10.1186/s12014-026-09616-1
“It is possible that the glycosylation profiles obtained from Expi293F cells may not fully reflect those of endogenous IgE produced by B cells, particularly at complex-type glycosylation sites, where cell-type-specific differences in galactosylation and sialylation are known to occur [25, 26].”
Fc and tailpiece determinants of IgM tailpiece-mediated IgG hexamerisationPreprint
Peters, S. J., Sopp, J. M., Ede, J., Ahdash, Z., Hailu, H., Hold, A., Holmes, B., Cragg, M. S., & Humphreys, D. (2026) · Research Square · doi:10.21203/rs.3.rs-10432191/v1
- Authors
- Blundell, P. A., Lu, D., Dell, A., Haslam, S., & Pleass, R. J.
- Affiliations
- Liverpool School of Tropical Medicine (GB)
- Imperial College London (GB)
- Corresponding author
- Pleass, Richard J
- Venue
- The Journal of Immunology
- Publisher
- Oxford University Press (OUP)
- Published
- 2020-02
- Volume
- 204
- Issue
- 4
- Pages
- 1022-1034
- ISSN
- 0022-1767 (ISSN-L)
- 1550-6606
- PMC
- PMC6994840
- OpenAlex
- W2999940487
- Cited by
- 29
- References
- 63
- Citations by year
- 2026: 4 · 2025: 2 · 2024: 7 · 2023: 5 · 2022: 3 · 2021: 7 · 2020: 1
- FWCI
- 1.30
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Locations
- The Journal of Immunology (publishedVersion, cc-by)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- LSTM Online Archive (Liverpool School of Tropical Medicine) (acceptedVersion)
- Funders
- Wellcome Trust (Z/17/Z, 109469/Z/15/Z, 097830/Z/11/Z, 208938, 208938/Z/17/Z, /Z/15/Z, 219576/Z/19/Z)
- International Seafood Sustainability Foundation
- Directorate for Biological Sciences
- Medical Research Council (MC_PC_12017, MC_PC_16078)
- Biotechnology and Biological Sciences Research Council (BB/F008309/1)
- 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
- Health Sciences › Medicine › Radiology, Nuclear Medicine and Imaging › Monoclonal and Polyclonal Antibodies Research
- Keywords
- Fc glycosylation, IgG1, influenza B, influenza antibodies, CHO-K1 cells, sialylation, N-glycans, Fc receptor binding, hemagglutination, antibody glycosylation
- MeSH terms
- Animals, Antibody Specificity, Cricetulus, Glycoproteins, Glycosylation, Cricetinae, Humans, Immunoglobulin G, Immunoglobulin Fc Fragments, Influenza, Human, Influenza B virus, CHO Cells, HEK293 Cells
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