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Cited by 220Monoclonal and Polyclonal Antibodies ResearchGlycosylation and Glycoproteins ResearchViral Infectious Diseases and Gene Expression in Insects

In vitro glycoengineering of IgG1 and its effect on Fc receptor binding and ADCC activity

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Thomann, Marco, Schlothauer, Tilman, Dashivets, Tetyana, Malik, Sebastian, Avenal, Cecile, Bulau, Patrick, Rüger, Petra, Reusch, Dietmar·2015·PLOS ONE
DOI 10.1371/journal.pone.0134949PMC PMC4534130
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Abstract

The importance and effect of Fc glycosylation of monoclonal antibodies with regard to biological activity is widely discussed and has been investigated in numerous studies. Fc glycosylation of monoclonal antibodies from current production systems is subject to batch-to-batch variability. If there are glycosylation changes between different batches, these changes are observed not only for one but multiple glycan species. Therefore, studying the effect of distinct Fc glycan species such as galactosylated and sialylated structures is challenging due to the lack of well-defined differences in glycan patterns of samples used. In this study, the influence of IgG1 Fc galactosylation and sialylation on its effector functions has been investigated using five different samples which were produced from one single drug substance batch by in vitro glycoengineering. This sample set comprises preparations with minimal and maximal galactosylation and different levels of sialylation of fully galactosylated Fc glycans. Among others, Roche developed the glycosyltransferase enzyme sialyltransferase which was used for the in vitro glycoengineering activities at medium scale. A variety of analytical assays, including Surface Plasmon Resonance and recently developed FcγR affinity chromatography, as well as an optimized cell-based ADCC assay were applied to investigate the effect of Fc galactosylation and sialylation on the in vitro FcγRI, IIa, and IIIa receptor binding and ADCC activity of IgG1. The results of our studies do not show an impact, neither positive nor negative, of sialic acid- containing Fc glycans of IgG1 on ADCC activity, FcγRI, and RIIIa receptors, but a slightly improved binding to FcγRIIa. Furthermore, we demonstrate a galactosylation-induced positive impact on the binding activity of the IgG1 to FcγRIIa and FcγRIIIa receptors and ADCC activity.

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  • In vitro galactosylation and sialylation of IgG1 Fc glycans did not show any impact on ADCC activity but showed a slight improvement of binding to FcγRIIa.

    “Besides, in vitro galactosylation and sialylation of IgG1 Fc glycans did not show any impact on ADCC activity but showed a slight improvement of binding to FcγRIIa (Thomann et al., 2015).”

    From Therapeutic glycoprotein production in mammalian cells (2017) · cites this paper as Thomann et al., 2015

    The Summary of results (Table 2) reports that increased sialylation had no impact on ADCC and that both galactosylation and sialylation increased FcγRIIa binding (slightly in the column assay). But it reports that increased galactosylation increased ADCC activity, contrary to the claim that neither modification affected ADCC. (LLM, conf=0.92)

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Authors
Thomann, M., Schlothauer, T., Dashivets, T., Malik, S., Avenal, C., Bulau, P., Rüger, P., & Reusch, D.
Affiliations
  • Roche Pharma AG (Germany) (DE)
  • Center for Integrated Protein Science Munich (DE)
Corresponding author
Thomann, Marco
ORCID
Venue
PLOS ONE
Publisher
Public Library of Science (PLoS)
Published
2015-08-12
Volume
10
Issue
8
Pages
e0134949
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0134949
PMC
PMC4534130
OpenAlex
W1191527629
Cited by
220
References
3
Citations by year
2026: 9 · 2025: 14 · 2024: 14 · 2023: 23 · 2022: 21 · 2021: 25 · 2020: 26 · 2019: 20 · 2018: 26 · 2017: 22 · 2016: 18 · 2015: 2
FWCI
18.37
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Topics
  • Health Sciences › Medicine › Radiology, Nuclear Medicine and Imaging › Monoclonal and Polyclonal Antibodies Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Glycosylation and Glycoproteins Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Viral Infectious Diseases and Gene Expression in Insects
Keywords
IgG1, Fc glycosylation, sialylation, galactosylation, sialyltransferase, antibody-dependent cellular cytotoxicity, Fc receptor binding, FcγRIIa, FcγRIIIa, FcγRI, monoclonal antibodies, glycan heterogeneity
MeSH terms
Antibody-Dependent Cell Cytotoxicity, Binding Sites, Galactose, Glycosylation, Humans, Immunoglobulin G, Protein Binding, Recombinant Proteins, Sialic Acids, Sialyltransferases, Protein Engineering, Gene Expression, Receptors, IgG, HEK293 Cells, ErbB Receptors

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