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PreprintCited by 37Mass Spectrometry Techniques and ApplicationsGlycosylation and Glycoproteins ResearchAdvanced Proteomics Techniques and Applications

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Exposing the molecular heterogeneity of glycosylated biotherapeutics

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Schachner, Luis, Mullen, Christopher, Phung, Wilson, Hinkle, Joshua, Beardsley, Michelle Irwin, Bentley, Tracy, Day, Peter, Tsai, Christina, Sukumaran, Siddharth, Baginski, Tomasz, DiCara, Danielle, Agard, Nicholas, Masureel, Matthieu, Gober, Joshua, ElSohly, Adel, Syka, John, Huguet, Romain, Marty, Michael, Sandoval, Wendy·2023·Nature Communications
DOI 10.21203/rs.3.rs-2928767/v1PMC PMC11021452

Preprint — published as 10.1038/s41467-024-47693-8 · Paper page

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Abstract

Glycosylated biotherapeutics are an emerging class of drugs with high molecular heterogeneity, which can affect their safety and efficacy. Characterizing this heterogeneity is crucial for drug development and quality assessment, but existing methods are limited in their ability to analyze intact glycoproteins. Here, we present a new approach to glycoform fingerprinting that uses proton-transfer charge-reduction with gas-phase fractionation to analyze intact glycoproteins by mass spectrometry. The method provides a detailed landscape of the intact molecular weights present in biotherapeutic protein preparations in a single experiment and offers insights into glycoform composition when coupled with a suitable bioinformatic strategy. We tested the approach on various biotherapeutic molecules, including Fc-fusion, VHH-fusion, and peptide-bound MHC class II complexes to demonstrate efficacy in measuring the proteoform-level diversity of biotherapeutics. Notably, we inferred the glycoform distribution for hundreds of molecular weights for the eight-times glycosylated fusion drug IL22-Fc, enabling correlations between glycoform sub-populations and the drug’s pharmacological properties. Our method is broadly applicable and provides a powerful tool to assess the molecular heterogeneity of emerging biotherapeutics.

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Metadata

Authors
Schachner, L., Mullen, C., Phung, W., Hinkle, J., Beardsley, M. I., Bentley, T., Day, P., Tsai, C., Sukumaran, S., Baginski, T., DiCara, D., Agard, N., Masureel, M., Gober, J., ElSohly, A., Syka, J., Huguet, R., Marty, M., & Sandoval, W.
Affiliations
  • Genentech (US)
  • Thermo Fisher Scientific (United States) (US)
  • University of Arizona (US)
Corresponding author
Sandoval, Wendy
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2023-05-25
Volume
15
Issue
1
Pages
3259-3259
ISSN
2041-1723 (ISSN-L)
DOI
10.21203/rs.3.rs-2928767/v1
PMC
PMC11021452
OpenAlex
W4394845195
Cited by
37
References
1
Citations by year
2026: 19 · 2025: 12 · 2024: 6
FWCI
4.59
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • Research Square (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • National Science Foundation (1845230)
  • Genentech
  • Division of Chemistry (CHE-1845230)
Topics
  • Physical Sciences › Chemistry › Spectroscopy › Mass Spectrometry Techniques and Applications
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Glycosylation and Glycoproteins Research
  • Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
Keywords
molecular heterogeneity, glycoproteins, gas-phase fractionation, proteoforms, Fc fusion protein, glycan composition
MeSH terms
Glycoproteins, Glycosylation, Polysaccharides, Mass Spectrometry

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