Exposing the molecular heterogeneity of glycosylated biotherapeutics
Open accessPublished version of a preprint
Abstract
The heterogeneity inherent in today’s biotherapeutics, especially as a result of heavy glycosylation, can affect a molecule’s 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 or other heterogeneous biotherapeutics. Here, we present an approach to the molecular assessment of biotherapeutics that uses proton-transfer charge-reduction with gas-phase fractionation to analyze intact heterogeneous and/or glycosylated proteins by mass spectrometry. The method provides a detailed landscape of the intact molecular weights present in biotherapeutic protein preparations in a single experiment. For glycoproteins in particular, the method may offer insights into glycan 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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Cites
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- Authors
- Schachner, L. F., Mullen, C., Phung, W., Hinkle, J. D., Beardsley, M. I., Bentley, T., Day, P., Tsai, C., Sukumaran, S., Baginski, T., DiCara, D., Agard, N. J., Masureel, M., Gober, J., ElSohly, A. M., Melani, R., Syka, J. E. P., Huguet, R., Marty, M. T., & Sandoval, W.
- Affiliations
- Genentech (US)
- Thermo Fisher Scientific (United States) (US)
- University of Arizona (US)
- Corresponding author
- Sandoval, Wendy
- ORCID
- Venue
- Nature Communications
- Published
- 2024-04-16
- Volume
- 15
- Issue
- 1
- Pages
- 3259-3259
- ISSN
- 2041-1723 (ISSN-L)
- PMC
- PMC11021452
- OpenAlex
- W4394845195
- Cited by
- 36
- References
- 11
- Citations by year
- 2026: 19 · 2025: 12 · 2024: 5
- FWCI
- 4.48
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- 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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