Exposing the molecular heterogeneity of glycosylated biotherapeutics
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 a...
| Main Authors: | , , , , , , , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2024-04-01
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| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-024-47693-8 |
| _version_ | 1797199313777983488 |
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| author | Luis F. Schachner Christopher Mullen Wilson Phung Joshua D. Hinkle Michelle Irwin Beardsley Tracy Bentley Peter Day Christina Tsai Siddharth Sukumaran Tomasz Baginski Danielle DiCara Nicholas J. Agard Matthieu Masureel Joshua Gober Adel M. ElSohly Rafael Melani John E. P. Syka Romain Huguet Michael T. Marty Wendy Sandoval |
| author_facet | Luis F. Schachner Christopher Mullen Wilson Phung Joshua D. Hinkle Michelle Irwin Beardsley Tracy Bentley Peter Day Christina Tsai Siddharth Sukumaran Tomasz Baginski Danielle DiCara Nicholas J. Agard Matthieu Masureel Joshua Gober Adel M. ElSohly Rafael Melani John E. P. Syka Romain Huguet Michael T. Marty Wendy Sandoval |
| author_sort | Luis F. Schachner |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-24T07:13:46Z |
| format | Article |
| id | doaj.art-1f51a60ae7254824a781e70ff1592428 |
| institution | Directory Open Access Journal |
| issn | 2041-1723 |
| language | English |
| last_indexed | 2024-04-24T07:13:46Z |
| publishDate | 2024-04-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Nature Communications |
| spelling | doaj.art-1f51a60ae7254824a781e70ff15924282024-04-21T11:24:36ZengNature PortfolioNature Communications2041-17232024-04-0115111310.1038/s41467-024-47693-8Exposing the molecular heterogeneity of glycosylated biotherapeuticsLuis F. Schachner0Christopher Mullen1Wilson Phung2Joshua D. Hinkle3Michelle Irwin Beardsley4Tracy Bentley5Peter Day6Christina Tsai7Siddharth Sukumaran8Tomasz Baginski9Danielle DiCara10Nicholas J. Agard11Matthieu Masureel12Joshua Gober13Adel M. ElSohly14Rafael Melani15John E. P. Syka16Romain Huguet17Michael T. Marty18Wendy Sandoval19Department of Microchemistry, Proteomics and Lipidomics, Genentech, Inc.Life Sciences Mass Spectrometry, Thermo Fisher Scientific, Inc.Department of Microchemistry, Proteomics and Lipidomics, Genentech, Inc.Life Sciences Mass Spectrometry, Thermo Fisher Scientific, Inc.Pharmaceutical Technical Development, Genentech, Inc.Pharmaceutical Technical Development, Genentech, Inc.Pharmaceutical Technical Development, Genentech, Inc.Pharmaceutical Technical Development, Genentech, Inc.Pharmaceutical Technical Development, Genentech, Inc.Pharmaceutical Technical Development, Genentech, Inc.Department of Antibody Engineering, Genentech, Inc.Department of Antibody Engineering, Genentech, Inc.Department of Structural Biology, Genentech, Inc.Department of Protein Chemistry, Genentech, Inc.Department of Protein Chemistry, Genentech, Inc.Life Sciences Mass Spectrometry, Thermo Fisher Scientific, Inc.Life Sciences Mass Spectrometry, Thermo Fisher Scientific, Inc.Life Sciences Mass Spectrometry, Thermo Fisher Scientific, Inc.Department of Chemistry and Biochemistry, University of ArizonaDepartment of Microchemistry, Proteomics and Lipidomics, Genentech, Inc.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.https://doi.org/10.1038/s41467-024-47693-8 |
| spellingShingle | Luis F. Schachner Christopher Mullen Wilson Phung Joshua D. Hinkle Michelle Irwin Beardsley Tracy Bentley Peter Day Christina Tsai Siddharth Sukumaran Tomasz Baginski Danielle DiCara Nicholas J. Agard Matthieu Masureel Joshua Gober Adel M. ElSohly Rafael Melani John E. P. Syka Romain Huguet Michael T. Marty Wendy Sandoval Exposing the molecular heterogeneity of glycosylated biotherapeutics Nature Communications |
| title | Exposing the molecular heterogeneity of glycosylated biotherapeutics |
| title_full | Exposing the molecular heterogeneity of glycosylated biotherapeutics |
| title_fullStr | Exposing the molecular heterogeneity of glycosylated biotherapeutics |
| title_full_unstemmed | Exposing the molecular heterogeneity of glycosylated biotherapeutics |
| title_short | Exposing the molecular heterogeneity of glycosylated biotherapeutics |
| title_sort | exposing the molecular heterogeneity of glycosylated biotherapeutics |
| url | https://doi.org/10.1038/s41467-024-47693-8 |
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