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Exposing the molecular heterogeneity of glycosylated biotherapeutics

Author

Listed:
  • Luis F. Schachner

    (Inc.)

  • Christopher Mullen

    (Inc.)

  • Wilson Phung

    (Inc.)

  • Joshua D. Hinkle

    (Inc.)

  • Michelle Irwin Beardsley

    (Inc.)

  • Tracy Bentley

    (Inc.)

  • Peter Day

    (Inc.)

  • Christina Tsai

    (Inc.
    Ascendis Pharma)

  • Siddharth Sukumaran

    (Inc.
    Janssen)

  • Tomasz Baginski

    (Inc.)

  • Danielle DiCara

    (Inc.)

  • Nicholas J. Agard

    (Inc.)

  • Matthieu Masureel

    (Inc.)

  • Joshua Gober

    (Inc.)

  • Adel M. ElSohly

    (Inc.)

  • Rafael Melani

    (Inc.)

  • John E. P. Syka

    (Inc.)

  • Romain Huguet

    (Inc.)

  • Michael T. Marty

    (University of Arizona)

  • Wendy Sandoval

    (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.

Suggested Citation

  • Luis F. Schachner & Christopher Mullen & Wilson Phung & Joshua D. Hinkle & Michelle Irwin Beardsley & Tracy Bentley & Peter Day & Christina Tsai & Siddharth Sukumaran & Tomasz Baginski & Danielle DiCa, 2024. "Exposing the molecular heterogeneity of glycosylated biotherapeutics," Nature Communications, Nature, vol. 15(1), pages 1-13, December.
  • Handle: RePEc:nat:natcom:v:15:y:2024:i:1:d:10.1038_s41467-024-47693-8
    DOI: 10.1038/s41467-024-47693-8
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    References listed on IDEAS

    as
    1. Therese Wohlschlager & Kai Scheffler & Ines C. Forstenlehner & Wolfgang Skala & Stefan Senn & Eugen Damoc & Johann Holzmann & Christian G. Huber, 2018. "Native mass spectrometry combined with enzymatic dissection unravels glycoform heterogeneity of biopharmaceuticals," Nature Communications, Nature, vol. 9(1), pages 1-9, December.
    2. Thapakorn Jaroentomeechai & Jessica C. Stark & Aravind Natarajan & Cameron J. Glasscock & Laura E. Yates & Karen J. Hsu & Milan Mrksich & Michael C. Jewett & Matthew P. DeLisa, 2018. "Single-pot glycoprotein biosynthesis using a cell-free transcription-translation system enriched with glycosylation machinery," Nature Communications, Nature, vol. 9(1), pages 1-11, December.
    3. Patrick A. Ott & Zhuting Hu & Derin B. Keskin & Sachet A. Shukla & Jing Sun & David J. Bozym & Wandi Zhang & Adrienne Luoma & Anita Giobbie-Hurder & Lauren Peter & Christina Chen & Oriol Olive & Todd , 2017. "An immunogenic personal neoantigen vaccine for patients with melanoma," Nature, Nature, vol. 547(7662), pages 217-221, July.
    4. Jasmine M. Hershewe & Katherine F. Warfel & Shaelyn M. Iyer & Justin A. Peruzzi & Claretta J. Sullivan & Eric W. Roth & Matthew P. DeLisa & Neha P. Kamat & Michael C. Jewett, 2021. "Improving cell-free glycoprotein synthesis by characterizing and enriching native membrane vesicles," Nature Communications, Nature, vol. 12(1), pages 1-12, December.
    5. Tomislav Čaval & Weihua Tian & Zhang Yang & Henrik Clausen & Albert J. R. Heck, 2018. "Direct quality control of glycoengineered erythropoietin variants," Nature Communications, Nature, vol. 9(1), pages 1-8, December.
    6. Yang Yang & Fan Liu & Vojtech Franc & Liem Andhyk Halim & Huub Schellekens & Albert J. R. Heck, 2016. "Hybrid mass spectrometry approaches in glycoprotein analysis and their usage in scoring biosimilarity," Nature Communications, Nature, vol. 7(1), pages 1-10, December.
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