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Analysis of recombinant human follicle-stimulating hormone (FSH) by mass spectrometric approaches

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Abstract

Recombinant human follicle stimulating hormone is an important drug in reproductive medicine. Thorough analysis of the heterodimeric heavily glycosylated protein is a prerequisite for the evaluation of production batches as well as for the determination of “essential similarity” of new biosimilars. The concerted application of different liquid chromatography-mass spectrometry methods enabled the complete depiction of the primary structure of this pituitary hormone. Sequence coverage of 100% for the α- as well as the β-chain was achieved with tryptic peptides. Most of these peptides could be verified by tandem mass spectrometry. Site-specific analysis of all four glycosylation sites was, however, not possible with tryptic but with chymotryptic peptides. Quantification of the glycoforms of each glycopeptide was accomplished with the software MassMap®. Both protein subunits gave interpretable mass spectra upon S-alkylation and separation on a C5 reversed-phase column. Glycan isomer patterns were depicted by separation on porous graphitic carbon, using mass spectrometric detection for the evaluation of the glycopeptide liquid chromatography-electrospray ionization data. The currently marketed product Gonal-f™ and a potential biosimilar were compared with the help of these procedures.

Schematic depiction of the glycoprotein nature of human follicle-stimulating hormone with the alfa chain in blue and the beta chain in purple and a mass spectrum of the alfa chain at the bottom.

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Acknowledgments

The authors gratefully acknowledge Dr. Richard Peck (Basel, Switzerland) for carefully reading the manuscript and Dr. Thomas Hemetsberger for valuable advice.

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Authors and Affiliations

  1. Department of Chemistry, University of Natural Resources and Life Sciences (BOKU), Muthgasse 18, 1190, Vienna, Austria

    Josephine Grass, Martin Pabst & Friedrich Altmann

  2. Polymun Scientific, 1190, Vienna, Austria

    Martina Chang

  3. MassMap GmbH & Co. KG, 82515, Wolfratshausen, Germany

    Manfred Wozny

Authors
  1. Josephine Grass
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  2. Martin Pabst
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  3. Martina Chang
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  4. Manfred Wozny
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  5. Friedrich Altmann
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Corresponding author

Correspondence to Friedrich Altmann.

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Published in the special issue Analytical Sciences in Austria with Guest Editors G. Allmaier, W. Buchberger, and K. Francesconi.

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Grass, J., Pabst, M., Chang, M. et al. Analysis of recombinant human follicle-stimulating hormone (FSH) by mass spectrometric approaches. Anal Bioanal Chem 400, 2427–2438 (2011). https://doi.org/10.1007/s00216-011-4923-5

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  • DOI: https://doi.org/10.1007/s00216-011-4923-5

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