Abstract
Intact mass analysis of proteins is simple, fast, and specific, and it effectively provides structural insight into the proteoforms or variants of the analyzed protein. For instance, the multiple glycoforms of recombinant monoclonal antibodies can be effectively analyzed by intact mass spectrometry (MS). A recent development in the Orbitrap technology has made this platform particularly well suited for analysis of large intact biomolecules, and here we describe procedures for performing intact mass analysis of intact glycoproteins using the Orbitrap platform, with the aim of identifying and quantitating the glycoforms. Emphasis is placed on the analysis of biopharmaceutical immunoglobulins (IgGs), but the procedures can be extended to other glycoproteins as needed.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Beck A, Wagner-Rousset E, Ayoub D et al (2013) Characterization of therapeutic antibodies and related products. Anal Chem 85:715–736. https://doi.org/10.1021/ac3032355
Kayser V, Chennamsetty N, Voynov V et al (2011) Glycosylation influences on the aggregation propensity of therapeutic monoclonal antibodies. Biotechnol J 6:38–44. https://doi.org/10.1002/biot.201000091
Liu L (2015) Antibody glycosylation and its impact on the pharmacokinetics and pharmacodynamics of monoclonal antibodies and Fc-fusion proteins. J Pharm Sci 104:1866–1884. https://doi.org/10.1002/jps.24444
CMC Biotech Working group (2009) A-Mab: a case study in bioprocess development. CASSS 2.1:
Zhang L, Luo S, Zhang B (2016) Glycan analysis of therapeutic glycoproteins. MAbs 8:205–215. https://doi.org/10.1080/19420862.2015.1117719
Makarov A, Denisov E (2009) Dynamics of ions of intact proteins in the Orbitrap mass analyzer. J Am Soc Mass Spectrom 20:1486–1495. https://doi.org/10.1016/j.jasms.2009.03.024
Bailey AO (2018) The quest for highly effective resolution: therapeutic protein characterization via separations-coupled native MS. Am Chem Soc:1–15
van de Waterbeemd M, Fort KL, Boll D et al (2017) High-fidelity mass analysis unveils heterogeneity in intact ribosomal particles. Nat Methods 14:283–286. https://doi.org/10.1038/nmeth.4147
Gault J, Donlan JAC, Liko I et al (2016) High-resolution mass spectrometry of small molecules bound to membrane proteins. Nat Methods 13:333–336. https://doi.org/10.1038/nmeth.3771
Belov ME, Damoc E, Denisov E et al (2013) From protein complexes to subunit backbone fragments: a multi-stage approach to native mass spectrometry. Anal Chem 85:11163–11173. https://doi.org/10.1021/ac4029328
Füssl F, Trappe A, Cook K et al (2018) Comprehensive characterisation of the heterogeneity of adalimumab via charge variant analysis hyphenated on-line to native high resolution Orbitrap mass spectrometry. MAbs 11(1):116–128. https://doi.org/10.1080/19420862.2018.1531664
Füssl F, Cook K, Scheffler K et al (2018) Charge variant analysis of monoclonal antibodies using direct coupled pH gradient cation exchange chromatography to high-resolution native mass spectrometry. Anal Chem 90:4669–4676. https://doi.org/10.1021/acs.analchem.7b05241
Kristensen DB, Jensen PF, Slot TM, et al (2019) Expanding applications of chromatographic techniques coupled to native mass spectrometry for routine analysis of large and complex biopharmaceutical molecules. In: HPLC 2019. Milan
Protein Metrics Intact Mass software. https://www.proteinmetrics.com/products/intact-mass/
Thermo BioPharma Finder software. https://www.thermofisher.com/dk/en/home/industrial/mass-spectrometry/liquid-chromatography-mass-spectrometry-lc-ms/lc-ms-software/multi-omics-data-analysis/biopharma-finder-software.html
Genedata Expressionist. https://www.genedata.com/products/expressionist/biotherapeutics/intact-proteins/
Fischer S, Hoernschemeyer J, Mahler H-C (2008) Glycation during storage and administration of monoclonal antibody formulations. Eur J Pharm Biopharm 70:42–50. https://doi.org/10.1016/j.ejpb.2008.04.021
Scheffler K, Damoc E (2017) Full characterization of heterogeneous antibody samples under denaturing and native conditions on the Q Exactive BioPharma mass spectrometer. Appl Note 72348
Skoog B, Wichman A (1986) Calculation of the isoelectric points of polypeptides from the amino acid composition. TrAC Trends Anal Chem 5:82–83. https://doi.org/10.1016/0165-9936(86)80045-0
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2021 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Kristensen, D.B., Sloth, T.M., Ørgaard, M., Jensen, P.F. (2021). Characterization of Protein Glycoforms at Intact Level by Orbitrap Mass Spectrometry. In: Delobel, A. (eds) Mass Spectrometry of Glycoproteins. Methods in Molecular Biology, vol 2271. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1241-5_2
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1241-5_2
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1240-8
Online ISBN: 978-1-0716-1241-5
eBook Packages: Springer Protocols