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Füssl F, Trappe A, Cook K, et al. Comprehensive characterisation of the heterogeneity of adalimumab via charge variant analysis hyphenated on-line to native high resolution Orbitrap mass spectrometry. MAbs. 2018;11(1):116-128doi: 10.1080/19420862.2018.1531664.
Füssl, F., Trappe, A., Cook, K., Scheffler, K., Fitzgerald, O., & Bones, J. (2019). 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, Florian, et al. "Comprehensive characterisation of the heterogeneity of adalimumab via charge variant analysis hyphenated on-line to native high resolution Orbitrap mass spectrometry." mAbs vol. 11,1 (2019): 116-128. doi: https://doi.org/10.1080/19420862.2018.1531664
Füssl F, Trappe A, Cook K, Scheffler K, Fitzgerald O, Bones J. Comprehensive characterisation of the heterogeneity of adalimumab via charge variant analysis hyphenated on-line to native high resolution Orbitrap mass spectrometry. MAbs. 2019 Jan;11(1):116-128. doi: 10.1080/19420862.2018.1531664. Epub 2018 Nov 11. PMID: 30296204; PMCID: PMC6343805.
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MAbs. 2019 Jan;11(1):116-128. doi: 10.1080/19420862.2018.1531664. Epub 2018 Nov 11.

Comprehensive characterisation of the heterogeneity of adalimumab via charge variant analysis hyphenated on-line to native high resolution Orbitrap mass spectrometry.

mAbs

Florian Füssl, Anne Trappe, Ken Cook, Kai Scheffler, Oliver Fitzgerald, Jonathan Bones

Affiliations

  1. a Characterisation and Comparability Lab , NIBRT - The National Institute for Bioprocessing Research and Training , Co , Dublin , Ireland.
  2. b School of Biotechnology , Dublin City University , Dublin 9 , Ireland.
  3. c Thermo Fisher Scientific , Hemel Hempstead , UK.
  4. d Thermo Fisher Scientific , Germering , Germany.
  5. e St. Vincent's University Hospital , Dublin 4 , Ireland.
  6. f Conway Institute of Biomolecular and Biomedical Research , University College Dublin , Dublin 4 , Ireland.
  7. g School of Chemical and Bioprocess Engineering, University College Dublin , Dublin 4 , Ireland.

PMID: 30296204 PMCID: PMC6343805 DOI: 10.1080/19420862.2018.1531664

Abstract

Charge variant analysis is a widely used tool to monitor changes in product quality during the manufacturing process of monoclonal antibodies (mAbs). Although it is a powerful technique for revealing mAb heterogeneity, an unexpected outcome, for example the appearance of previously undetected isoforms, requires further, time-consuming analysis. The process of identifying these unknowns can also result in unwanted changes to the molecule that are not attributable to the manufacturing process. To overcome this, we recently reported a method combining highly selective cation exchange chromatography-based charge variant analysis with on-line mass spectrometric (MS) detection. We further explored and adapted the chromatographic buffer system to expand the application range. Moreover, we observed no salt adducts on the native protein, also supported by the optimal choice of MS parameters, resulting in increased data quality and mass accuracy. Here, we demonstrate the utility of this improved method by performing an in-depth analysis of adalimumab before and after forced degradation. By combining molecular mass and retention time information, we were able to identify multiple modifications on adalimumab, including lysine truncation, glycation, deamidation, succinimide formation, isomerisation, N-terminal aspartic acid loss or C-terminal proline amidation and fragmentation along with the N-glycan distribution of each of these identified proteoforms. Host cell protein (HCP) analysis was performed using liquid chromatography-mass spectrometry that verified the presence of the protease Cathepsin L. Based on the presence of trace HCPs with catalytic activity, it can be questioned if fragmentation is solely driven by spontaneous hydrolysis or possibly also by enzymatic degradation.

Keywords: Orbitrap; adalimumab; cation exchange chromatography; charge variant analysis; high resolution mass spectrometry; monoclonal antibodies; native mass spectrometry

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