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Issue 9, 2019
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Hybrid mass spectrometry methods reveal lot-to-lot differences and delineate the effects of glycosylation on the tertiary structure of Herceptin®

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Abstract

To quantify the measurable variations in the structure of a biopharmaceutical product we systematically evaluate three lots of Herceptin®, two mAb standards and an intact Fc-hinge fragment. Each mAb is examined in three states; glycan intact, truncated (following endoS2 treatment) and fully deglycosylated. Despite equivalence at the intact protein level, each lot of Herceptin® gives a distinctive signature in three different mass spectrometry approaches. Ion mobility mass spectrometry (IM-MS) shows that in the API, the attached N-glycans reduce the conformational spread of each mAb by 10.5–25%. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) data support this, with lower global deuterium uptake in solution when comparing intact to the fully deglycosylated protein. HDX-MS and activated IM-MS map the influence of glycans on the mAb and reveal allosteric effects which extend far beyond the Fc domains into the Fab region. Taken together, these findings and the supplied interactive data sets establish acceptance criteria with application for MS based characterisation of biosimilars and novel therapeutic mAbs.

Graphical abstract: Hybrid mass spectrometry methods reveal lot-to-lot differences and delineate the effects of glycosylation on the tertiary structure of Herceptin®

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Publication details

The article was received on 12 Nov 2018, accepted on 12 Jan 2019 and first published on 17 Jan 2019


Article type: Edge Article
DOI: 10.1039/C8SC05029E
Chem. Sci., 2019,10, 2811-2820
  • Open access: Creative Commons BY license
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    Hybrid mass spectrometry methods reveal lot-to-lot differences and delineate the effects of glycosylation on the tertiary structure of Herceptin®

    R. Upton, L. G. Migas, K. J. Pacholarz, R. G. Beniston, S. Estdale, D. Firth and P. E. Barran, Chem. Sci., 2019, 10, 2811
    DOI: 10.1039/C8SC05029E

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