ChemRxiv
These are preliminary reports that have not been peer-reviewed. They should not be regarded as conclusive, guide clinical practice/health-related behavior, or be reported in news media as established information. For more information, please see our FAQs.
1/1
2 files

Hybrid Mass Spectrometry Methods Reveal Lot-to-Lot Differences and Delineate the Effects of Glycosylation on the Structure of Herceptin®

preprint
submitted on 27.07.2018 and posted on 30.07.2018 by Rosie Upton, Lukasz G. Migas, Kamila J. Pacholarz, Richard G. Beniston, David Firth, Sian Estdale, Perdita E. Barran

To consider the measurable variations in biopharmaceuticals we use mass spectrometry and 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 protein level, each lot of Herceptin® gives a distinctive signature in three different mass spectrometry analyses. 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 supports 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 could be used to provide acceptance criteria with application for MS based characterisation of biosimilars and novel therapeutic mAbs.

Funding

BBSRC

History

Email Address of Submitting Author

rosie.upton@postgrad.manchester.ac.uk

Institution

University of Manchester

Country

United Kingdom

ORCID For Submitting Author

0000-0001-7204-2910

Declaration of Conflict of Interest

No conflict of interest

Exports

Logo branding

Exports