Hybrid mass spectrometry methods reveal lot-tolot differences and delineate the effects of glycosylation on the tertiary structure of Herceptin

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.


Rosie Upton, a Lukasz G. Migas, a Kamila J. Pacholarz, a Richard G. Beniston,b
Sian Estdale, b David Firth b and Perdita E. Barran *a

a Manchester Institute of Biotechnology, Michael Barber Centre for Collaborative Mass
Spectrometry, University of Manchester, 131 Princess Street, Manchester, M1 7DN, UK.
E-mail: Perdita.barran@manchester.ac.uk
b Covance Laboratories Ltd., Otley Road, Harrogate, HG3 1PY, UK


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