Issue 9, 2019

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

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®

Supplementary files

Article information

Article type
Edge Article
Submitted
12 nov 2018
Accepted
12 jan 2019
First published
17 jan 2019
This article is Open Access

All publication charges for this article have been paid for by the Royal Society of Chemistry
Creative Commons BY license

Chem. Sci., 2019,10, 2811-2820

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

This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. You can use material from this article in other publications without requesting further permissions from the RSC, provided that the correct acknowledgement is given.

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