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

Rosie Upton,Perdita E Barran,Richard G Beniston,David Firth,Sian Estdale,Kamila J Pacholarz,Lukasz G Migas

doi:10.1039/c8sc05029e

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.

Highlights

As many as 70 monoclonal antibody products are predicted to be on the market by 2020.1 To date, the majority of commercial mAb products are expressed in mammalian cell lines[2] which are responsible for transcribing post-translational modi cations (PTMs) such as glycosylation
We have shown that native mass spectrometry can be usefully employed to distinguish between lots of an active biopharmaceutical and demonstrated a method by which data can be compared with that obtained from a standard
The results present signi cant conformational similarities between two of the Herceptin® lots lot C exists in a wider range of conformational states demonstrated by both Hydrogen/deuterium exchange mass spectrometry (HDX-MS) and IM

Summary

Introduction

As many as 70 monoclonal antibody (mAb) products are predicted to be on the market by 2020.1 To date, the majority of commercial mAb products are expressed in mammalian cell lines[2] which are responsible for transcribing post-translational modi cations (PTMs) such as glycosylation. Subtle differences in the cell cultures and manufacturing conditions including pH and temperature can lead to variations in the glycan pro les and structure of the mAbs.[3] This process-related variability can lead to changes in the biological activity and pharmacokinetics. One critical assessment to make, with direct relevance to the development and licencing of new biopharmaceuticals as well as to biosimilar production, is a comparison between lots of a given mAb product

Methods

Results

Conclusion