Monoclonal antibody N-glycosylation profiling using capillary electrophoresis – Mass spectrometry: Assessment and method validation

Abstract

A B S T R A C TCharacterization of therapeutic proteins represents a major challenge for analytical sciences due to their heterogeneity caused by post-translational modifications (PTM). Among these PTM, glycosylation which is possibly the most prominent, require comprehensive identification because of their major influence on protein structure and effector functions of monoclonal antibodies (mAbs). As a consequence, glycosylation profiling must be deeply characterized. For this application, several analytical methods such as separation-based or MS-based methods, were evaluated. However, no CE-ESI-MS approach has been assessed and validated. Here, we illustrate how the use of CE-ESI-MS method permits the comprehensive characterization of mAbs N-glycosylation at the glycopeptide level to perform relative quantitation of N-glycan species. Validation of the CE-ESI-MS method in terms of robustness and reproducibility was demonstrated through the relative quantitation of glycosylation profiles for ten different mAbs produced in different cell lines. Glycosylation patterns obtained for each mAbs were compared to Hydrophilic Interaction Chromatography of 2-aminobenzamide labelled glycans with fluorescence detector (HILIC-FD) analysis considered as a reference method. Very similar glycoprofiling were obtained with the CE-ESI-MS and HILIC-FD demonstrating the attractiveness of CE-ESI-MS method to characterize and quantify the glycosylation heterogeneity of a wide range of therapeutic mAbs with high accuracy and precision.

Recent advances in capillary electrophoresis‐mass spectrometry: Instrumentation, methodology and applications

“…Giorgetti et al. used CZE‐MS on the glycopeptide level for relative quantification of glycosylation profiles of various mAbs, including the discrimination of Infliximab‐Remicade ® and its biosimilar . Although being important for many biopharmaceuticals and in the context of clinical diagnostics, O ‐glycosylation is usually less studied than N ‐glycosylation.…”

“…In a subsequent study, the same group demonstrated an offline RPLC-CZE-ESI-MS method, including RPLC fraction collection with subsequent CZE-ESI-MS analysis [79]. In this way, 268 site-specific N-glycopeptides of ␣-1-acid glycoprotein could be identified, which represented [80]. Although being important for many biopharmaceuticals and in the context of clinical diagnostics, O-glycosylation is usually less studied than N-glycosylation.…”

Modern separation techniques coupled to high performance mass spectrometry for glycolipid analysis

“…Because of such difficulties, though in the last few years important developments of CE ESI MS approaches, some even introducing chips and automation or combining the on‐line separation with top‐down fragmentation for protein characterization and metabolomics were reported, much less was achieved in innovations dedicated to glycoconjugate analysis. Still a number of original publications presenting excellent protocols and several reviews highlighting in details the advancements of the method for general glycomics are available .…”

Revealing the potential of capillary electrophoresis/mass spectrometry: the tipping point

“…That separation selectivity is definitely an excellent asset in order to improve the sensitivity and enable the detection of low‐abundance glycosylations. In addition, the MS/MS spectra allowed the identification of the structure of the glycan present on the protein and to localize its position on the protein by identification of the fragments corresponding to the peptide backbone (Figure ) …”

“…(C) MS/MS fragmentation spectra of [EEQFNSTYR] + G0F. Adapted from Giorgetti et al Copyright (2017) Elsevier BV…”