Monoclonal antibodies (mAbs) and their related products as antibody-drug-conjugates (ADCs) or biosimilars represent a constantly growing class of molecules therapeutic proteins used as treatment against numerous diseases. These compounds can undergo several modifications which could alter the efficiency of treatments. In this context, several analytical methods were designed to deliver a comprehensive structural characterization and guarantee the quality of biotherapeutics. Capillary electrophoresis (CE) is considered today as a major technique for the analysis of biotherapeutics due to benefic characteristics as high resolution separation and miniaturized format. Different CE modes have been developed to characterize mAbs at different levels such as capillary gel electrophoresis (CGE), capillary isoelectric focusing (cIEF), and capillary zone electrophoresis (CZE). Recent developments in CE-mass spectrometry (MS) coupling assessed this technology as a promising tool to obtain high level structural characterization of biopharmaceuticals. Moreover, upcoming techniques such as 2D CE-MS and microfluidic systems are now emerging to offer new possibilities beyond actual limits. This review will be dedicated to discuss the state-of-the-art CE-based methods for the characterization of mAbs and ADCs in the period 2016-2018.

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.

Capillary zone electrophoresis-mass spectrometry (CE-MS) is a mature analytical tool for the efficient profiling of (highly) polar and ionizable compounds. However, the use of CE-MS in comparison to other separation techniques remains underrepresented in metabolomics, as this analytical approach is still perceived as technically challenging and less reproducible, notably for migration time. The latter is key for a reliable comparison of metabolic profiles and for unknown biomarker identification that is complementary to high resolution MS/MS. In this work, we present the results of a Metabo-ring trial involving 16 CE-MS platforms among 13 different laboratories spanning two continents. The goal was to assess the reproducibility and identification capability of CE-MS by employing effective electrophoretic mobility (μ eff ) as the key parameter in comparison to the relative migration time (RMT) approach. For this purpose, a representative cationic metabolite mixture in water, pretreated human plasma, and urine samples spiked with the same metabolite mixture were used and distributed for analysis by all laboratories. The μ eff was determined for all metabolites spiked into each sample. The background electrolyte (BGE) was prepared and employed by each participating lab following the same protocol. All other parameters (capillary, interface, injection volume, voltage ramp, temperature, capillary conditioning, and rinsing procedure, etc.) were left to the discretion of the contributing laboratories. The results revealed that the reproducibility of the μ eff for 20 out of the 21 model compounds was below 3.1% vs 10.9% for RMT, regardless of the huge heterogeneity in experimental conditions and platforms across the 13 laboratories. Overall, this Metabo-ring trial demonstrated that CE-MS is a viable and reproducible approach for metabolomics.