The improvement of separation efficiency for protein analysis in capillary electrophoresis (CE) is a challenging topic in which protein adsorption onto the capillary wall plays a crucial role. In this work, a simple method allowing the quantification of the adsorption of proteins onto the coated or untreated inner surface of the fused silica capillary was developed based on the determination of the retention factor by measuring separation efficiency of individual proteins at different separation voltages (i.e., different linear velocities). This approach was applied to the quantification of the residual adsorption of four test proteins on five-layer polyelectrolyte coatings and bare fused silica capillary. It allows to get a fair ranking of the coating performances toward protein adsorption, whatever their apparent electrophoretic mobilities (migration times) are. Due to the existence of (even low) residual adsorption, the electrophoretic operating conditions (electric field, capillary length, and internal diameter) can be optimized to improve the separation performances resulting in experimental separation efficiency up to ∼600 000 plates.m–1 in conditions compatible with MS coupling. This approach represents a crucial step in the course to get antifouling coatings for protein separation in CE. It can be used for the evaluation and ranking of virtually any coating (neutral or charged) in CE.

Monoclonal antibody (mAb) therapeutics attract the largest concern due to their strong therapeutic potency and specificity. The Fc region of mAbs is common to many new biotherapeutics as biosimilar, antibody drug conjugate or fusion protein. Fc region has consequences for Fc-mediated effector functions that might be desirable for therapeutic applications. As a consequence, there is a continuous need for improvement of analytical methods to enable fast and accurate characterization of biotherapeutics. Capillary zone electrophoresis-Mass spectrometry couplings (CZE-MS) appear really attractive methods for the characterization of biological samples. In this report, we used CZE-MS systems developed in house and native MS infusion to allow precise middle-up characterization of Fc/2 variant of cetuximab. Molecular weights were measured for three Fc/2 charge variants detected in the CZE separation of cetuximab subunits. Two Fc/2 C-terminal lysine variants were identified and separated. As the aim is to understand the presence of three peaks in the CZE separation for two Fc/2 subunits, we developed a strategy using CZE-UV/MALDI-MS and CZE-UV/ESI-MS to evaluate the role of N-glycosylation and C-terminal lysine truncation on the CZE separation. The chemical structure of N-glycosylation expressed on the Fc region of cetuximab does not influence CZE separation while C-terminal lysine is significantly influencing separation. In addition, native MS infusion demonstrated the characterization of Fc/2 dimers at pH 5.7 and 6.8 and the first separation of these dimers using CZE-MS.