Complementary middle‐down and intact monoclonal antibody proteoform characterization by capillary zone electrophoresis – mass spectrometry

Abstract

High-resolution capillary zone electrophoresis – mass spectrometry (CZE-MS) has been of increasing interest for the analysis of biopharmaceuticals. In this work, a combination of middle-down and intact CZE-MS analyses has been implemented for the characterization of a biotherapeutic monoclonal antibody (mAb) with a variety of post-translational modifications (PTMs) and glycosylation structures. Middle-down and intact CZE separations were performed in an acidified methanol-water background electrolyte on a capillary with a positively charged coating (M7C4I) coupled to an Orbitrap mass spectrometer using a commercial sheathless interface (CESI). Middle-down analysis of the IdeS-digested mAb provided characterization of PTMs of digestion fragments. High resolution CZE enabled separation of charge variants corresponding to 2X-deamidated, 1X-deamidated, and non-deamidated forms at baseline resolution. In the course of the middle-down CZE-MS analysis, separation of glycoforms of the FC/2 fragment was accomplished due to hydrodynamic volume differences. Several identified PTMs were confirmed by CZE-MS2. Incorporation of TCEP-HCl reducing agent in the sample solvent resulted in successful analysis of reduced forms without the need for alkylation. CZE-MS studies on the intact mAb under denaturing conditions enabled baseline separation of the 2X-glycosylated, 1X-glycosylated, and aglycosylated populations as a result of hydrodynamic volume differences. The presence of a trace quantity of dissociated light chain was also detected in the intact protein analysis. Characterization of the mAb under native conditions verified identifications achieved via intact analysis and allowed for quantitative confirmation of proteoforms. Analysis of mAbs using CZE-MS represents a complementary approach to the more conventional liquid-chromatography – mass spectrometry-based approaches.

Combination of intact, middle-up and bottom-up levels to characterize 7 therapeutic monoclonal antibodies by capillary electrophoresis – Mass spectrometry

“…are identified for the 1X-glycosyled forms. As expected, no separation of mAbs isoforms has been observed whereas separation obtained between 2X-glycosyled and 1X-glycosylated forms [20,22].…”

“…However, strategies with optical detection didn't allow to obtain structural information for the basic and/or acidic variants representing the limitation of these methodologies. These last years, some reports revealed the usefulness of CZE-MS for the assessment of macro-heterogeneity [13,20,22]. MS resolving power coupled CZE efficiency exhibited as well the baseline separation of 2X-glycosylated, 1X-glycosylated and aglycosylated population as the characterization of various other PTMs such as deamidation, oxidation or lysine clipped forms.…”

“…Indeed, due to the negative charge surface of the silanol groups, electrostatic interactions imply peak tailing phenomena degrading separation efficiency. Different strategies as positive polyethylenimine (PEI) or uncharged polyacrylamide coatings have been investigated [20,22]. In a previous study, our group detailed a CZE-MS methodology to perform the analysis of mAbs at the intact level.…”

The impact of standard accelerated stability conditions on antibody higher order structure as assessed by mass spectrometry

“…Alternatively, or in combination with the above separation techniques, steps could be made to reduce the mass complexity of the analyte using limited or complete proteolysis with middle-down and bottom-up proteomics approaches. 71,72 While no new protein modifications were observed, we were able to identify proteolytic degradation products for bevacizumab. These proteolytic degradation products, centered within the heavy chain V H /C 1 inter-domain region, are supportive of a 'proteolytic hot-spot' which is consistent with previously published data.…”

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

“…In a similar strategy, CE/MS was recently implemented for the characterization of mAbs and ADCs showing in particular the separation of intact charge variants. Indeed, the electrophoretic separation showed, on the intact protein level, the possibility to separate isoforms exhibiting PTMs or also the separation of glycoforms …”