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

Electrophoresis
Arseniy M BelovAlexander R Ivanov

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 fo...Continue Reading

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Citations

Jul 20, 2018·Rapid Communications in Mass Spectrometry : RCM·Rabah GahoualYannis-Nicolas François
Oct 24, 2018·European Journal of Mass Spectrometry·Jérémie GiorgettiEmmanuelle Leize-Wagner
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Feb 1, 2020·Journal of Pharmaceutical and Biomedical Analysis·Jérémie GiorgettiYannis-Nicolas François
Nov 2, 2019·Journal of Pharmaceutical and Biomedical Analysis·Julien CamperiNathalie Delaunay
May 28, 2019·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Antony LechnerYannis-Nicolas François
May 16, 2021·Journal of Chromatography. a·Meriem DadouchCatherine Perrin
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Nov 9, 2019·Analytical Chemistry·Courtney J KristoffLisa A Holland
Nov 13, 2018·Analytical Chemistry·Huiming YuanYukui Zhang
Aug 27, 2021·Current Opinion in Biotechnology·Elli MakrydakiCleo Kontoravdi

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