Cutting-edge capillary electrophoresis characterization of monoclonal antibodies and related products

Abstract

Out of all categories, monoclonal antibodies (mAbs), biosimilar, antibody-drug conjugates (ADCs) and Fc-fusion proteins attract the most interest due to their strong therapeutic potency and specificity. Because of their intrinsic complexity due to a large number of micro-heterogeneities, there is a crucial need of analytical methods to provide comprehensive in-depth characterization of these molecules. CE presents some obvious benefits as high resolution separation and miniaturized format to be widely applied to the analysis of biopharmaceuticals. CE is an effective method for the separation of proteins at different levels. capillary gel electrophoresis (CGE), capillary isoelectric focusing (cIEF) and capillary zone electrophoresis (CZE) have been particularly relevant for the characterization of size and charge variants of intact and reduced mAbs, while CE-MS appears to be a promising analytical tool to assess the primary structure of mAbs and related products. This review will be dedicated to detail the current and state-of-the-art CE-based methods for the characterization of mAbs and related products.

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

“…Analysis of intact mAbs is typically performed to determine protein molecular mass, the extent of oligomerization, and additional structural information, such as the presence of free chains , glycosylation , as well as various PTM combinations . On conventional Orbitrap™ instruments, MS resolution is inversely proportional to the square root of m/z , which makes baseline resolution of higher molecular weight species challenging.…”

Top-down proteomic analysis of protein pharmaceuticals, mixtures of protein complexes, and ribosomal protein extracts by capillary zone electrophoresis-mass spectrometry

“…Use of uncoated bare-fused silica capillaries under acidic BGE conditions can result in significant band broadening and poor recovery due to adsorption of positively-charged proteins to the negatively-charged silanol groups on the capillary surface [274]. Numerous neutral and positively-charged capillary coatings have been developed to overcome the adsorption issue [268,274]. On a capillary with a positively-charged coating and an acidic BGE, the drag force exerted on positively-charged analytes by the electroosmotic bulk flow (EOF) and the electrostatic field force, causing analyte migration towards the cathode, act in opposite directions.…”

“…Analysis of intact mAbs is typically performed in order to determine protein molecular mass, extent of oligomerization, and additional structural information, such as the presence of free chains [266], glycosylation [1, 25,267], as well as various PTM combinations [1,268]. On conventional Orbitrap™ instruments, MS resolution is inversely proportional to the square root of m/z, which makes baseline resolution of higher molecular weight species challenging.…”

Recent advances in protein analysis by capillary and microchip electrophoresis

“…152 In the highlighted work from 2015 and early 2016 CE-ESI-MS analysis was developed for the analysis of reduced mAb with heavy and light chains 153 and analysis of intact IgG1. 149 A CZE method with cationic capillary coating for the identification of mAb in routine mAb for cancer therapy in hospitals was reported.…”