Integrated Microfluidic Capillary Electrophoresis-Electrospray Ionization Devices with Online MS Detection for the Separation and Characterization of Intact Monoclonal Antibody Variants

Abstract

Here, we demonstrate an integrated microfluidic capillary electrophoresis-electrospray ionization (CE-ESI) device for the separation of intact monoclonal antibody charge variants with online mass spectrometric (MS) identification. The need for dynamic coating and zwitterionic background electrolyte (BGE) additives has been eliminated by utilizing surface chemistry within the device channels to control analyte adsorption and electroosmotic flow (EOF) while maintaining separation efficiency. The effectiveness of this strategy was illustrated with the separation of charge variants of Infliximab. Three major species corresponding to C-terminal lysine variants were separated with an average resolution of 0.80 and identified by mass difference. In addition to the lysine variants, masses were determined for minor acidic and basic species. The separation of these variants prior to MS analysis facilitated the identification of glycosylation patterns for each of the variants. The general applicability of this method was demonstrated by analyzing two additional monoclonal antibody species: an IgG2 antibody and an IgG1 antibody conjugate. The IgG2 proved to have similar modifications to Infliximab with lower relative abundances of the lysine variants. Analysis of the IgG1 drug conjugate further exemplified the advantages of MS detection; differences in the extent of antibody conjugation were detectable despite limited CE resolution. The CE-ESI-MS methodology described here is a rapid and generic strategy for the separation of intact mAb charge variants and facilitates the identification of variants through MS detection.

Glycoform Separation and Characterization of Cetuximab Variants by Middle-up Off-Line Capillary Zone Electrophoresis-UV/Electrospray Ionization-MS

“…41,42 Gahoual and co-workers demonstrated that particular glycopeptides having a difference of one galactose could be baseline separated 13 whereas Redman et al did not observe mobility shifts between intact mAb glycoforms due to the low impact of 162 Da on the mass of intact mAb (≈0.1%). 28 Our work demonstrated that, concerning glycoform with a difference of one galactose residue, the middle-up approach does not allow one to observe baseline separations. The mass of galactose moiety accounts for ≈0.6% of the Fc/2 domain and ≈0.2% of the F(ab′) 2 domain.…”

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

“…One example involves commercial microchips sold through 908 Devices (Boston, MA). A number of publications involving this interface have been reported [71][72][73]. A distinct advantage of such systems includes administration of capillary coating in the gas phase through chemical vapor deposition [71].…”

Recent Developments of Microchip Capillary Electrophoresis Coupled with Mass Spectrometry

“…Ramsey et al used the MCE-MS device described in [85], for the separation and characterization of charge variants of intact monoclonal antibodies (Infliximab) [87].…”