Middle-Up Characterization of the Monoclonal Antibody Infliximab by Capillary Zone Electrophoresis-Mass Spectrometry
Abstract
Capillary zone electrophoresis-electrospray ionization time-of-flight mass spectrometry (CZE-ESI-TOF-MS) for the characterization of the monoclonal antibody (mAb) infliximab and its variants is presented. Infliximab was analyzed using a middle-up approach involving either reduction or digestion with the enzyme IdeS.
A multilayer capillary coating of Polybrene-dextran sulfate-Polybrene (PB-DS-PB) in combination with a background electrolyte of 40% acetic acid provided efficient separation of the obtained antibody fragments, that is, light chain (LC) and heavy chain (HC), as well as F(ab')2 and Fc/2 parts. C-terminal lysine variants were also resolved.
Recorded mass spectra of HC and Fc/2 fragments permitted assignment of 13 glycoforms and provided a quantitative profile, with GOF the most abundant glycoform (similar to 50%). CZE-ESI-TOF-MS represents an efficient means for the straightforward analysis of a monoclonal antibody and its proteoforms.