Middle-up characterization of monoclonal antibodies by online reduction liquid chromatography-mass spectrometry.

This study describes the fully automated middle-up characterization of monoclonal antibodies (mAbs) and next-generation variants by online reduction liquid chromatography-mass spectrometry (LC-MS). Proteins were trapped on-column and subjected to online desalting, denaturation and reduction prior to reversed phase elution of the created subunits in the MS. The evaluation of more than 20 different therapeutic proteins including full length mAbs (subclasses IgG1, IgG2 and IgG4), bispecific antibodies, antibody fragments, fusion proteins and antibody-drug conjugates (ADC) revealed that the online reduction method is as powerful as the widely applied offline sample preparation with dithiothreitol (DTT) as reducing agent and guanidine hydrochloride (Gnd.