Full Characterization of Heterogeneous Antibody Samples Under Denaturing and Native Conditions on a Hybrid Quadrupole-Orbitrap Mass Spectrometer

MS analysis of antibodies at the protein and peptide levels is critical during development and production of biopharmaceuticals. The compositions of current generation therapeutic proteins are often complex due to their heterogeneity caused by various modifications which are relevant for their efficacy. Intact proteins analyzed by ESI-MS are detected in higher charge states that also provide more complexity in mass spectra. Protein analysis in native or nativelike conditions with zero or minimal organic solvent and neutral or weakly acidic pH decreases charge state value resulting in mAb detection at higher m/z ranges with more spatial resolution. Here we have analyzed the profiles of three monoclonal antibodies under denaturing and native conditions by direct infusion with offline desalting and with on-line desalting via size exclusion and reversed phase type columns. The samples were analyzed with three different workflows (Figure 1): 1) the analysis on the intact level under native and denaturing conditions; 2) the analysis of subunits following IdeS digestion and or reduction; and 3) peptide mapping following sample preparation applying the Thermo ScientificTM SMART DigestTM kit. The mass spectrometer used for all experiments was a commercially available hybrid quadrupole-Orbitrap mass spectrometer with modified instrument control software to allow for improved high mass transmission and mass detection up to 8000 m/z. This modification is necessary for the analysis of antibody samples on the intact level under native conditions requiring the detection of masses beyond the standard mass range of up to 6000 m/z.