Characterization of Monoclonal Antibodies Oxidation Variants Using Middle-downApproach and Hydrogen/Deuterium Exchange Mass Spectrometry

Both non-oxidized and oxidized mAb were diluted (1 to 9 ratio) with labeling buffer and incubated for multiple time points. The samples were then quenched with 4M guanidine, 200 mM citric acid (pH 2.7) at 0.5 °C and subject to online pepsin digest at 8 °C for three minutes at 50 μL/min flow rate in a fully automated manner using H/D-X PAL (LEAP Technologies). The digested peptides were injected into a PepMap trapping column washed for one minute and eluted to a Hypersil GOLD C18 reverse phase column. An UltiMate 3000 nano pump system was employed to separate the digested peptides with 5% to 40% mobile phase B in 6 minutes gradient at flow rate of 40 μL/min. The separated peptides MS analysis was performed with Thermo ScientificTM Orbitrap FusionTM TribridTM mass spectrometer at 60K resolution.