Normality tests

Abstract

Recombinant monoclonal antibodies (MAbs) can be heterogeneous due to modifications that can occur during expression, purification or during storage. These large multichain proteins (approximately 150 kDa) are structurally challenging for detailed characterization to identify the sites of modifications. We report the use of LTQ Orbitrap mass spectrometry to accurately measure the average masses of individual glycoforms by direct infusion of an intact antibody. To identify the site-specific modification of methionines in the antibody caused by forced oxidation, we used a 'middle-down' approach. The antibody was subjected to limited digestion using the endoproteinase Lys-C and reduced to generate Fab heavy chain, single chain Fc and light chain fragments (approximately 25 kDa each). These species were subjected to on-line liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) analysis using an LTQ Orbitrap, where these large precursors were dissociated by higher-energy collisions in the C-trap. High resolution and accuracy achieved for resulting fragments allowed us to show in a site-specific manner that only the methionines in the Fc heavy chain were oxidized under the studied conditions.