Middle-down analysis of monoclonal antibodies with electron transfer dissociation Orbitrap FTMS

The rapid growth of approved biotherapeutics, e.g., monoclonal antibodies or immunoglobulins G (IgGs), demands improved techniques for their quality control. Traditionally, proteolysis-based bottom-up mass spectrometry (MS) has been employed. However, the long, multistep sample preparation protocols required for bottom-up MS are known to potentially introduce artifacts in the original sample. For this reason, a top-down MS approach would be preferable. The current performance of top-down MS of intact monoclonal Ig Gs, though, enables reaching only up to similar to 30% sequence coverage, with incomplete sequencing of the complementarity determining regions which are fundamental for IgG's antigen binding. Here, we describe a middle-down MS protocol based on the use of immunoglobulin G-degrading enzyme of Streptococcus pyogenes (IdeS), which is capable of digesting IgGs in only 30 min. After chemical reduction, the obtained similar to 25 kDa proteolytic fragments were analyzed by reversed phase liquid chromatography (LC) coupled online with an electron transfer dissociation (ETD)-enabled hybrid Orbitrap Fourier transform mass spectrometer (Orbitrap Elite FTMS). Upon optimization of ETD and product ion transfer parameters, results show that up to similar to 50% sequence coverage for selected IgG fragments is reached in a single LC run and up to similar to 70% when data obtained by distinct LC MS runs are averaged. Importantly, we demonstrate the potential of this middle-down approach in the identification of oxidized methionine residues. The described approach shows a particular potential for the analysis of IgG mixtures.