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Middle-Down Analysis of Monoclonal Antibodies with Electron Transfer Dissociation Orbitrap Fourier Transform Mass Spectrometry

Fornelli, Luca, Ayoub, Daniel, Aizikov, Konstantin, Beck, Alain, Tsybin, Yury O.
Analytical chemistry 2014 v.86 no.6 pp. 3005-3012
Streptococcus pyogenes, antigens, chemical reduction, dissociation, electron transfer, immunoglobulin G, mass spectrometry, methionine, monoclonal antibodies, proteolysis, quality control, reversed-phase liquid chromatography, spectrometers
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 IgGs, though, enables reaching only up 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 ∼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 ∼50% sequence coverage for selected IgG fragments is reached in a single LC run and up 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.