title: Middle-down analysis of monoclonal antibodies with electron transfer dissociation orbitrap fourier transform mass spectrometry.
abstract: 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.
authors.affiliation: Biomolecular Mass Spectrometry Laboratory, Ecole Polytechnique Fédérale de Lausanne , 1015 Lausanne, Switzerland.
authors.forename: Luca
authors.initials: L
authors.lastname: Fornelli
authors.forename: Daniel
authors.initials: D
authors.lastname: Ayoub
authors.forename: Konstantin
authors.initials: K
authors.lastname: Aizikov
authors.forename: Alain
authors.initials: A
authors.lastname: Beck
authors.forename: Yury O
authors.initials: YO
authors.lastname: Tsybin
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D004579:Electron Transport; D005583:Fourier Analysis; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D013297:Streptococcus pyogenes / Q000737:chemistry
chemical_list: D000911:Antibodies, Monoclonal