title: Conformational changes of recombinant monoclonal antibodies by limited proteolytic digestion, stable isotope labeling, and liquid chromatography-mass spectrometry.
abstract: Limited proteolytic digestion is a method with a long history that has been used to study protein domain structures and conformational changes. A method of combining limited proteolytic digestion, stable isotope labeling, and mass spectrometry was established in the current study to investigate protein conformational changes. Recombinant monoclonal antibodies with or without the conserved oligosaccharides, and with or without oxidation of the conserved methionine residues, were used to test the newly proposed method. All of the samples were digested in ammonium bicarbonate buffer prepared in normal water. The oxidized deglycosylated sample was also digested in ammonium bicarbonate buffer prepared in (18)O-labeled water. The sample from the digestion in (18)O-water was spiked into each sample digested in normal water. Each mixed sample was subsequently analyzed by liquid chromatography-mass spectrometry (LC-MS). The molecular weight differences between the peptides digested in normal water versus (18)O-water were used to differentiate peaks from the samples. The relative peak intensities of peptides with or without the C-terminal incorporation of (18)O atoms were used to determine susceptibility of different samples to trypsin and chymotrypsin. The results demonstrated that the method was capable of detecting local conformational changes of the recombinant monoclonal antibodies caused by deglycosylation and oxidation.
authors.affiliation: Product Characterization, Alexion Pharmaceuticals, Cheshire, CT 06410, USA.
authors.forename: Gomathinayagam
authors.initials: G
authors.lastname: Ponniah
authors.affiliation: Product Characterization, Alexion Pharmaceuticals, Cheshire, CT 06410, USA.
authors.forename: Christine
authors.initials: C
authors.lastname: Nowak
authors.affiliation: Product Characterization, Alexion Pharmaceuticals, Cheshire, CT 06410, USA.
authors.forename: Adriana
authors.initials: A
authors.lastname: Kita
authors.affiliation: Product Characterization, Alexion Pharmaceuticals, Cheshire, CT 06410, USA.
authors.forename: Guilong
authors.initials: G
authors.lastname: Cheng
authors.affiliation: Product Characterization, Alexion Pharmaceuticals, Cheshire, CT 06410, USA.
authors.forename: Yekaterina
authors.initials: Y
authors.lastname: Kori
authors.affiliation: Product Characterization, Alexion Pharmaceuticals, Cheshire, CT 06410, USA. Electronic address: liuh@alxn.com.
authors.forename: Hongcheng
authors.initials: H
authors.lastname: Liu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism; D016466:CHO Cells; D002853:Chromatography, Liquid; D002918:Chymotrypsin / Q000378:metabolism; D003412:Cricetulus; D006031:Glycosylation; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry* / Q000378:metabolism; D007553:Isotope Labeling; D013058:Mass Spectrometry; D008715:Methionine / Q000032:analysis / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010455:Peptides / Q000032:analysis / Q000378:metabolism; D059748:Proteolysis; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D010455:Peptides; D011994:Recombinant Proteins; D008715:Methionine; D002918:Chymotrypsin; D014357:Trypsin
keywords: Deglycosylation; Limited proteolytic digestion; Mass spectrometry; Methionine oxidation; Recombinant monoclonal antibodies; Stable isotope