title: Identification and characterization of asparagine deamidation in the light chain CDR1 of a humanized IgG1 antibody.
abstract: Despite technological advances, detection of deamidation in large proteins remains a challenge and the use of orthogonal methods is needed for unequivocal assignment. By a combination of cation-exchange separation, papain digestion, and a panel of mass spectrometry techniques we identified asparagine deamidation in light chain complementarity determining region 1 (CDR1) of a humanized IgG1 monoclonal antibody. The reaction yields both Asp and isoAsp, which were assigned by Edman degradation and by isoAsp detection using protein isoaspartate methyltransferase. The deamidated antibody variants were less potent in antigen binding compared to the nondegraded antibody. Changes in near-UV CD spectra, susceptibility to papain cleavage in an adjacent CDR2 loop, and the tendency of the newly formed isoAsp to undergo isomerization suggest local perturbations in the structure of the isoAsp-containing antibody.
authors.affiliation: Merck & Co. Inc, West Point, PA, USA.
authors.forename: Josef
authors.initials: J
authors.lastname: Vlasak
authors.forename: Marie C
authors.initials: MC
authors.lastname: Bussat
authors.forename: Shiyi
authors.initials: S
authors.lastname: Wang
authors.forename: Elsa
authors.initials: E
authors.lastname: Wagner-Rousset
authors.forename: Mark
authors.initials: M
authors.lastname: Schaefer
authors.forename: Christine
authors.initials: C
authors.lastname: Klinguer-Hamour
authors.forename: Marc
authors.initials: M
authors.lastname: Kirchmeier
authors.forename: Nathalie
authors.initials: N
authors.lastname: Corvaïa
authors.forename: Roxana
authors.initials: R
authors.lastname: Ionescu
authors.forename: Alain
authors.initials: A
authors.lastname: Beck
mesh_terms: D000941:Antigens / Q000276:immunology; D001216:Asparagine / Q000032:analysis* / Q000737:chemistry; D002152:Calorimetry, Differential Scanning; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D022801:Complementarity Determining Regions / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D018360:Crystallography, X-Ray; D003641:Deamination; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D007147:Immunoglobulin Light Chains / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D007536:Isomerism; D013058:Mass Spectrometry; D008958:Models, Molecular; D015394:Molecular Structure; D010206:Papain / Q000378:metabolism
chemical_list: D000941:Antigens; D022801:Complementarity Determining Regions; D007074:Immunoglobulin G; D007147:Immunoglobulin Light Chains; D001216:Asparagine; D010206:Papain