Identification and characterization of asparagine deamidation in the light chain CDR1 of a humanized IgG1 antibody.
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.
Vlasak
Josef
J
Merck & Co. Inc, West Point, PA, USA.
Bussat
Marie C
MC
Wang
Shiyi
S
Wagner-Rousset
Elsa
E
Schaefer
Mark
M
Klinguer-Hamour
Christine
C
Kirchmeier
Marc
M
Corvaïa
Nathalie
N
Ionescu
Roxana
R
Beck
Alain
A
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
D000941:Antigens; D022801:Complementarity Determining Regions; D007074:Immunoglobulin G; D007147:Immunoglobulin Light Chains; D001216:Asparagine; D010206:Papain