Anal Biochem. 2009 Sep 15;392(2):145-54. Epub 2009 Jun 2.

Identification and characterization of asparagine deamidation in the light chain CDR1 of a humanized IgG1 antibody.

Vlasak J, Bussat MC, Wang S, Wagner-Rousset E, Schaefer M, Klinguer-Hamour C, Kirchmeier M, Corvaïa N, Ionescu R, Beck A.

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Merck & Co. Inc, West Point, PA, USA.

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.

PMID:: 19497295; [PubMed - indexed for MEDLINE]

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Identification and characterization of asparagine deamidation in the light chain CDR1 of a humanized IgG1 antibody.

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