title: Structural effect of deglycosylation and methionine oxidation on a recombinant monoclonal antibody.
abstract: Methionine (Met) is one of the most susceptible amino acids to oxidation. Met256 (CH2-Met15.1) and Met432 (CH3-Met107) of a recombinant humanized monoclonal IgG1 antibody are located in the CH2 and CH3 domains, respectively. In three-dimensional structure, these two Met residues are close to the CH2-CH3 interface. In close proximity, oligosaccharides on the conserved asparagine (Asn) residues are enclosed in the CH2 domains. The relationship of Met oxidation with oligosaccharides and their effect on the structure of the antibody was investigated. Removal of oligosaccharides did not alter the oxidation rates of Met256 and Met432, however it caused significant structural changes as evidenced by the susceptibility of the deglycosylated antibody to trypsin and chymotrypsin. Oxidation of Met256 and Met432 did not cause significant conformational changes of the antibody with oligosaccharides, however oxidation of these Met residues accelerated degradation of the deglycosylated antibody. Analysis by mass spectrometry indicated that most of the protease cleavage sites were in the CH2 domains, which suggested that conformational changes induced by the removal of oligosaccharides and further by Met oxidation were local to the CH2 domains.
authors: Process Sciences Department, Abbott Bioresearch Center, 100 Research Drive, Worcester, MA 01605, United States. Hongcheng.liu@abbott.com
authors: Hongcheng
authors: H
authors: Liu
authors: Georgeen
authors: G
authors: Gaza-Bulseco
authors: Tao
authors: T
authors: Xiang
authors: Chris
authors: C
authors: Chumsae
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D008715:Methionine / Q000737:chemistry*; D051379:Mice; D009844:Oligosaccharides / Q000737:chemistry*; D010084:Oxidation-Reduction; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry
chemical_list: D000911:Antibodies, Monoclonal; D009844:Oligosaccharides; D011994:Recombinant Proteins; D008715:Methionine