Human IgG1 hinge fragmentation as the result of H2O2-mediated radical cleavage.
Hinge cleavage of a recombinant human IgG1 antibody, generated during production in a Chinese hamster ovary cell culture, was observed in the purified material. The cleavage products could be reproduced by incubation of the antibody with H(2)O(2) and featured complementary ladders of the C- and N-terminal residues (Asp(226)-Lys(227)-Thr(228)-His(229)-Thr(230)) in the heavy chain of the Fab domain and the upper hinge of one of the Fc domains, respectively. Two adducts of +45 and +71 Da were also observed at the N-terminal residues of some Fc fragments and were identified as isocyanate and alpha-ketoacyl derivatives generated by radical cleavage at the alpha-carbon position through the diamide and alpha-amidation pathways. We determined that the hinge cleavage was initiated by radical-induced breakage of the disulfide bond between the two hinge cysteines at position 231 (Cys(231)-Pro-Pro-Cys-Pro), followed by the formation of a thiyl radical (Cys(231)-S(*)) on one cysteine and sulfenic acid (Cys(231)-SOH) on the other. The location of the initial radical attack and the critical role of Cys(231) were demonstrated by the observation that 5,5-dimethyl-1-pyrroline N-oxide only reacted with the Cys(231) radical and completely blocked hinge cleavage, suggesting the necessity of an electron/radical transfer from the Cys(231) radical to the hinge residues where cleavage was observed. As a precursor of hydroxyl radicals, H(2)O(2) is widely produced in healthy cells and tissues and therefore could be the source for the radical-induced fragmentation of human IgG1 antibodies in vivo.
Department of Analytical and Formulation Science, Amgen Incorporated, Seattle, Washington 98119, USA. byan@amgen.com
Boxu
B
Yan
Zac
Z
Yates
Alain
A
Balland
Gerd R
GR
Kleemann
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry*; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry* / Q000235:genetics; D007074:Immunoglobulin G / Q000737:chemistry* / Q000235:genetics; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D006861:Hydrogen Peroxide