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 H₂O₂ and featured complementary ladders of the C- and N-terminal residues (Asp²²⁶–Lys²²⁷–Thr²²⁸–His²²⁹–Thr²³⁰) 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 α-ketoacyl derivatives generated by radical cleavage at the α-carbon position through the diamide and α-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 (Cys²³¹-Pro-Pro-Cys-Pro), followed by the formation of a thiyl radical (Cys²³¹-S^•) on one cysteine and sulfenic acid (Cys²³¹-SOH) on the other. The location of the initial radical attack and the critical role of Cys²³¹ were demonstrated by the observation that 5,5-dimethyl-1-pyrroline N-oxide only reacted with the Cys²³¹ radical and completely blocked hinge cleavage, suggesting the necessity of an electron/radical transfer from the Cys²³¹ radical to the hinge residues where cleavage was observed. As a precursor of hydroxyl radicals, H₂O₂ 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.