Human IgG1 Hinge Fragmentation as the Result of H2O2-mediated Radical Cleavage

Boxu Yan,Zac Yates,Alain Balland,Gerd R Kleemann

doi:10.1074/jbc.m109.064147

Abstract

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.

Highlights

Chance for introducing undesirable modifications such as oxidation, proteolytic cleavage, deamidation, and isomerization
The thiyl radical initializes an electron transfer/radical transfer to the hinge residues, which gives rise to backbone cleavage on a one radical cleavage per molecule basis to generate one free Fab domain fragment with a ladder of the C-terminal heavy chain residues complementary to the N-terminal ladder of one of the heavy chains of the Fc domain in the truncated IgG1 mAb
The results showed that P1 was a heavily oxidized partial molecule, which was lacking one of the Fab domains and contained a truncated Fc domain, in which one of the Fc domain heavy chains (Fc-HC) were characterized by a unique N-terminal ladder consisting of the hinge residues DKTHT (Table 1 and Fig. 2)

Summary

To whom correspondence should be addressed

Chance for introducing undesirable modifications such as oxidation, proteolytic cleavage, deamidation, and isomerization. A recent study indicated that antibodies have the intrinsic capacity to convert molecular oxygen into hydrogen peroxide (H2O2) [8] and in this process to produce some short lived hydroxyl radical species (HO1⁄7) at the interface of the light and heavy chains (9 –12) These observations were further supported by a more recent observation that the light chains (three ␬ and three ␭ types) from the urine of six patients who had multiple myeloma and light chain proteinuria were found capable of generating H2O2 [13]. We describe the degradation of a recombinant human IgG1 antibody that was purified from CHO cells cultured in a bioreactor, and we present evidence for a specific H2O2-mediated radical cleavage in the hinge region of the antibody. We propose that the observed sequential hinge cleavage of an IgG1 resulted from a hydroxyl radical reaction mechanism

EXPERIMENTAL PROCEDURES

RESULTS

16 Da at Pro232 Trace

DISCUSSION