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
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
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