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Background: Radical reactions result in breakage of the heavy-light chain linkage and hinge cleavage of an IgG1. Results: The degraded products are generated by different reaction pathways and mechanisms. Conclusion: A His 229 /Tyr substitution improves stability and effector function of an IgG1. Significance: A mechanism based strategy to engineer the upper hinge to improve multiple properties of an IgG1 is feasible.

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We report that the production of hydrogen peroxide by radical chain reductions of molecular oxygen into water in buffers leads to hinge degradation of a human IgG1 under thermal incubation conditions. The production of the hydrogen peroxide can be accelerated by superoxide dismutase or redox active metal ions or inhibited by free radical scavengers. The hydrogen peroxide production rate correlates well with the hinge cleavage. In addition to radical reaction mechanisms described previously, new degradation pathways and products were observed. These products were determined to be generated via radical reactions initiated by electron transfer and addition to the interchain disulfide bond between Cys 215 of the light chain and Cys 225 of the heavy chain. Decomposition of the resulting disulfide bond radical anion breaks the C-S bond at the side chain of Cys, converting it into dehydroalanine and generating a sulfur radical adduct at its counterpart. The hydrolysis of the unsaturated dehydropeptides removes Cys and yields an amide at the C terminus of the new fragment. Meanwhile, the competition between the carbonyl (-C ␣ ONH-) and the side chain of Cys allows an electron transfer to the ␣ carbon, forming a new intermediate radical species (-⅐ C ␣ (O ؊ )NH-) at Cys 225 . Dissociative deamidation occurs along the N-C ␣ bond, resulting in backbone cleavage. Given that hydrogen peroxide is a commonly observed product of thermal stress and plays a role in mediating the unique degradation of an IgG1, strategies for improving stability of human antibody therapeutics are discussed.Recombinant monoclonal antibodies (mAbs) represent one of the fastest growing segments of biotechnology. More than 20 mAbs have been approved by the United States Food and Drug Administration, and a volume of information is available in the research and development of therapeutic mAbs. Despite this progress, there are still unmet demands in improving the stability, efficacy, pharmacokinetic profile, and production yield of mAbs (1, 2). New mechanisms that govern these characteristics, in particular new mechanisms underlying the instability of mAbs, remain to be elucidated. As one of the product quality criteria, the stability needs to be monitored in the entire development course because it can profoundly affect mAb properties relevant to their therapeutic application. One of the common observations in the stability testing program is the so-called "hinge cleavage," which generates a Fab domain and a partial IgG1 that is missing the Fab. These products contain ladder cleavage sites of the upper hinge residues at new termini of the products (3-7), which were found, interestingly, as the same as those observed in H 2 O 2 -mediated radical-induced hinge cleavage of an IgG1 (8). The observation of the same cleavage sites under different conditions (e.g. thermal incubation conditions versus cell culture production) implies that a common mechanism may exist to drive this unique degradation.Two types of interchain disulfide bonds are present in the hing...