Breaking the Light and Heavy Chain Linkage of Human Immunoglobulin G1 (IgG1) by Radical Reactions

Abstract

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...

Assessment of disulfide and hinge modifications in monoclonal antibodies

“…In general, hinge modifications are induced by exposure to heat [, ], H 2 O 2 , acidic/basic pH , or UV light . Disulfide bonds are strongly involved in these processes that also comprise different cleavage processes , thioether bond formation and Cys racemization . Also iso‐Asp formation is an often observed variant of these modification processes .…”

“…These investigations elucidated the underlying reaction mechanisms that lead to diverse hinge modification and fragmentation (see also Fig. ): Disulfide ‐linked Fab fragments were described as a result of radical transfer between the LC‐HC disulfide bond and the first HC‐HC disulfide bond including the upper hinge His as radical center. This resulted in cleavage of any one peptide bond between these two disulfide bonds (formation of ragged termini or ladder formation as denoted above).…”

“…Such cleavage can be attributed to the low steric hindrance by the small side chain. Other fragmentation processes are facilitated by different amino acid side chains like for aspartic acid (Asp), Ser, Thr, Cys, or Asn and also by the occurrence of free radicals . A total of 47 potential fragmentation sites have been summarized previously .…”

Metal ion interactions with mAbs: Part 1

“…Hydroxyl radicals generated in the presence of Cu 2C sulfate have been implicated in the cleavage of the IgG1 hinge region. 12 Our data using mannitol showed that there was no impact on fragmentation in the presence of this hydroxyl radical scavenger. Nonetheless, it has been shown that a hydroxyl radical generated in close proximity to the site of oxidation may be consumed before it can diffuse, which would render mannitol unable to quench the reaction.…”

“…11 Non-enzymatic fragmentation, on the other hand, can be observed by direct hydrolysis, 6 b-elimination, 7 and free-radical catalysis of peptide bond cleavage in mAbs, 12 and metal-mediated oxidative cleavage in peptides. 13 Fragmentation kinetics by non-enzymatic methods has been shown to vary with pH; pH 6 has the lowest rate of cleavage and rates increase in both more acidic and basic conditions, 14 and storage temperature.…”