Assessment of disulfide and hinge modifications in monoclonal antibodies (1)
Clear focus of pharmaceutical development is on the IgG subtype whereby IgG1, IgG2, and IgG4 are most relevant. In contrast to IgG1 and IgG2, IgG4 does not exhibit complement activation. In addition, IgG2 and IgG4 have lower FcγRI, FcγRII, FcγRIIIa/b receptor affinities than IgG1. IgG3 is not used as therapeutic, due to its short half‐life (approximately 7 days for IgG3 versus approximately 21 days for IgG1).
Formation of antibody fragment that results from loss of one Fab (Fab/c) with disulfide‐linked LC and Fab fragment of the HC was attributed to a nucleophilic attack of the serine hydroxyl on the lysine carbonyl with subsequent N‐terminal cleavage (mechanism 1). 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). Disulfide‐linked Fab fragments did also result from C‐terminal Asp cleavage under acidic conditions (mechanisms 2 and 5). In case of IgG1, thioether bond (lanthionine) formation was observed between the C‐terminal Cys of the LC and its counterpart in the HC (mechanisms 3 and 4). Formation of a cyclic imidazoline intermediate (mechanism 3). Noncovalent Fab (Fab part of the cleaved HC without disulfide bridge to adjacent LC) and Fc (Fc part of the cleaved HC) were described as a result of β‐elimination at the LC‐HC disulfide bond with subsequent hydrolysis under basic conditions (mechanism 4).Formation of dehydroalanine by β‐elimination at the LC‐HC disulfide bond with subsequent Michael‐like addition (mechanism 4).
IgG2 disulfide bond shuffling in serum leads to IgG2‐A, IgG2‐B, and IgG2‐A/B isoforms. By using a flow‐through dialysis system, conversion kinetics of the IgG2‐A, IgG2‐A/B, and IgG2‐B isoforms were studied in vitro under physiological‐like conditions. the steady state ratio between IgG2‐A, IgG2‐B, and IgG2‐A/B would have been shifted in comparison to the in vitro ratio. In IgG2κ antibodies, formation of IgG2‐B from IgG2‐A/B was faster than the same conversion of IgG2λ antibodies.
Endogenous and therapeutic IgG4 antibodies can exchange HC/LC parts in circulation that results in undesired bispecific antibodies. This can be attributed to an equilibrium between inter‐ and intrachain cystines of the two HCs that are not always linked covalently. In vivo, this mechanism requires reducing conditions in blood or at cell surfaces. It was also described that interactions between the CH3 domains play a role in Fab arm exchange between two MAbs.
