Assessment of disulfide and hinge modifications in monoclonal antibodies

Abstract

During the last years there was a substantial increase in the use of antibodies and related proteins as therapeutics. The emphasis of the pharmaceutical industry is on IgG1, IgG2, and IgG4 antibodies, which are therefore in the focus of this article. In order to ensure appropriate quality control of such biopharmaceuticals, deep understanding of their chemical degradation pathways and the resulting impact on potency, pharmacokinetics, and safety is required. Criticality of modifications may be specific for individual antibodies and has to be assessed for each molecule. However, some modifications of conserved structure elements occur in all or at least most IgGs. In these cases, criticality assessment may be applicable to related molecules or molecule formats. The relatively low dissociation energy of disulfide bonds and the high flexibility of the hinge region frequently lead to modifications and cleavages. Therefore, the hinge region and disulfide bonds require specific consideration during quality assessment of mAbs. In this review, available literature knowledge on underlying chemical reaction pathways of modifications, analytical methods for quantification and criticality are discussed. The hinge region is prone to cleavage and is involved in pathways that lead to thioether bond formation, cysteine racemization, and iso‐Asp (Asp, aspartic acid) formation. Disulfide or sulfhydryl groups were found to be prone to reductive cleavage, trisulfide formation, cysteinylation, glutathionylation, disulfide bridging to further light chains, and disulfide scrambling. With regard to potency, disulfide cleavage, hinge cleavage, disulfide bridging to further light chains, and cysteinylation were found to influence antigen binding and fragment crystallizable (Fc) effector functionalities. Renal clearance of small fragments may be faster, whereas clearance of larger fragments appears to depend on their neonatal Fc receptor (FcRn) functionality, which in turn may be impeded by disulfide bond cleavage. Certain modifications such as disulfide induced aggregation and heterodimers from different antibodies are generally regarded critical with respect to safety. However, the detection of some modifications in endogenous antibodies isolated from human blood and the possibility of in vivo repair mechanisms may reduce some safety concerns.

Fluctuations in dissolved oxygen concentration during a CHO cell culture process affects monoclonal antibody productivity and the sulfhydryl‐drug conjugation process

“…20,38 Although trisulfides have no clinical relevance, their presence is known to affect the reduction step of conjugation. 21,38 As shown in Table 1, the amount of measurable trisulfides in Lots 1 and 5 ranged from 1.3 to 1.5% while Lots 2-4 contained trisulfides ranging from 2.5 to 3.0%. Although we acknowledge the small difference between the lots, as discussed later, we suspect only a fraction of trisulfides in our IgG 2 molecule were measurable by available methods and technology.…”

CE‐SDS method development, validation, and best practice—An overview

“…1), it was further observed that F(ab') 2 migrated before HH, although it has a higher Mw than HH, and that Fab migrated before HC and Fc. This can be explained by the effect of glycosylation and consequently reduced SDS binding on the hydrodynamic size of the protein, which is the actual ground for separation rather than the Mw, and was confirmed by comparing with PNGase-F deglycosylated mAb [22][23][24]. Deglycosylation shifted all species with a glycan linking site to earlier migration times.…”

Impact of enzymatic reduction on bivalent bispecific antibody fragmentation and loss of product purity upon reoxidation

“…The most pronounced difference between BisAb formats is the reduction pathway for BisAb‐C, which produced significant levels of LH and LHH fragments. Half antibody formation is also characteristic of IgG4 mAbs due to hinge flexibility (Bloom, Madanat, Marriott, Wong, & Chan, 1997; Moritz & Stracke, 2017; Silva, Vetterlein, Jose, Peters, & Kirby, 2015). The additional scFv fragments and (Gly 4 /Ser) 2 peptide linkers above the hinge region for the BisAb‐C format likely result in highly dynamic Fab arms, which would make the hinge disulfides more accessible to thioredoxin as illustrated in Figure 4.…”