Abstract:Human IgG2 consists of disulfide-mediated structural isoforms, classified by the number of Fab arms disulfide-linked to the heavy chain hinge. In the IgG2-B isoform, both Fab arms are linked to the hinge region, and in IgG2-A, neither Fab arm are linked to the hinge. IgG2-A/B is a hybrid between these two forms, with only one Fab arm disulfide-linked to the hinge. Within each of these isoform types are subtypes, with subtle disulfide-linkage differences. Here we explored the structural basis for the A 1 and A … Show more
“…40 Literature reported IgG 2 κ to vary between A1, A2, B, and A/B isoforms, favoring the A/B and B isoforms. 37,41 As expected, the majority of our product was in the A/B and B isoforms, and consisted over 60% of total product as determined by RP-HPLC (data not shown).…”
Section: Trisulfide Detection In An Igg 2 κsupporting
confidence: 80%
“…Pritatsky et al reported trisulfides in IgG 2 hinge region, but their tested antibody was an IgG 2 λ and the majority of their product was in the A isoform . Literature reported IgG 2 κ to vary between A1, A2, B, and A/B isoforms, favoring the A/B and B isoforms . As expected, the majority of our product was in the A/B and B isoforms, and consisted over 60% of total product as determined by RP‐HPLC (data not shown).…”
“…40 Literature reported IgG 2 κ to vary between A1, A2, B, and A/B isoforms, favoring the A/B and B isoforms. 37,41 As expected, the majority of our product was in the A/B and B isoforms, and consisted over 60% of total product as determined by RP-HPLC (data not shown).…”
Section: Trisulfide Detection In An Igg 2 κsupporting
confidence: 80%
“…Pritatsky et al reported trisulfides in IgG 2 hinge region, but their tested antibody was an IgG 2 λ and the majority of their product was in the A isoform . Literature reported IgG 2 κ to vary between A1, A2, B, and A/B isoforms, favoring the A/B and B isoforms . As expected, the majority of our product was in the A/B and B isoforms, and consisted over 60% of total product as determined by RP‐HPLC (data not shown).…”
“…By nonreducing peptide mapping and Edman sequencing even more disulfide subvariants of the IgG2‐A/B and IgG2‐B structures could be identified . IgG2‐A1 and IgG2‐A2 isoforms with slight differences in disulfide linkages are also described elsewhere . A2 was more resistant to reduction and had a lower Fc glycan flexibility.…”
Section: Disulfide‐ and Hinge‐related Modificationsmentioning
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.
“… 30 The A’ species has the same disulfide configuration as the A-form, but contains a hinge disulfide that is protected from reduction. 39 Figure 2. nrRP-UHPLC chromatograms of the starting material (A), redox-enriched A and B isoforms (B), and CEX-fractionated IgG2-A and IgG2-B mAbs (C).…”
Cysteine-linked antibody-drug conjugates (ADCs) produced from IgG2 monoclonal antibodies (mAbs) are more heterogeneous than ADCs generated from IgG1 mAbs, as IgG2 ADCs are composed of a wider distribution of molecules, typically containing 0 – 12 drug-linkers per antibody. The three disulfide isoforms (A, A/B, and B) of IgG2 antibodies confer differences in solvent accessibilities of the interchain disulfides and contribute to the structural heterogeneity of cysteine-linked ADCs. ADCs derived from either IgG2-A or IgG2-B mAbs were compared to better understand the role of disulfide isoforms on attachment sites and distribution of conjugated species. Our characterization of these ADCs demonstrated that the disulfide configuration affects the kinetics of disulfide bond reduction, but has minimal effect on the primary sites of reduction. The IgG2-A mAbs yielded ADCs with higher drug-to-antibody ratios (DARs) due to the easier reduction of its interchain disulfides. However, hinge-region cysteines were the primary conjugation sites for both IgG2-A and IgG2-B mAbs.
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