In agreement with earlier observations,21,22 the B isoform eluted earlier than the A isoform in both the non-reduced and partially-reduced mAbs generated at low pH. Open in a separate window Figure 5. Disulfide isoform distribution of IgG2-A (gray) and IgG2-B (black) mAb incubated in the reaction buffer for 3?hours at 37C. highly potent cytotoxic molecule to the prospective cells, most of which are developed for oncology indications.1C3 The mechanism of action of an ADC relies on the internalization of the antibody-drug complex, followed by intracellular degradation and release of the cytotoxic drug that disrupts an essential cellular process and leads to cell death. Endogenous cysteine and lysine residues are currently the most popular conjugation locations for ADCs in medical development, but other locations such as GW806742X glycans and manufactured antibodies with solitary amino acid substitutions will also be being developed.1,4 Currently, four ADCs have gained US Food and Drug Administration market approvals (Adcetris?, Kadcyla?, Mylotarg?, and Besponsa?) and over 50 ADCs were in active medical tests in 2016.1,5 The majority of ADCs GW806742X use monoclonal antibodies (mAbs) of the IgG1 subclass, and therefore the majority of published information related to physicochemical and course of action characterization pertains to IgG1 ADCs. However, at least 9 clinical-stage ADCs use an IgG2 or IgG4 mAb, and knowledge gained from IgG1 ADCs may not apply to these molecules.1,6 Cysteine-linked ADCs that target drug-linker conjugation to endogenous cysteines involved in interchain disulfide bonds constitute approximately half of all active clinical ADCs.5 Thiol-maleimide coupling is a common approach for attaching the drug-linkers (DL) to the free cysteines generated by a controlled partial reduction of the antibody interchain disulfide bonds.7,8 This approach results in a heterogeneous mixture of molecules with variable levels of conjugation, ranging from 0C8 medicines for IgG1 and IgG4 ADCs or 0C12 medicines for IgG2 ADCs. Furthermore, the main conjugation locations differ based on the antibody subclass, with IgG1s preferentially conjugating at cysteines in the antigen-binding fragment (Fab), but in the hinge cysteines in IgG2s.6 The distribution of drug-loaded varieties and the locations of drug-linker attachment are critical quality attributes (CQAs) because these properties affect the medicines product stability and their pharmacokinetic/pharmacodynamics (PK/PD) profiles.9C14 The four IgG subclasses (IgG1, IgG2, IgG3, IgG4) differ structurally with respect to their conserved amino GW806742X acid sequences, the space of the core hinge region, and the number and configurations of interchain disulfide bonds.15,16 IgG1 mAbs have been the most popular subclass utilized for therapeutic mAbs because they possess favorable biological and physicochemical characteristics that enhance their developability as clinical drug product candidates.17C19 The cumulative knowledge of therapeutic IgG1 mAbs and their structural simplicity likely led to their selection as the preferred isotype for ADC development. However, IgG1 mAbs can elicit strong effector functions that discord with the desired mechanism of action (MOA) of an ADC.16,19,20 IgG2 and IgG4 mAbs are weak complement cascade activators and have weaker affinities for the Fc receptors than IgG1s, and therefore are potentially more compatible with the MOA of an ADC because of the increased possibility of cellular internalization.16,19 The IgG2 subclass includes the A, A/B, and B disulfide isoforms that are formed under common large-scale bioprocess conditions, where the isoform distribution depends on the cell culture parameters and the space of exposure of the antibody to the cell culture fluid.21C25 Each IgG2 disulfide isoform differs in the linkages between the light chain-heavy chain (LC-HC) interchain disulfide, thereby affecting the higher-order structures, overall charge, hydrophobicity, and binding affinities.21,22 The higher-order structural differences in the Fab/CH2 interface of the IgG2 isoforms affect the solvent accessibilities of the interchain disulfides.26,27 One thought for the ADC manufacturing process is the potential for disulfide relationship rearrangement, which can occur at the preferred pH range for the partial reduction and PPP2R1B conjugation reactions.28C32 The pace of intramolecular thiol-disulfide exchange is accelerated when the conditions favor the degradation of disulfides and the formation of the thiolate anion, such as under the presence of a reducing reagent or in neutral or alkaline environments.28,33 IgG2 mAbs are particularly susceptible to disulfide relationship scrambling, and have been reported to spontaneously rearrange from your A A/B B configurations under reducing conditions.25,34 Antibodies of the IgG4 subclass have also been reported to undergo disulfide relationship scrambling that results in formation of intrachain disulfides and subsequent dissociation of the half-antibody.35C38 GW806742X We previously reported the IgG2-A isoform interchain disulfide bonds reduce and conjugate at.