Abstract
Purpose
Accurate assessment of heavy/light chain pairing is essential for asymmetric bispecific antibodies (BsAbs), because low-level mispairing (including light-chain swapping) can be difficult to resolve at the intact IgG level. We sought to enable robust Fab-level pairing assessment with a workflow broadly applicable across human IgG subclasses.Methods
IgG-like BsAbs were digested with FabRICATOR (IdeS) to generate uniform F(ab)₂ and Fc fragments, followed by selective hinge reduction using 2-mercaptoethylamine (2-MEA) to produce discrete Fab fragments. Reduction conditions were optimized to minimize over-reduction and suppress 2-MEA-derived Fab adducts. Fab-level species were evaluated by LC-MS, and an RP-LC method was developed to separate Fc from Fab to reduce spectral interference.Results
Compared with DTT and TCEP, 2-MEA preferentially reduced hinge-region disulfides to efficiently convert F(ab)₂ to Fab under practical conditions (e.g., 10 mM, 37 °C, 30-60 min). A key analytical artifact was formation of 2-MEA-related Fab adducts (Δmass ~ 76 Da) that can suppress or overlap Fab signals. Adduct formation was reduced in PBS relative to Tris buffer (pH 8) and was further suppressed to near-background levels by inclusion of EDTA (5 mM). Under optimized conditions, Fab-level mispairing was detected across multiple BsAb case studies, including low-concentration early-stage samples analyzed without pre-treatment, and the workflow was demonstrated for IgG1/IgG4-based BsAbs and bispecific modalities (altibodies), as well as IgG2/IgG3 antibodies.Conclusions
IdeS digestion combined with selective hinge reduction and controlled solution chemistry (PBS + EDTA) provides a practical, scalable LC-MS framework for routine Fab-level chain pairing assessment to support BsAb development and characterization.