ArticlePharmaceutical research2026
Fab-Level LC-MS Assessment of Chain Pairing in Asymmetric Bispecific Antibodies Enabled by IdeS Digestion and Selective Hinge Reduction.
Article in Pharmaceutical research, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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5 authors.
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Abstract
purposeAccurate 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.
methodsIgG-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.
resultsCompared 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.
conclusionsIdeS 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.
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