Bioanalysis Zone

Poster: an immunoaffinity capture/UPLC-MS/MS method for multiplexed quantitation of two highly homologous antibodies in human serum



Monoclonal antibody (mAb) based biotherapeutics have emerged as a dominant class of approved medicines during the past decade in both market share and variety of target diseases. Ligand binding assays (LBAs), such asenzyme-linked immunosorbent assays (ELISA), have been the gold standard for the bioanalysis of mAbs in support of non-clinical and clinical trials. While conventional LBAs provide highly sensitive and specific detection for the quantitation of mAbs, combining an LBA with liquid chromatography tandem mass spectrometry (LC-MS/MS) detection offers additional orthogonal dimensions of selectivity/specificity, extending the assay utility to a multiplexed format otherwise not achievable by LBA alone.
Herein, we present a hybrid LBA/LC-MS/MS method for the concurrent quantitation of co-administered mAbs, A and B in human serum. The two mAbs of interest have >99% sequence homology and the same therapeutic target. Due to their similarity, development of a multiplexed LBA assay was rendered extremely challenging. However, using the hybrid assay approach, the two mAbs were isolated by immunoaffinity capture, digested, and simultaneously quantified using unique signature tryptic peptides from each mAb as surrogates for the target antibodies. The method has shown optimal compatibility with individual LBAs for mAbs, A and B.

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