LC-MS/MS method for quantification of dupilumab in human serum using Protein G enrichment.

Abstract

Dupilumab, a monoclonal antibody targeting IL-4Rα, is used for the treatment of type 2 inflammatory diseases. Recent studies have revealed a nonlinear relationship between its serum concentration and clinical response, particularly a "biological tipping point" during dose interval prolongation, highlighting the need for individualized therapeutic drug monitoring (TDM). In this study, a robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for dupilumab quantification in human serum was developed and validated. Specifically, dupilumab was enriched from 50 µL serum using Protein G magnetic beads, followed by denaturation, reduction, alkylation, and tryptic digestion. A surrogate peptide SGQSPQLLIYLGSNR was selected for quantification, and its stable isotope-labeled (SIL) analog served as an internal standard. Separation was performed on an Acquity UPLC BEH C8 column (50 mm × 2.1 mm, 1.7 µm) with detection using positive electrospray ionization in multiple reaction monitoring (MRM) mode. Excellent linearity was achieved from 1.00 to 400 µg/mL (r² > 0.99), with a lower limit of quantification of 1.00 µg/mL. Intra- and inter-run precision (%CV) and accuracy (%Bias) were within 9.27%. The validated method was successfully applied to analyze clinical serum samples. The established LC-MS/MS method provides a reliable alternative to immunoassays, with a wide dynamic range suitable for TDM and personalized dose interval adjustments in patients receiving dupilumab.