Kevin Ray , Yue Lu , Pegah Jalili , Jeffrey Turner , Nicolas Caffarelli , Tom Juehne
MilliporeSigma, a business of Merck KGaA, Darmstadt, Germany
Monoclonal antibodies are the largest class of biotherapeutics approved for a variety of clinical indications, particularly in oncology and autoimmune diseases. During preclinical drug development, assays are required to understand the absorption, distribution, metabolism and excretion of monoclonal antibodies, which is crucial for their design and selection. Hereby, we demonstrate a robust, high-throughput workflow for quantification of human IgG1 antibodies in animal sera by LC-MS/MS utilizing a stable isotope labeled universal monoclonal antibody internal standard which is introduced prior to immunoaffinity enrichment and tryptic digestion on an easy-to-use 96-well plate based kit format.
Figure 1. Comparison of universal plate-based and magnetic bead-based workflow for quantification IgG1 antibodies.
Figure 2. Total ion chromatogram of three selected signature peptides, one quantitative (TTPP) and two qualitative (VVSV and FNWY), used for universal analysis of IgG1 therapeutic drug antibodies in preclinical studies
Figure 3. The calibration curves for standards of Infliximab over the concentration range of 0.1-12.5 μg/mL and Rituximab over the extended range of 0.8 - 200 μg/mL using 5PL regression.
Figure 4. Reproducibility of universal TTPP signature peptide from Adalimumab at LLOQ of 0.1 μg/mL concentration.