Effects of Calibration Approaches on the Accuracy for LC−MS Targeted Quantification of Therapeutic Protein

ABSTRACT: LC−MS provides a promising alternative to ligand-binding assays for quantification of therapeutic proteins and biomarkers. As LC−MS methodology is based on the analysis of proteolytic peptides, calibration approaches utilizing various calibrators and internal standards (I.S.) have been developed. A comprehensive assessment of the accuracy and reliability of these approaches is essential but has yet been reported. Here we performed a well-controlled and systematic comparative study using quantification of monoclonal-antibody in plasma as the model system. Method development utilized a high-throughput orthogonal-array-optimization, and two sensitive and stable signature-peptides (SP) from different domains were selected based on extensive evaluations in plasma matrix. With the purities of all protein/peptide standards corrected by quantitative amino acid analysis (AAA), five calibration approaches using stable-isotope-labeled (SIL) I.S. were thoroughly compared, including those at peptide, extended-peptide, and protein levels and two “hybrid ” approaches (i.e., protein calibrator with SIL-peptide or SIL-extended-peptide I.S.). These approaches were further evaluated in parallel for a 15 time point, preclinica