Institution: | 1. School of Chemical Engineering, Tianjin University, Tianjin, China;2. State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing, China
Beijing United-Power Pharma Tech Co., Ltd., Beijing, China;3. State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing, China;4. School of Chemical Engineering, Tianjin University, Tianjin, China
State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing, China
Beijing United-Power Pharma Tech Co., Ltd., Beijing, China |
Abstract: | We present a simple and robust LC–MS/MS assay for the simultaneous quantitation of an antibody cocktail of trastuzumab and pertuzumab in monkey serum. The LC–MS/MS method saved costs, decreased the analysis time, and reduced quantitative times relative to the traditional ligand-binding assays. The serum samples were digested with trypsin at 50°C for 60 min after methanol precipitation, ammonium bicarbonate denaturation, dithiothreitol reduction, and iodoacetamide alkylation. The tryptic peptides were chromatographically separated using a C18 column (2.1 × 50 mm, 2.6 μm) with mobile phases of 0.1% formic acid in water and acetonitrile. The other monoclonal antibody, infliximab, was used as internal standards to minimize the variability during sample processing and detection. A unique peptide for each monoclonal antibody was simultaneously quantified using LC–MS/MS in the multiple reaction monitoring mode. Calibration curves were linear from 2.0 to 400 μg/mL. The intra- and inter-assay precision (%CV) was within 8.9 and 7.4% (except 10.4 and 15.1% for lower limit of quantitation), respectively, and the accuracy (%Dev) was within ±13.1%. The other validation parameters were evaluated, and all results met the acceptance criteria of the international guiding principles. Finally, the method was successfully applied to a pharmacokinetics study after a single-dose intravenous drip administration to cynomolgus monkeys. |