title: Validated LC-MS/MS analysis of immune checkpoint inhibitor Nivolumab in human plasma using a Fab peptide-selective quantitation method: nano-surface and molecular-orientation limited (nSMOL) proteolysis.
abstract: We previously reported the nano-surface and molecular-orientation limited (nSMOL) proteolysis, which is a novel method for selective quantitation of monoclonal antibody Fab. The nSMOL strategy is a Fab-selective limited proteolysis which utilizes the size difference between the protease nanoparticle (200nm) and the antibody resin pore (100nm). Here, we applied this method to a fully validated LCMS analysis of Nivolumab in human plasma. The immunoglobulin fraction was collected using Protein A resin, which was then followed by nSMOL reaction using the FG nanoparticle surface-immobilized trypsin under a nondenaturing physiological condition at 50°C for 7h. After removal of resin and nanoparticles by filter centrifugation, signature peptides were separated using the ODS column liquid chromatography. The signature peptide ASGITFSNSGMHWVR from Nivolumab complementarity-determining region (CDR) and the P14R internal standard were simultaneously quantified by multiple-reaction monitoring (MRM) LCMS, with parent m/z 550.8>fragment m/z 661.5 (y11 2+). The lower limit of quantification (LLOQ) of Nivolumab using the nSMOL method was 0.977μg/ml, with a linear dynamic range of from 0.977 to 250μg/ml. The intra- and inter-assay precision of LLOQ, low quality control (LQC), middle quality control (MQC), and high quality control (HQC) were 7.56-17.9% and 15.6%, 6.99-9.25% and 7.51%, 2.51-8.85% and 8.01%, and 4.78-7.33% and 6.75%, respectively. Our study demonstrates that the nSMOL bioanalysis can be utilized as a reliable method for clinical pharmacokinetic studies of Nivolumab and other antibody drugs.
authors.affiliation: Life Science Research Center, SHIMADZU Corporation, Japan.
authors.forename: Noriko
authors.initials: N
authors.lastname: Iwamoto
authors.affiliation: Life Science Research Center, SHIMADZU Corporation, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Shimada
authors.affiliation: Department of Pharmacy, National Cancer Center Hospital, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Terakado
authors.affiliation: Division of Clinical Pharmacology & Translational Research, Exploratory Oncology Research & Clinical Trial Center, National Cancer Center, Japan. Electronic address: akhamad@ncc.go.jp.
authors.forename: Akinobu
authors.initials: A
authors.lastname: Hamada
mesh_terms: D000911:Antibodies, Monoclonal / Q000097:blood* / Q000737:chemistry; D002853:Chromatography, Liquid / Q000379:methods*; D004355:Drug Stability; D005260:Female; D006801:Humans; D016014:Linear Models; D008297:Male; D036103:Nanotechnology; D000077594:Nivolumab; D059748:Proteolysis; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D000911:Antibodies, Monoclonal; D000077594:Nivolumab
keywords: Bioanalysis; Immune checkpoint inhibitor; LCMS; Monoclonal antibody; Nivolumab; nSMOL