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

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Analytical technologies in the biomedical and life sciences, 2016-06, Vol.1023-1024, p.9-16
• Main Authors: Iwamoto, Noriko, Shimada, Takashi, Terakado, Hiroyuki, Hamada, Akinobu
• Format: Article
• Language: English
• Subjects: Antibodies; Antibodies, Monoclonal - blood; Antibodies, Monoclonal - chemistry; Bioanalysis; Blood plasma; Chromatography, Liquid - methods; Drug Stability; Female; Humans; Immune checkpoint inhibitor; LCMS; Linear Models; Male; Monoclonal antibody; Nanostructure; Nanotechnology; Nivolumab; nSMOL; Peptides; Polymers; Proteolysis; Quality control; Reproducibility of Results; Resins; Sensitivity and Specificity; Signatures; Tandem Mass Spectrometry - methods
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2016.04.038

•The nSMOL is a Fab-selective limited proteolysis for LCMS bioanalysis of antibody.•Fully validated LCMS bioanalysis of Nivolumab in human plasma was developed.•The signature peptide ASGITFSNSGMHWVR was selected from Nivolumab CDR.•The nSMOL bioanalysis can be applicable as a method for clinical PK and PK-guided cancer therapy for Nivolumab. 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.