An LC-MS/MS Method for Absolute Quantification of Nivolumab in Human Plasma: Application to Clinical Therapeutic Drug Monitoring

Background: Nivolumab is a fully humanized IgG4 monoclonal antibody that targets the programmed death-1 (PD-1) receptor, disrupting PD-1-mediated signaling and restoring antitumor immunity. The objective of this study was to develop a nivolumab quantification method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and to evaluate its application in clinical therapeutic drug monitoring. Methods: Nivolumab was purified from human plasma using rProtein A resin and then digested with trypsin. The AS-GITFSNSGMHWVR peptide (multiple reaction monitoring transition: m/z 550.6 -> 661.4) was detected as a surrogate peptide of nivolumab by triple quadrupole mass spectrometry. Plasma samples (126) were collected from 14 patients with non-small cell lung cancer who were undergoing clinical dosing regimen with nivolumab. The pharmacokinetic data were analyzed using Phoenix NLME software (Version 7.0, Certara, St. Louis, MO) based on a previously reported population pharmacokinetics (PPK) model of nivolumab. Results: Nivolumab was selectively detected in human plasma and the linear range was 5-200 mcg/mL (R-2 = 0.99). The accuracy and intraday and interday imprecision were within +/- 15% of the quality control values of 5 (lower limit of quantification), 10 (low), 80 (medium), and 160 (high) mcg/mL. The nivolumab concentrations measured using LC-MS/MS were consistent with those of previously reported PPK models, and the pharmacokinetic parameters could be adequately predicted from a single trough concentration using a Bayesian approach. Conclusions: An absolute quantification method for nivolumab using LC-MS/MS was successfully developed and validated. Combined with PPK analysis, this method should be useful for the therapeutic drug monitoring of nivolumab in clinical practice.