Development of Simple and Rapid Method for Emicizumab Quantification by LC-MS/MS in Human Plasma

Emicizumab is a new therapeutic monoclonal antibody indicated for prophylaxis in severe haemophilia A pa-tients. Pharmacokinetic variability has been reported in clinical studies, thus dose optimisation based on quantification of plasma drug concentration could be considered to reduce this variability. Therefore, a reliable and accurate quantification of emicizumab is required. In this study, we developed a method for absolute quantification of emicizumab using liquid chromatography coupled to triple quadrupole mass spectrometry (LC-MS/MS). Sample preparation was based on organic solvent precipitation of proteins followed by trypsin diges-tion. A signature peptide of emicizumab was used for quantification by LC-MS/MS. A stable isotope-labelled peptide was used as an internal standard. Finally, 6 samples from patients treated with emicizumab were quantified by LC-MS/MS and compared with those obtained with the modified one-stage activated partial pro -thrombin time technique (aPTT) based FVIII activity. The LC-MS/MS method was validated according to FDA recommendations. Good linearity of the calibration curves was observed over the range 5-150 mu g/mL. The cross -validation showed an acceptable correlation of the developed LC-MS/MS method with the modified aPTT-based FVIII activity assay, and the Bland-Altman analysis did not show any significant bias.