Simultaneous quantification of rituximab and eculizumab in human plasma by liquid chromatography-tandem mass spectrometry and comparison with rituximab ELISA kits.

OBJECTIVES

Specific and sensitive analytical techniques to quantify therapeutic monoclonal antibodies (mAbs) are required for therapeutic drug monitoring. The quantification of mAbs has been historically performed using enzyme-linked immunosorbent assays (ELISAs), for which the limitations in terms of specificity have led to the development of alternative analytical strategies.

METHODS

Here, we describe the validation of a liquid chromatography tandem mass-spectrometry (LC-MS/MS) method for the simultaneous quantification of rituximab (RTX - anti-CD20) and eculizumab (ECU - anti-C5). Sample preparation was based on our previously published method, using protein G purification and trypsin digestion. A new specific peptide for RTX, containing an N-terminal pyroglutamine and a trypsin miss-cleavage, enables better sensitivity, while peptide of ECU was chosen thanks to an in silico trypsin digestion and the Skyline® software. Full-length stable-isotope-labeled adalimumab was added to plasma samples as an internal standard. RTX in 50 human serum samples was quantified by LC-MS/MS and the concentrations obtained compared to those obtained with two commercial ELISA kits (Lisa Tracker® and Promonitor®).

RESULTS

Calibration curves were linear from 1 to 200 µg.mL for RTX and 5 to 200 µg.mL for ECU, and within-day and between-day accuracy and precision fulfilled Food and Drug Administration validation criteria. Comparison of the LC-MS/MS method with ELISA showed a negligible bias with the Lisa Tracker® kit (4%), but significant bias with the Promonitor® assay (mean underestimation of 69% for the Promonitor® assay).

CONCLUSIONS

This new LC-MS/MS method allows the simultaneous quantification of RTX and ECU in human samples and could be used for therapeutic drug monitoring.