Development and validation of an LC-MS/MS method for simultaneous quantification of co-administered trastuzumab and...

Given the increasing use of combination therapy with multiple monoclonal antibodies (mAbs), there is a clinical need for multiplexing assays. For the frequently co-administered anti-human epidermal growth factor receptor 2 (HER2) mAbs trastuzumab and pertuzumab, we developed a high-throughput and robust hybrid ligand-binding liquid chromatography-mass spectrometry (LC-MS)/MS quantitative assay. Nanomolar concentrations of trastuzumab and pertuzumab were determined in 10 µL serum samples after extraction by affinity purification through protein A beads, followed by on-bead reduction, alkylation, and trypsin digestion. After electrospray ionization, quantification was obtained by multiple reaction monitoring LC-MS/MS using SILuMab as an internal standard. The method was validated according to the current guidelines from the US Food and Drug Administration and the European Medicines Agency. Assay linearity was established in the ranges 0.250-250 μg/mL for trastuzumab and 0.500-500 μg/mL for pertuzumab. The method was accurate and selective for the simultaneous determination of trastuzumab and pertuzumab in clinical samples, thereby overcoming the limitation of ligand binding assays that cannot quantify mAbs targeting the same receptor. Furthermore, this method requires a small blood volume, which reduces blood collection time and stress for patients. The assay robustness was verified in a clinical trial where trastuzumab and pertuzumab concentrations were determined in 670 serum samples. As we used commercially available reagents and standards, the described generic bioanalytical strategy can easily be adapted to multiplex quantifications of other mAb combinations in non-clinical and clinical samples.