Abstract
Background
Cetuximab, IgG1 monoclonal antibody, is used for the treatment of head and neck cancer. Proteomic approaches using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) without an immunopurification technique have not been applied to the quantitation of serum cetuximab. This study developed a simple and rapid LC-MS/MS method for the absolute quantitation of cetuximab in human serum and applied it to clinical settings.
Methods
Surrogate peptides derived from cetuximab digests were selected using a Fourier transform mass spectrometer. Reduced-alkylated serum cetuximab without immunopurification was digested for 20 minutes using an immobilized trypsin. The digestion products were purified by solid-phase extraction. The surrogate peptide and its stable isotope labeled-peptide as an internal standard were quantified by the triple quadrupole mass spectrometer. The present method was applied to the quantitation of serum samples in head and neck cancer patients treated with intravenous cetuximab. Regression analysis and Bland-Altman plots were used to compare the serum concentrations of cetuximab between LC-MS/MS and commercially available ELISA in cancer patients.
Results
The ASQSIGTNIHWYQQR peptide contains cetuximab complementarity-determining region 1 and was determined as the surrogate peptide for the quantification of cetuximab. The chromatographic run time was 10 minutes and no peaks interfering with surrogate peptides in serum digestion products were observed. The calibration curve of absolute cetuximab in serum was linear over the concentration range of 4-200 µg/mL and its correlation coefficient was 0.998. The lower limit of quantification of cetuximab in human serum was 4 µg/mL. The intra-assay and inter-assay precision and accuracy were less than 13.2% and 88.0-100.7%, respectively. The serum concentration range of cetuximab was 19-140 µg/mL in patients, and the median was 68 µg/mL. The serum cetuximab concentrations in LC-MS/MS were correlated with those in ELISA (r = 0.899, P < 0.01) and the mean bias was 1.5% in cancer patients.
Conclusions
The present simple and rapid proteomics-based analytical method with acceptable analytical performance can be helpful for evaluating the absolute concentration of serum cetuximab in clinical settings.
