Analysis of Pembrolizumab in Human Plasma by LC-MS/HRMS. Method Validation and Comparison with Elisa

Pembrolizumab is a humanized immunoglobulin G4-kappa anti-PD1 antibody used in the treatment of different solid tumors or haematological malignancies. A liquid chromatography coupled with a high resolution mass spectrometry (orbitrap technology) method was fully developed, optimized, and validated for quantitative analysis of pembrolizumab in human plasma. A mass spectrometry assay was used for the first time a full-length stable isotope-labelled pembrolizumab-like (Arginine C-13(6)-N-15(4) and Lysine C-13(6)-N-15(2)) as an internal standard; the sample preparation was based on albumin depletion and trypsin digestion and, finally, one surrogate peptide was quantified in positive mode. The assay showed good linearity over the range of 1-100 mu g/mL, a limit of quantification at 1 mu g/mL, excellent accuracy from 4.4% to 5.1%, and also a between-day precision below 20% at the limit of quantification. In parallel, an in-house ELISA was developed with a linearity range from 2.5 to 50 mu g/mL. Then, results were obtained from 70 plasma samples of cancer patients that were treated with pembrolizumab and quantified with both methods were compared using the Passing-Bablok regression analysis and Bland-Altman plotting. The LC-MS/HRMS method is easy to implement in the laboratory for use in the context of PK/PD studies, clinical trials, or therapeutic drug monitoring.

Automated summary

A method for quantifying Pembrolizumab in human plasma was developed using stable isotope-labeled internal standards and a surrogate peptide quantified in positive mode, showing good linearity and accuracy. Comparison of results from cancer patient plasma samples analyzed by two methods showed that the LC-MS/HRMS method is suitable for clinical trials and therapeutic drug monitoring.