Quantification of the anti-murine PD-1 monoclonal antibody RMP1-14 in BALB/c mouse plasma by liquid...

RMP1-14 is a monoclonal antibody that targets the murine PD-1 protein, and has been used extensively to probe the effects of PD-1 inhibition in preclinical murine models. However, to date, no quantitative analytical methods have been published for RMP1-14. To evaluate its anti-tumor activity in BALB/c mice inoculated with CT26.WT murine colon cancer cells, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify RMP1-14 in BALB/c mouse K₃EDTA plasma was developed and validated. The methodology used a signature peptide (GFYPPDIYTEWK) as a surrogate for RMP1-14 quantitation and an isotopically labeled analog of the signature peptide as the internal standard. Initial method development focused on a hybrid LC-MS/MS assay involving Protein G immunoprecipitation, but this strategy was abandoned due to lack of selectivity. The final validated method consisted of dilution with Tris-buffered saline, trypsin digestion, and desalting using micro solid-phase extraction. Analytical run time was 3.50 min, and the method demonstrated linearity between 0.500 and 50.0 μg/mL of intact RMP1-14. Accuracy, precision, and robustness were all acceptable, and the method was demonstrated to be comparable to a commercially available fit-for-purpose enzyme-linked immunosorbent assay (ELISA) capable of measuring RMP1-14. The validated method was used to generate pharmacokinetic parameters from tumor-bearing BALB/c mice dosed with RMP1-14 at either 2.50 or 7.50 mg/kg. Overall, the validated method represents a novel tool that can be used to evaluate RMP1-14 activity in future immuno-oncology studies.