Biotherapeutics quantification in tissues remains a challenge for PK/PD studies due to matrix complexities and low concentrations. To increase sensitivity and robustness, optimized immunoextraction (IE) conditions of Pembrolizumab were combined with nano-liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS).

The LC-HRMS method was developed on a digest of the monoclonal antibody Pembrolizumab. Pembrolizumab peptides were injected on a C18 nano-LC column (15 cm * 75 µm) at a flow rate of 1 µL/min and analyzed on an Exploris 480 instrument.

IE robustness and sensitivity were optimized and evaluated by capturing spiked Pembrolizumab in mouse plasma. Several types of magnetic beads and ligands were compared to define the best conditions. Pembrolizumab was eluted with 1% FA, before digestion by trypsin.

We firstly established a DDA method to select Pembrolizumab reporter peptides, then a PRM method was developed based on the most intense signals. By the DDA method, sequence coverage of Pembrolizumab was 69%. A total of 28 precursors from 22 peptides were selected for PRM. To ensure the integrity of Pembrolizumab during quantification, selected peptides were both in light and heavy chains and covered the Fab and Fc parts.

Tosylactivated, Streptavidin and Protein A beads are evaluated along with different types of capture ligands, such as anti-Fc or anti-F(ab’)² Human. Different ratios of immobilized ligands-to-proteins are also compared. Best conditions regarding signal of the targeted peptides are combined and evaluated regarding LOD and repeatability.

The best conditions will be selected for full analytical validation of the quantitative workflow and further applied to tissues (liver, brain, heart, tumor…) from mice treated by Pembrolizumab. In the future, a DIA method will be developed to detect and quantify unusual modifications or degradations of the therapeutic antibody.