Mass spectrometry as a quantitative methodology to assess the biodistribution of monoclonal antibodies

Over the past thirty years, various naked and armed monoclonal antibodies (mAbs) have been clinically utilized as targeted therapeutics in the field of oncology. Understanding the quantitative biodistribution of mAbs is crucial to accurately evaluate the accumulation of the drug at the site of disease and in healthy tissues. In this study, we describe a hybrid platform based on affinity enrichment and LC-MS for the ex vivo quantification of tumor-targeting mAbs in cancer lesions and in healthy organs upon systemic administration in tumor-bearing mice. To reduce sample complexity and increase the analyte concentration, different enrichment modalities were evaluated and compared for their efficiency of antibody recovery. A homologous mAb with a different light chain isotype, a stable-isotope-labeled peptide, or a stable isotope-labeled protein were evaluated as putative internal standards. This was done by comparing their reliability in normalising errors introduced during the sample preparation or the LC-MS analysis, with the stable-isotope labelled protein outperforming the others. After enrichment and protein digestion, tryptic peptides were subjected to nanoHPLC-HRMS analysis on an Orbitrap Q-Exactive mass spectrometer coupled to an EASY nanoLC 1000 system via a Nano Flex ion source. Chromatographic separation was carried out at room temperature on an Acclaim PepMAp RSLC column (50 µm x 15 cm, particle size 2 µm, pore size, 100 Å). Different MS acquisition modes (i.e., DDA-top12, a SIM-dda and a PRM) were evaluated comparing the peptides" total signals and signals to noise ratio. As a case study, the ex vivo biodistribution of a fully human monoclonal antibody targeting the extra domain B of fibronectin in 129sv mice bearing teratocarcinoma tumor was carried out 24 h after a single dose administration. Data obtained with the mass spectrometer were compared to biodistributions performed in the same condition using radiolabelled antibody (i.e., ⁸⁹Zr or ¹²⁵I).