LAUSR

Characterization of antibody-drug conjugates (ADCs) using mass spectrometry (MS) is important in drug discovery, formula-tion development and as part of the quality control processes. To facilitate the electrospray ionization (ESI) and produce high-quality mass spectra, common components of storage solutions for monoclonal antibodies (mAbs) and ADCs such as non-volatile phosphate-buffered saline (PBS), should be replaced before analysis. Centrifugal spin-type kits are extensively used for the desalting or buffer-exchange of mAbs and ADCs samples. The commercially available kits commonly require at least 100µL of a sample at 1 mg/mL for optimal recovery. However, most ESI-MS based analyzes require no more than 25 µg of protein for triplicate injection. In this study, we present a novel method for desalting of ADCs and mAbs building on the SP3 approach with non-functionalized carboxylate coated magnetic beads without affinity ligands. The analytes bind to the hydro-philic beads upon the addition of organic solvent, and various solutions of volatile salts or acids can be used in the elution step. The optimized protocol allowed for 88 % recovery of ADC at 25 µL sample volume and 90 % recovery at 100 µL. More than 90 % of the salts were removed using a process of 20 minutes. The intra- and inter-day precision showed little variation with an RSD of 1 % and 2 %, respectively. The compatibility of this new workflow with low sample volumes is highly valuable since a smaller fraction of the sample is wasted for analysis of the expensive analytes, without compromising recovery.