Géry Van Vyncht

Despite all of the recent advances in analytical technologies dedicated to biotherapeutics, accurate protein quantification remains a challenge for the biopharmaceutical industry. UV spectrophotometry is commonly used for batch testing, but it requires the knowledge of the extinction coefficient of the protein, whose experimental determination requires the accurate concentration of a reference standard obtained by an absolute quantification method. To address the need for a fast analytical method capable of accurately quantifying a protein without any specific reference substance, an isotope dilution ICP-MS method was developed and validated, based on sulfur determination, allowing very accurate determination of a single protein in solution after microwave digestion.

We have developed a range of analytical workflows using mass spectrometry, in a regulated environment, to support pharmaceutical companies in the development and control of their monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs). High-resolution mass spectrometry is a powerful tool for the analysis of antibodies, but is not readily compatible with a number of chromatographic techniques using high-salt mobile phases. Herein, we present the development and use for marketed mAbs and ADCs of 2D LC–MS via an online desalting step. We demonstrate the importance of such a setup for the determination of drug:antibody ratio (DAR), and the analysis of molecularity, fragmentation, and charge variants (deamidation, oxidation), notably under stress conditions. We discuss the advantages of 2D LC–MS in a regulated environment.