Alcohol-based solvents as mobile phases for LC-MS characterization of therapeutic proteins

Acetonitrile (ACN) is currently the preferred solvent in reversed phase (RP) chromatography for protein characterization through peptide mapping. Despite its effective performance, ACN poses toxicity risks to humans and has adverse effects on environmental sustainability. In the current work, we developed a novel alcohol-based solvent system for peptide mapping by systematic evaluation of parameters such as organic eluent composition, solvent gradient, flow rate, and column temperature. We compared the chromatographic performance and MS response of peptides between the standard (ACN based) and the new developed solvent systems (EtOH/IPA based). The results of our study show that the EtOH/IPA based solvent system improves selectivity factor (α) and resolution (R), while the standard ACN based solvent system provides a lower peak width and hence a higher peak height. The majority of the analysed peptides exhibited shorter retention times, whereas hydrophobic peptides eluted later when using the EtOH/IPA solvent system. Several critical quality attributes (CQA) of a monoclonal antibody (mAb) were successfully characterized by this method without compromising the chromatographic separations and MS response of the peptides. Furthermore, the suitability of the new approach for LC-UV assessment of a mAb as part of an identity test of therapeutic proteins was demonstrated. Our proposed approach, which prioritizes safety, non-toxicity, and compatibility with all LC-MS instruments, offers significant support to a broad community of academic and biopharmaceutical scientists in their pursuit of a bottom-up green strategy.