Replace, reduce, and reuse organic solvents in peptide downstream processing: the benefits of dimethyl carbonate over acetonitrile

Preparative liquid chromatography under reversed phase conditions (RPLC) is the most common method for the purification of therapeutic peptides in industrial downstream processing. Routine methods typically involve the use of aqueous buffers and acetonitrile (ACN) as the organic modifier. This choice is driven by the unique chemico-physical properties of ACN. However, ACN poses health and environmental risks, which downgrades it among the preferred choices from the greenness point of view. In this work, a step towards the greening of the downstream purification process is presented for three polypeptides following the three Rs of green analytical chemistry: replace, reduce, and reuse. This is achieved by using a mixture of isopropanol (IPA) and dimethyl carbonate (DMC) as green alternative solvents to ACN. A DMC/IPA mixture allows the reduction of the toxicological impact and amount of waste generated while preserving comparable process performance. Moreover, the possibilities of distilling and reusing the solvent waste derived from the chromatographic operations and lyophilizing the peptide solution without affecting the finite product quality have been explored.