Counter-Current Chromatography Enables Use of Green Solvents for Productive Peptide Purification Processes

Peptide purification by preparative reversed-phase liquid chromatography remains one of the most resource-intensive stages in synthetic peptide manufacturing. Production processes commonly rely on acetonitrile/trifluoroacetic acid (ACN/TFA) mobile phases mainly because of their high chromatographic resolution. However, both components raise significant environmental and safety concerns related to toxicity, flammability, waste generation, and, in the case of TFA, environmental persistence as a per- and polyfluoroalkyl substance (PFAS, known as a “forever chemical”). Green alternatives based on ethanol, dimethyl carbonate, and sustainable acidic additives such as acetic acid have been proposed, but their industrial adoption remains limited due to reduced chromatographic performance, often resulting in lower yield and productivity under conventional batch operation. In this work, multi-column counter-current solvent gradient purification (MCSGP) was investigated as a strategy to integrate the use of green eluent systems without compromising process performance. Two therapeutic peptides, Tirzepatide and Tetracosactide, were selected as representative case studies with different structural complexity. Using ethanol/acetic acid for Tirzepatide and dimethyl carbonate/acetic acid for Tetracosactide, the MCSGP process achieved purity levels equivalent to those obtained with conventional ACN/TFA batch chromatography, with 88.1% yield at 89.0% purity for Tirzepatide and 93.8% yield at 94.0% purity for Tetracosactide. Productivity for Tirzepatide was improved, reaching 6.3 g/Lresin/h. These results demonstrate that MCSGP can compensate for the reduced separation efficiency typically associated with green eluent systems, enabling sustainable peptide purification without compromising process performance. By combining green solvents with the MCSGP process, this work paves the way for more sustainable peptide purification processes while maintaining high yield and productivity.