The Chromatography Forum

of Delaware Valley

Therapeutic peptides are considered one of the most promising class of biopharmaceuticals. Their industrial production (upstream processing) has exceptionally advanced in the last ten years, especially for what regards solid-phase synthesis. However, these advancements have not been matched by equivalent improvements in purification procedures (downstream processing) which still represents the bottleneck, in terms of both cost, time and sustainability in the entire production process.

Single-column preparative liquid chromatography in reversed-phase (RPLC) conditions is the most widely used approach to purify the target peptide from its impurities. Solid-phase synthesis, indeed, do not lead only to the target peptide but also to a series of unwanted product-related impurities which have very similar chemo-physical properties, as well as chromatographic behavior, to that of the target. This means that very often more than one chromatographic step is required, thus leading to a relevant volume of waste solvent, mainly composed by acetonitrile (ACN) which need to be disposed. This solvent is toxic for humans and the environment; therefore (bio)pharmaceutical industries are claiming for the development of greener approaches to purify peptides as well as other biopharmaceuticals.

This lecture will show the latest results obtained by our group in this field. In the last years we have worked on two approaches to increase the sustainability of biopharmaceutical purification. The first one involves the replacement of ACN with greener alternatives. We have tested not only alcohols (ethanol and isopropanol) but also dimethyl carbonate (DMC), which is ranked among the greenest solvents by Solvent Selection Guides, but it has been barely applied as organic modifier in liquid chromatography [1,2].

The second approach relies on the use of advanced multicolumn countercurrent preparative liquid chromatography platforms which allows for the recycle of overlapping regions of the chromatogram inside the system. This mechanism is beneficial not only for automating the purification process, but also for reducing the solvent consumption. It will be shown that by employing Multicolumn Countercurrent Solvent Gradient Purification (MCSGP) for the purification of Icatibant (a small therapeutic peptide composed of 10 amino acids), it can be reduced by more than 80% with respect to the correspondent single-column process [3].

References

[1] S. Felletti, M. Spedicato, M. Catani et al. “Dimethyl carbonate as a green alternative to acetonitrile in reversed-phase liquid chromatography. Part I: Separation of small molecules”, 2023, J. Chromatogr. A, 1712, 464477.

[2] D. Bozza, C. De Luca, M. Catani, et al. “Dimethyl carbonate as a green alternative to acetonitrile in reversed-phase liquid chromatography. Part II: Purification of a therapeutic peptide”, 2023, J. Chromatogr. A, 1713, 464530.

[3] C. De Luca, S. Felletti, M. Catani, et al., “Process Intensification for the Purification of Peptidomimetics: The Case of Icatibant through Multicolumn Countercurrent Solvent Gradient Purification (MCSGP)”, 2021, Ind. Eng. Chem. Res., 60, 6826-6834.