Process intensification of the polishing step of a bioactive peptide through Multicolumn Countercurrent Solvent Gradient Purification

Pharmaceutical products, including peptides, must satisfy very strict purity specifications, because of quality and safety reasons. Therefore, the necessity to operate one or more purification steps to obtain high quality drugs is indisputable. Critical impurities chemically very similar to the target product are generated during the synthesis and are generally removed by means of preparative single-column chromatographic techniques (=batch methods) [1,2]. Batch methods struggle to separate completely the peptide of interest from other groups of impurities, because of their similarity and of high loading of sample processed in preparative conditions, which cause peaks overlapping [3]. The typical situation encountered in these cases is the so-called center-cut separation, where the target elutes as intermediate between two other groups of impurities less and more retained respectively. The direct consequence of this apparently insurmountable overlapping is a yield-purity trade-off, a limit intrinsic to batch chromatography according to which it is possible to obtain either high purity or high recovery of the peptide of interest, depending on whether the overlapping windows are collected or not [4]. This trade-off leads to drawbacks in the overall economy of the process. Multicolumn chromatographic processes, operating in continuous and countercurrent mode, can alleviate this limitation by performing internal recycling of the overlapping portions of the chromatogram [5]. The technique used in the frame of this research is twin-column Multicolumn Countercurrent Solvent Gradient Purification (MCSGP), which has been applied to the purification of an industrial crude of a bioactive decapeptide. It has been demonstrated that MCSGP leads to promising results, including a remarkable improvement in process performance (up to 6 times higher) from the point of view of recovery, productivity and solvent consumption, with respect to the corresponding batch run. The automation of the process on industrial scale would lead to great reproducibility which would reflect in improved consistency in product quality. References [1] De Luca, C.; Felletti, S.; Lievore, G.; Buratti, A.; Vogg, S.; Morbidelli, M.; Cavazzini, A.; Catani, M.; Macis, M.; Ricci, A.; Cabri, W. J Chromatogr A 2020, 1625, 1-7. [2] De Luca, C.; Felletti, S.; Lievore, G.; Chenet, T.; Morbidelli, M.; Sponchioni, M.; Cavazzini, A.; Catani, M. Trends Analyt Chem 2020, 132, 1-8. [3] Vogg, S.; Ulmer, N.; Souquet, J.; Broly, H.; Morbidelli, M. Biotechnol J 2019, 1800732, 1-8. [4] Müller-Späth, T.; Ströhlein, G.; Lyngberg, O.; Maclean, D. Chem Today 2013, 31, 56-60. [5] Steinebach, F.; Müller-Späth, T.; Morbidelli, M. Biotechnol J 2016, 11, 1126-1141.