STUDY OF THE ADSORPTION EQUILIBRIA OF A PHARMACEUTICAL RELEVANT PEPTIDE IN RP-LC THROUGH ADVANCED NUMERICAL MEANS

Due to their biologic activity, many peptides are used in cosmetic, nutraceutical and pharmaceutical fields [1]. Usually, peptides are obtained by means of solid phase synthesis which, unfortunately, doesn’t lead only to the main target molecule but to very complex mixtures [2]. The purification process is the bottleneck in the synthetic production of peptides, so it has to be optimized. Moreover, to purify industrial quantities of peptides, it is necessary to implement separation processes in a continuous manner, and this is feasible only knowing the optimal separation conditions in batch method. RP-LC is one of the most employed techniques for isolating the peptide of interest from the impurities and by-products deriving from synthesis [3]. In this work, a crude (=not purified) mixture of one octapeptide has been considered. Some preliminary gradient tests have been conducted to find out the best conditions for the separation of the peptide and the main impurity. Different mixtures of two mobile phases (0.02% TFA in MilliQ and 0.02% TFA in ACN) have been used during elution. Afterwards, the chromatographic profiles have been obtained also in isocratic conditions at different mobile phase compositions, chosen depending on gradient experiments. The shape of the profiles suggests that the adsorption isotherm is a convex upward type. Using inverse method, an advanced numerical method which is particularly suitable when only small amounts of sample are available, experimental peaks have been fitted with different isotherm models; Langmuir isotherm gave the best results. The Langmuir isotherm parameters have been estimated for each fraction of organic modifier. It is known that the retention factor of a peptide, and therefore the isotherm parameters, are heavily affected by the organic solvent percentage [4]. This dependence has been evaluated; it has been found out that b (=adsorption equilibrium constant) follows an exponential trend and decreases if the organic modifier fraction increases. [1] D. Agyei, C. M. Ongkudon, C. Y. Wei, A. S. Chan, M. K. Danquah, Bioprocess challenges to the isolation and purification of bioactive peptides. Food and Bioprod. Proc. 98 (2016) 244-256. [2] S. Bernardi, D. Gétaz, N. Forrer, M. Morbidelli, Modeling of mixed-mode chromatography of peptides. Jour. Chrom. A 1283 (2013) 46-52. [3] D. Gétaz, G. Stroelhein, A. Butté, M. Morbidelli, Model-based design of peptide chromatographic purification processes. Jour. Chrom. A 1284 (2013) 69-79. [4] N. Marchetti, F. Dondi, A. Felinger, R. Guerrini, S. Salvadori, A. Cavazzini, Modeling of overloaded gradient elution of nociceptin/orphanin FQ in revesed-phase liquid chromatography. Jour. Chrom. A 1079 (2005) 162-172