Modeling the nonlinear behavior of a bioactive peptide in reversed-phase gradient elution chromatography

The interest around the use of peptides for therapeutic scopes is continuously increasing. Thanks to their high specificity and inherent affinity for target molecules, peptides represent an effective solution to unresolved medical issues avoiding typical toxic side effects of exogenous chemical drugs. From an industrial point of view, they are produced mainly by means of solid phase synthesis. However, during the synthesis, many undesired impurities (truncated or modified peptides, by-products, scavengers, etc.) are produced. Purification is therefore needed to obtain the target peptide at the desired degree of purity for pharmaceutical and therapeutic scopes. Preparative liquid chromatography is the most widely used technique for the purification of therapeutic peptides, but its major drawback is that experimental conditions are usually optimized through trial-and-error approach, causing significant waste of compound and time. The investigation of adsorption behaviour and phase equilibria involved in the separation of the target peptide using a model-based approach, is the basis not only to investigate the feasibility of purification process via preparative chromatography but also to possibly provide information on e.g., maximum loading or affinity for the stationary phase, that may help to optimize large-scale purification and continuous processes, increasingly employed in pharmaceutical manufacturing to alleviate the trade-off between yield and purity. In this work, the adsorption behaviour of a therapeutic peptide, octreotide, has been investigated and modeled under reversed-phase liquid chromatography (RP-LC) gradient elution conditions under a variety of water/acetonitrile + 0.02% (v/v) trifluoroacetic acid (TFA) mixtures. Adsorption isotherms have been determined by means of Inverse Method, injecting only 5 µL of peptide solutions. The linear solvent strength (LSS) model was used to find the correlation between isotherm parameters and mobile phase composition. This work demonstrates how the modelling of adsorption behaviour of pharmaceutically relevant peptides can be performed with only few milligrams of compound. This is particularly important when the amount of available peptide is reduced, or its cost is elevated