A New Methodology for Synthetic Peptides Purification and Counterion Exchange in One Step Using Solid-Phase Extraction Chromatography

Synthetic peptides are commonly obtained by means of solid-phase peptide synthesis (SPPS), in which separation of the peptide from the solid support requires treatment with 92.5% v/v trifluoroacetic acid (TFA); therefore, peptides are obtained as trifluoroacetate salts. For promising anticancer/antibacterial peptides it is essential to exchange the counterion from trifluoroacetate to hydrochloride or acetate, since the former are more widely studied in biological activity assays. In this research, RP-SPE-based methodologies were designed, developed, and implemented for simultaneous counterion exchange and peptide purification. Critical process steps were identified and parameters such as mobile phase composition, elution, and program were optimized. Analysis of the counterion exchange reaction and characterization of the final products was performed using high-performance liquid chromatography, attenuated total reflectance, nuclear magnetic resonance, and mass spectrometry. Peptides with purities between 82–97% and a trifluoroacetate ion content less than 0.36% were obtained. This novel counterion exchange proved efficient for peptides with different characteristics such as length, polarity, polyvalency, and presence of non-natural amino acids or non-protein molecules, therefore showing a wide range of applications in the field of therapeutic peptides. The methods developed are fast, efficient, low-cost, and do not require robust instrumentation and can be routinely implemented in SPPS.