Evaluating Electrostatic Repulsion-Reversed Phase Methods for Peptide Impurities

A recent publication in the European Journal of Pharmaceutical Sciences presents a comparison of two chromatographic methods—static and dynamic electrostatic repulsion-reversed phase (ERRP)—for the separation of peptide impurities (1). The study, authored by researchers from La Sapienza University of Rome, University of Bologna, and Fresenius Kabi iPSUM, explores the performance of these approaches using the glucagon-like peptide-1 (GLP-1) liraglutide as a case study.

The analysis of GLP-1 and its analogues is crucial due to their significant therapeutic role in managing conditions like type 2 diabetes and obesity. Precise characterization ensures the safety, efficacy, and quality of these peptide-based drugs, particularly given their complex structures and potential impurities, offering a significant challenge for the pharmaceutical industry. Detecting and quantifying process-related impurities, stereoisomers, and degradation products are essential to prevent adverse effects and to comply with stringent regulatory standards. Therefore, advanced analytical techniques are vital to help safeguard clinical outcomes and maintain the integrity of GLP-1 therapeutics.

ERRP chromatography is based on exploiting electrostatic interactions between charged stationary phases and analytes, primarily to improve the separation of basic peptides and their impurities. The technique involves two modes: static-ERRP (s-ERRP), where positive charges are covalently bonded to the stationary phase, and dynamic-ERRP (d-ERRP), where surface charges are generated by adsorbing ion-pairing agents onto the mobile phase.

In s-ERRP, the stationary phase contains permanently anchored positive charges, creating a consistent electrostatic environment that facilitates the separation of peptides with subtle stereochemical differences, such as epimers. Conversely, d-ERRP dynamically introduces positive charges via ion-pairing agents like tetrabutylammonium salts, providing a flexible approach that can be optimized according to specific analytical needs.

In s-ERRP mode, the stationary phases were functionalized with positively charged groups, and acid additives were used to stabilize analytes. The choice of acids affected the compatibility with mass spectrometry (MS): difluoroacetic acid (DFA) was favored for MS compatibility, while trifluoroacetic acid (TFA) provided increased resolution but caused ion suppression. This balancing act between resolution and MS sensitivity was a key aspect of the method development. Applying d-ERRP involved dynamically generating positive charges during chromatography, which improved the separation of liraglutide and its impurities, including stereoisomers such as [D-Ser]⁸-Liraglutide and [D-Allo-Thr]⁵-Liraglutide. The optimized gradient conditions allowed for clearer resolution of these closely related compounds, demonstrating the potential of d-ERRP for detailed impurity profiling.

The study highlights that both ERRP modes, when combined with advanced stationary phases and tailored mobile phases, can be effective for separating complex peptide impurities, with the choice between s-ERRP and d-ERRP guided by specific analytical goals, such as the need for higher resolution or MS compatibility. In this study, the ability to distinguish epimeric impurities with high precision is important for the pharmaceutical development and quality control of peptide drugs. The compatibility of s-ERRP with MS presents advantages in workflows that require sensitive detection and quantification. However, it is important to note that the dynamic approach was the superior method.

This work demonstrates the applicability of ERRP chromatography for the separation of challenging peptide impurities. The authors concluded that further optimization of ERRP may enhance analytical performance in future applications in peptide analysis, contributing to improved analytical capabilities within pharmaceutical research and quality assurance processes.

Reference

(1) Manetto, S.; Mazzoccanti, G.; Bassan, M.; et al. Comparing the Performance of Electrostatic Repulsion-Reversed Phase Chromatography Approaches in the Resolution of Complex Peptide Mixture: Liraglutide as Case Study. Eur. J. Pharm. Sci. 2025, 211, 107120. DOI: 10.1016/j.ejps.2025.107120