Complementary Chromatographic Strategies for Enhanced Impurity Analysis and Purification of Peptides: A comparative study of HILIC, SFC, and RP-LC

Sammanfattning

Introduction: Peptide therapeutics are becoming increasingly important across multiple disease areas, bringing increased structural diversity and a broader range of physicochemical properties. This diversity, together with more complex synthetic- and product-related impurities, raises the bar for liquid chromatography methods that should be efficient and sustainable. Reversed-phase liquid chromatography (RP-LC) is widely used; however, it may have limited resolving power for closely related impurities. Hydrophilic interaction liquid chromatography (HILIC) and supercritical fluid chromatography (SFC) offer complementary selectivity that may improve impurity resolution and workflow efficiency. Background: Despite recent studies on the topic, systematic guidance for applying HILIC and SFC to peptide samples is limited; the influence of organic modifier, buffer conditions, and stationary-phase chemistry on selectivity and robustness remains greatly unexplored. Aim(s): To compare HILIC and SFC with RP-LC for peptide analysis and purification, establish orthogonality, and identify practical method-development guidelines to apply to diverse peptide profiles. Methods: A set of nine commercially available peptide drugs were analysed by RP-LC, HILIC and SFC for comparison. The effects of chromatographic variables on retention, elution order, peak shape and resolution were investigated. Results: HILIC exhibited distinct and often inverted elution orders relative to RP-LC, consistent with their different retention mechanisms. Some of the HILIC methods resolved several synthesis-related deletion impurities that co-eluted in RP-LC. In SFC, additive identity governed retention via ion pairing: average retention followed TFA < ESA < MSA for most peptides. The columns CN, GSBC and GSFS provided the most balanced SFC performance across additives; highly basic and polar peptides (e.g., ACTH, liraglutide) retained strongly across phases. Conclusion and future perspective: HILIC provided robust, orthogonal selectivity to RP-LC for peptide mixtures, potentially improving impurity detection and purification. In SFC, peptide retention and peak quality were influenced by the additives, as well as peptide and column chemistries.