Ion-exchange chromatography for impurity profiling of therapeutic peptides

Despite the growing therapeutic importance of peptides such as glucagon-like peptide-1 (GLP-1) analogues, their analytical characterization has received comparatively limited attention. Reversed-phase liquid chromatography (RPLC) remains the reference technique for peptide purification and analysis; however, the pronounced structural and physicochemical diversity of therapeutic peptides, spanning linear, macrocyclic, and disulfide-rich scaffolds, underscores the need for complementary separation strategies. This study aimed to expand the analytical toolbox for therapeutic peptide characterization by evaluating ion-exchange chromatography (IEX). Four IEX columns were assessed: two strong cation exchangers (ProPac 3R SCX and BioPro IEX SF Accura) and two strong anion exchangers (ProPac 3R SAX and BioPro IEX QF Accura), all tested in 2.1 × 50 mm formats to reduce sample consumption and analysis time. Initial evaluations using standard protein IEX methods revealed they were unsuitable for the target therapeutic peptides. To optimize the separation, salt gradients at pH 2.0 (cation exchange chromatography) and pH 10.0 (anion exchange chromatography) were evaluated alongside alternative pH-gradients, specifically a classic phosphate-citrate mixture and a polyamine-based tri-buffer mixture (pH 2.5-9.0). Among the approaches tested, salt-gradient elution consistently provided superior performance. The prevalence of secondary hydrophobic interactions significantly impacted chromatographic performance, necessitating the addition of at least 30% acetonitrile (ACN) to mitigate peak broadening and improve peptide recovery. To evaluate stability-indicating capability, the developed IEX methods were benchmarked against an in-house reference RPLC purity method through forced degradation studies. Finally, we explored the use of IEX retention behavior to estimate peptide pI values, illustrating both the potential and the current limitations of ion-exchange chromatography for the characterization of therapeutic peptides.