Benchmarking size-exclusion chromatography columns for the analysis of therapeutic peptides and model oligonucleotides

Eleven modern size-exclusion chromatography (SEC) columns, including prototype columns designed to minimize non-specific interactions, were systematically evaluated for the analysis of peptides and oligonucleotides. Column physical properties and chromatographic performance were assessed under various mobile phase conditions. Notably, a reduced plate height close to 1 was achieved for one prototype column, representing a marked improvement over typical SEC performance (2 < h_min < 3). Mobile phase composition was optimized to balance chromatographic efficiency and analyte denaturation. The most denaturing conditions (30 % acetonitrile, 0.1 % trifluoroacetic acid) provided the best performance for linear, macrocyclic, disulfide-constrained, and lipid-conjugated peptides by effectively suppressing hydrophobic and ionic interactions. Columns with pore sizes ≥ 100 Å showed optimal performance, with UP-SW2000, Biozen dSEC-1, and BioCore SEC-120 columns (100-125 Å) yielding the best results. Column and mobile phase selection were particularly critical for hydrophobic peptides such as liraglutide and semaglutide; for these analytes, a phosphate-based mobile phase containing 20 % isopropanol was proposed to limit denaturation and potentially enable the characterization of non-covalent aggregates. For oligonucleotides, mobile phase composition had a limited impact, whereas stationary phase chemistry was decisive. Only two columns (UP-SW2000 and ACQUITY Premier SEC 125 Å) provided acceptable separations, enabling resolution of n-2 and n-3 shortmer impurities of 20-mer linear and structured oligonucleotides with resolutions close to 1. Coupling two 150-mm SEC columns in series further enhanced shortmer separation within 30 min.