Evaluation of micropillar array columns for chromatographic separation of phosphorothioated oligonucleotides and their diastereomers

Chromatographic analysis of therapeutic oligonucleotides is challenging due to the presence closely related impurities, degradants or metabolites and phosphorothioate bonds, which introduce chiral centers. In present study, ion pair reversed phase chromatography on micropillar array columns was investigated. Two commonly used mobile conditions were included. With 16.3 mM triethylamine 400 hexafluoroisopropanol, separation 16-mer differing in number positions linkages as well some n-1 n-2 truncations demonstrated complete suppression diastereoselectivity. Although positional isomers evaluated could not be resolved, an increase bonds resulted more retention. A 19-mer RNA sequence with 2′-fluor 2′-O-methyl modifications showed partial very close impurities. When using 15 triethyl ammonium acetate phase, diastereomer selectivity clearly observed for all analytes. The best result obtained mimic four since 16 theoretical diastereomers under tested. limited benchmark exercise improved capability new columns. Therefore, these can positioned a valuable alternative when oligonucleotide separations are expected.