Impact of column selection for the analysis of 3mer phosphorothioated oligonucleotides in hydrophilic interaction...

With advancing development of therapeutic oligonucleotides, the demand for chromatographic methods for analysis and identification of oligonucleotide impurities and metabolites increases. 3mer oligonucleotides selected for this study represent common metabolites of nusinersen (drug Spinraza), in terms of length, sequence and phosphorothioate modifications. The aim of this work is to investigate the hydrophilic interaction liquid chromatography applicability to analysis of short 3mer oligonucleotides. Their ability to suppress diastereomeric separation and to resolve oligonucleotides differing in one nucleobase at different position in the sequence (3´end, 5´end, middle) was evaluated. The effects of the 3mer sequence and chromatographic conditions (pH of the aqueous part of the mobile phase, buffer concentration, column temperature) on the oligonucleotides' separation according to their sequence ("sequence selectivity") and peak broadening due to diastereomeric separation were investigated for selected zwitterionic columns, diol column, amide column, and two chiral columns (cyclofructan- and quinine-based). The zwitterionic stationary phases showed a great potential for analysis of short oligonucleotides. Diol column showed partial separation of diastereomers, interfering with the sequence-based oligonucleotides resolution due the peak broadening under all tested conditions.