Improving the Chromatographic Separation of Phosphorothioate Oligonucleotide from Impurities by Optimizing Selectivity through Mobile-phase Conditions in Ion-pair Reversed-phase High-performance Liquid Chromatography

A diastereomer peak broadening has been observed in phosphorothioate (PS) oligonucleotide analysis in several modes of high-performance liquid chromatography (HPLC). PS oligonucleotide impurities are of various types, and their physicochemical properties are similar to those of PS oligonucleotides. Consequently, quality control requires the chromatographic separation of PS oligonucleotide from impurities and separation of impurities from one another, which is challenging. In this study, to optimize the selectivity for effectively separating PS oligonucleotide from its impurities such as phosphodiester impurities (POs) and n − 1 truncated sequences (n − 1 s) in ion-pair reversed-phase (IP-RP) HPLC, the effect of mobile-phase conditions on the selectivity was investigated. Most importantly, it was demonstrated that the selectivity for the diastereomers of PS oligonucleotides is optimally reduced by the ion-pair (IP) system using alkylamine with a tertiary or quaternary structure and alkyl chain lengths of ≥4, using a method of observing the peak widths of PS oligonucleotides. Alternatively, using counterion species, such as hydrophobic alkyl carboxylic acid, improves the selectivity between the PS oligonucleotide and its impurities. Consequently, the IP system using tributylamine and heptanoic acid is proposed as the system that has optimal selectivity. This system can separate PS oligonucleotides from spiked PO and n − 1 groups involving those with different positions and has outstanding quantitative performance at the 0.2 %–5 % range. This study provides a guidance for optimizing the selectivity for IP-RP HPLC, and the proposed IP system could be useful for ensuring the quality control of antisense oligonucleotides including PS linkages.