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

Abstract

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

Structured microgroove columns as a potential solution to obtain perfectly ordered particle beds

“…The same holds for the multi-capillary column with diffusive bridging concept recently introduced by Parmentier [18]. One other approach, based on using photolithography and plasma etching to produce silicon-based micro-pillar array columns has made it to commercialization in the area of nano-LC for proteomics [8,[19][20][21][22][23]. Although the induced order and the enlarged flow resistance of the micro-pillar array columns indeed lead to groundbreaking performances, these columns are silicon-based and currently offer a very limited choice of stationary phases.…”