Separation of oligonucleotide phosphorothioate diastereoisomers by pellicular anion-exchange chromatography

Synthetic oligonucleotides (ONs) are often prepared for development of therapeutic candidates. Among the modifications most often incorporated into therapeutic ONs are phosphorothioate (PT) linkages. The PT linkage introduces an additional chiral center at phosphorus to the chiral centers in D-ribose (and 2-deoxy-D-ribose) of the nucleic acid. Therefore, modified linkages can produce a diastereoisomer pair ([Rp] and [Sp]) at each PT linkage. These isomers are of identical length, sequence, charge and mass, and are not reliably separated by most chromatographic approaches (e.g., reversed phase chromatography) unless the ON is very short. Further these isomers are not distinguishable by single-stage mass spectrometry. During chromatography of a purified anti-NGF (nerve growth factor) aptamer containing 37 bases with 2 PT linkages by monolithic pellicular anion-exchange (pAE) column, we observed four components. The four components were postulated to be: (i) distinct folding conformations; (ii) fully and partially athioated aptamers; or (iii) PT diastereoisomers. Fractionation of the components, followed by de- and re-naturation failed to produce the original forms by refolding, eliminating option (i). Mass spectrometry of the fractionated, desalted samples revealed no significant mass differences, eliminating option (ii). Oxidative conversion of the PT to phosphodiester (PO) linkages in each of the purified components produced a single chromatographic peak, co-eluting with authentic PO aptamer, and having the PO aptamer mass. We conclude that the components resolved by pAE chromatography are diastereoisomers arising from the two PT linkages. Hence, pAE chromatography further enhances characterization of ON therapeutics harboring limited PT linkages and having up to 37 bases.