Evaluating orthogonality between ion-pair reversed phase, anion exchange, and hydrophilic interaction liquid chromatography for the separation of synthetic oligonucleotides

The orthogonality of separation between ion-pair reversed phase (IP-RP), anion exchange (AEX), and hydrophilic interaction liquid chromatography (HILIC) was evaluated for oligonucleotides. A polythymidine standard ladder was first used to evaluate the three methods and showed zero orthogonality, where retention and selectivity were based on oligonucleotide charge/size under all three conditions. Next, a model 23-mer synthetic oligonucleotide containing 4 phosphorothioate bonds with 2′ fluoro and 2′-O-methyl ribose modifications typical of small interfering RNA was used for evaluating orthogonality. The resolution and orthogonality were evaluated between the three modes of chromatography in terms of selectivity differences for nine common impurities, including truncations (n-1, n-2), addition (n + 1), oxidation, and de-fluorination. We first evaluated different ion-pairing reagents that provided the best separation of the key impurities while suppressing diastereomer separation due to phosphorothioate linkages. Although different ion-pairing reagents affected resolution, very little orthogonality was observed. We then compared the retention times between IP-RP, HILIC, and AEX for each impurity of the model oligonucleotide and observed various selectivity changes. The results suggest that coupling HILIC with either AEX or IP-RP provide the highest degree of orthogonality due to the differences in retention for hydrophilic nucleobases and modifications under HILIC conditions. IP-RP provided the highest overall resolution for the impurity mixture, whereas more co-elution was observed with HILIC and AEX. The unique selectivity patterns offered by HILIC provides an interesting alternative to IP-RP or AEX, in addition to the potential for coupling with multidimensional separations. Future work should explore orthogonality for oligonucleotides with subtle sequence differences such as nucleobase modifications and base flip isomers, longer strands such as guide RNA and messenger RNA, and other biotherapeutic modalities such as peptides, antibodies, and antibody-drug-conjugates.