Separation of deaminated impurities from the desired oligonucleotides using supercritical fluid chromatography

With recent advancements concerning the optimization of the analytical conditions, it is feasible to analyze polar molecules using supercritical fluid chromatography (SFC). In this study, the applicability of SFC is evaluated for analyzing 5-, 10-, 15-, and 18-mer oligonucleotides, and SFC is then applied to analyze deaminated products, which are side products generated during oligonucleotide synthesis. These side products are difficult to separate from the target oligonucleotide, with the difficulty varying depending on the deamination position and sequences, even when using ion-pair reversed-phase liquid chromatography (IP-RPLC), a common method for oligonucleotide analysis. Our results demonstrate that SFC, with octylamine as a modifier additive, can achieve sharp chromatographic peaks for 5-, 10-, 15-, and 18-mer oligonucleotides modified with 2'-O-methoxyethyl RNA (2'-MOE), regardless of the presence of the hydrophobic 4,4'-dimethoxytrityl (DMTr) group on the sequence. After optimization of the column oven temperature, modifier additive, and stationary phase, SFC successfully separated oligonucleotides with various numbers and positions of deamination from the target oligonucleotide. SFC exhibited different selectivities for DMTr-on and DMTr-off oligonucleotides compared with those for IP-RPLC, which indicates that SFC can serve as a valuable alternative tool for the purification and analysis of oligonucleotides.