Separating Impurities from Oligonucleotides Using Supercritical Fluid Chromatography

Researchers optimized supercritical fluid chromatography (SFC) for analyzing 5-, 10-, 15-, and 18-mer oligonucleotides (ONs) and assessed its effectiveness in separating impurities from ONs. The study was published in the Journal of Chromatography A (1) with a research team comprised of scientists from Osaka University (Osaka, Japan) and Shimadzu.

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Oligonucleotides are short DNA or RNA molecules used to modulate gene and protein expression and can accumulate in various tissues after administration. These molecules can be single- or double-stranded and include antisense oligonucleotides (ASOs), RNA interference, and aptamer RNAs (2). ON therapeutics are actively being researched and developed as new modalities for drug discovery, with the number of products in the market increasing each year. ASOs, a type of therapeutic ON consisting of single-stranded oligonucleotides approximately 20-mer in length, selectively bind via complementary base-pairing to messenger RNA (mRNA) and are the basis for one type of RNA-based therapeutic being explored for treating cancer and genetic disorders (3).

To increase their activity and safety, ASOs can be chemically modified with sugars, nucleobases, and phosphodiester moieties. They primarily synthesize these molecules using a solid-phase phosphoramidite method, where nucleotides are individually added to the growing chain through a coupling reaction with phosphoramidite on a solid support. Completely removing impurities generated through this synthesis remains challenging, making it essential to quantify and characterize these impurities in active pharmaceutical ingredients (APIs).

In this study, the team evaluated SFC for the analysis of 5-, 10-, 15-, and 18-mer oligonucleotides, before applying it to deaminated products or side products generated during oligonucleotide synthesis. Separating the side products from the target oligonucleotide proved difficult and depended on the deamination position and sequences; this is also the case when using ion-pair reversed-phase liquid chromatography (IP-RPLC), which is a common method for oligonucleotide analysis.

Researchers demonstrated that SFC, which incorporated octylamine as a modifier additive, could generate 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 the column oven temperature, modifier additive, and stationary phase were optimized, SFC successfully separated oligonucleotides with various numbers and positions of deamination from the target oligonucleotide. The technique also exhibited different selectivities for DMTr-on and DMTr-off oligonucleotides compared to IP-RPLC, indicating that SFC is a valuable alternative tool for the purification and analysis of oligonucleotides.

According to the team, this study is the first to report on using SFC to analyze long-chain ONs, such as 10-, 15-, and 18-mers, and to separate structurally similar sequences. For DMTr-on ONs, SFC separated the sequences that co-eluted in IP-RPLC. The trend proved consistent for sequences with different numbers and positions of deamination, likely because of the distinct interactions between the stationary phase and the nucleobase moieties of the analytes. Overall, the study highlights SFC’s significant potential for impurity analysis in ON therapeutics, particularly for those conjugated with hydrophobic organ-targeting ligands, beyond just DMTr groups.

References

(1) Hayashida, M.; Suzuki, R.; Horie, S.; et al. Separation of Deaminated Impurities from the Desired Oligonucleotides Using Supercritical Fluid Chromatography. J. Chromatogr. A 2025, 1744, 465731. DOI: 10.1016/j.chroma.2025.465731

(2) Oligonucleotide. ScienceDirect 2013. https://www.sciencedirect.com/topics/neuroscience/oligonucleotide (accessed 2025-2-20)

(3) Antisense Oligonucleotide. Toolkit 2025. https://toolkit.ncats.nih.gov/glossary/antisense-oligonucleotide/ (accessed 2025-2-19)