Separating Oligonucleotides Using Ion-Pair Reversed-Phase Liquid Chromatography

Szabolcs Fekete, Mateusz Imiolek, and Matthew Lauber of Waters Corporation recently demonstrated a new approach for separating oligonucleotides (Ons) using ion-pair reversed-phase liquid chromatography (IP-RPLC). Their findings were published in the Journal of Chromatography Open (1).

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IP-RPLC is considered the method of choice for analytical scale separations of ONs. According to the scientists, IP-RPLC is advantageous over different chromatographic methods due to its ability to be readily coupled with mass spectrometry, providing high kinetic performance (peak capacity) for both ONs and smaller nucleic acids. Typically, alkylamines are used as ion-pairing (IP) agents, with mobile phases buffered with weak volatile acids. Oligonucleotide retention in IP-RPLC involves several processes. The cationic group of the IP agent forms pairs (complexes) with the negatively charged phosphate groups of ONs. The IP agent can also adsorb to the stationary phase ligands via its hydrophobic moieties. The ion pairs are then adsorbed onto the surface of the stationary phase. Finally, the adsorbed ion pairs can be eluted by a gradient of organic co-solvent, typically acetonitrile or methanol. This complex process can involve a multi-step, mixed-mode mechanism, involving both hydrophobic and electrostatic interactions.

The current practice of IP-RPLC of ONs involves one of two approaches. The first is using the same IP agent(s) and buffer in both mobile phases A and B, only changing the proportion of water and organic co-solvent by running an organic solvent gradient. The second approach is used when mobile phase B is a dilution of A in organic co-solvent. In the latter approach, the later eluting solvents will experience a lower concentration of IP agents. Choosing between maintaining IP concentration or diluting the IP system is usually based on practical considerations, such as the simplicity of making mobile phases. While this approach may have practical advantages, it is likely to cause reduced selectivity through a narrow elution window.

In this study, a novel ion-pair reversed-phase liquid chromatography (IP-RPLC) method was combined with a dual gradient approach to enhance ON separation. In combining a weak ion-pairing (IP) agent in the starting (weaker) mobile phase with a strong IP agent in the final (stronger) mobile phase, the method is said to widen the elution window, improving the resolution and selectivity for complex ON mixtures. The approach was claimed to outperform traditional single IP systems, notable in separating sequence and size variants of ONs. Theoretically, the approach should create new separation options for ON therapeutics, where there is need to better resolve increasingly diverse modified residues and their related impurities.

In all case studies, the scientists noticed improved selectivity and resolution compared to commonly used single IP system methods. It was predicted that in combining the novel weak to strong IP gradient method with a concave (logarithmic) gradient program, the capabilities of platform ON IP-RP methods would systematically improve. By adjusting the composition of the weak IP and strong IP mobile phases (the ratio or nature of the two IP agents), it is predicted that the degree of freedom can be significantly improved to map selectivity to an even larger design space. The opposed dual IP gradient method can provide new possibilities for tuning ON separations, especially when handling complex ON mixtures. In the future, the scientists hope their method will prove helpful to the pharmaceutical industry by supporting a wave of new genetic medicines.

References

(1) Fekete, S.; Imiolek, M.; Lauber, M. Weak to Strong Ion-Pair Gradients to Expand the Selectivity of Oligonucleotide Separations in Reversed Phase Liquid Chromatography - A Proof of Concept. J. Chromatogr. Open 2025, 7, 100200. DOI: 10.1016/j.jcoa.2024.100200