Investigating Antisense Oligonucleotide Separation Kinetics Using Hydrophilic Interaction Liquid Chromatography

LCGC International spoke with Daniel Meston and Dwight Stoll from Gustavus Adolphus College in St. Peter, Minnesota, USA, about a project they worked on with Todd Maloney from Eli Lilly in Indianapolis, Indiana, USA, to investigate the optimal performance conditions of antisense oligonucleotides (ASOs) when using hydrophilic interaction liquid chromatography (HILIC).

Why are antisense oligonucleotides (ASOs) important therapeutic molecules and what treatments are they used for?

Daniel Meston: ASOs are short oligonucleotides (ONs) that bind ribonucleic acid (RNA) and modulate protein translation. They offer a promising approach to treat diseases that have been considered “undruggable” by conventional means because they may not respond to treatments involving monoclonal antibodies (mAbs), or small molecule therapies.

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What are the main challenges of analyzing ASOs from an analytical perspective and what separation techniques have been used?

Dwight Stoll: The main chromatographic challenge is insufficient separation power for the level of complexity of the analytes involved. The processes used to synthesize ASOs produce chemically-similar impurities, leading to peak overlap. Wide peaks can result from slow analyte diffusion and partial separation of diastereomers. Additionally, the details related to the retention mechanism for these molecules have not been fully worked out, which can complicate method optimization. These are some of the analysis challenges motivating our work to investigate ASOs.

The major modes of liquid chromatography (LC) used to separate ASOs are ion-pair reversed-phase high performance liquid chromatography (IP-RP–HPLC), hydrophilic interaction liquid chromatography (HILIC), and ion‑exchange chromatography (IEX) (1,2,3). These particular modes are effective for retaining the highly charged analytes that are otherwise too water-soluble to be well retained under conventional reversed-phase conditions.

In the paper you published recently you highlighted that there has not been much published on the factors that dictate these separations under HILIC conditions (4). How does the adjustment of flow rate in
HILIC separations impact the plate height and resolution for ASOs, and what trade-offs should be considered for method development?

Meston: It is generally understood that there is a mobile phase velocity that corresponds to a minimum in curves that relate plate height to mobile phase velocity (typically referred to as van Deemter curves). We have been working towards understanding what factors control this dependence for ASOs; however, we have found that these studies are complicated by the fact that the retention factors of ASOs decrease dramatically with increasing pressure under HILIC conditions. Therefore, when doing these experiments the change in retention caused by the change in pressure that accompanies changes in flow rate must be considered. One way to compensate for this is to change the mobile phase composition such that the retention factor can be held constant even when the flow rate is changed.

With a minimum reduced plate height of around two observed at low flow rates, how does this compare to benchmarks in other chromatographic techniques, and what insights does this provide for improving column efficiency in HILIC separations?

Stoll: It is generally understood that well-made columns exhibit a minimum reduced plate height of two or less, so the minimum plate heights of around two that we observed are consistent with this.

How does the dependence of resolution on flow rate observed in one-dimensional separations translate to the second dimension in two-dimensional liquid chromatography (2D-LC)?

Meston: We found that the best resolution of closely related ASOs is obtained at low flow rates. As far as we know this is a broadly applicable observation—at least for the HILIC columns and conditions we have studied. It is possible that we will observe different trends with other conditions, but this is what we have seen so far. An important advantage of 2D-LC is the ability to run a relatively fast first dimension separation that addresses compounds that are easy to resolve, and then transfer overlapping peaks to a low-flow second dimension for improved resolution.

What is novel about this research?

Stoll: This work has demonstrated that plate height for ASOs around 20 bases increases very strongly with increasing flow rate. However, excellent reduced plate heights in the order of three can be obtained at very low flow rate (for example, 0.1 mL/min with a 4.6-mm internal diameter [i.d.] column), if one is willing to wait. Such low flow rates are not used commonly in practice, which is understandable due to the long separation times involved; however, if there is no other way to separate the analytes of interest, then this could be an attractive option.

Given that resolution improves at lower flow rates, how could these findings be applied to optimizing the second dimension of a 2D-LC workflow for ASO analysis?

Meston: With respect to heart-cutting 2D-LC, if you have an area of coelution in the first dimension, it may be possible to utilize a low flow rate in the second dimension to achieve the necessary resolution without completely re-developing the first‑dimension separation.

What areas of research or technological developments would help to advance the use of HILIC to analyze ASOs?

Stoll: HILIC is a more “environmentally friendly” alternative to IP-RP–HPLC, despite the use of high levels of acetonitrile, but the mechanism that controls retention and selectivity for ASOs is not fully understood. Further work aimed at understanding the factors that control retention and selectivity will give users more tools to systematize method development. HILIC is particularly useful for separating charged analytes, such as ASOs, without the need for ion-pairing conditions. The outcomes of this work raise a lot of questions that will be interesting to address, including the ways in which mobile phase chemistry, stationary phase chemistry, and particle size affect the dependence of plate height on flow rate. With this knowledge, analysts will have a better understanding of the levers available to them during method development.

References

1. Stoll, D.; Sylvester, M.; Meston, D.; Sorensen, M.; Maloney, T. D. Development of Multiple Heartcutting Two-Dimensional Liquid Chromatography with Ion-Pairing Reversed-Phase Separations in Both Dimensions for Analysis of Impurities in Therapeutic Oligonucleotides. J. Chrom. A 2023, 1714, 464574. DOI: 10.1016/j.chroma.2023.464574
2. Goyon, A.; Zhang, K. Characterization of Antisense Oligonucleotide Impurities by Ion-Pairing Reversed-Phase and Anion Exchange Chromatography Coupled to Hydrophilic Interaction Liquid Chromatography/Mass Spectrometry Using a Versatile Two-Dimensional Liquid Chromatography Setup. Anal. Chem. 2020,
   92 (8), 5944–5951. DOI: 10.1021/acs.analchem.0c00114
3. Vanhinsbergh, C.; Hook, E. C.; Oxby, N.; Dickman, M. J.
   Optimization of Orthogonal Separations for the Analysis of Oligonucleotides Using 2D-LC. J. Chrom. B 2023, 1227, 123812.
   DOI: 10.1016/j.jchromb.2023.123812
4. Meston, D.; Maloney, T. D.; Stoll, D. R. Effect of Flow Rate on
   Plate Height and Resolution for Antisense Oligonucleotides
   Under Hydrophilic Interaction Liquid Chromatography
   Conditions. J. Chromatogr. A 2025, 1742, 465643.
   DOI: 10.1016/j.chroma.2024.465643