New research published in the Journal of Chromatography A examines the effect of flow rate on plate height and resolution for antisense oligonucleotides (ONs) under hydrophilic interaction liquid chromatography (HILIC).
Antibody-oligonucleotide conjugates or AOC use in antibody arrays; 3d rendering | Image Credit: © Love Employee - stock.adobe.com
The study was authored by Dwight Stoll, professor in chemistry at Gustavus Adolphus College, Daniel Meston, Associate Research Professor at Gustavus Adolphus College, and Todd D. Maloney, Associate Vice President at Eli Lilly. Stoll is the "LC Troubleshooting" columnist for LCGC International, and hosts the Analytically Speaking podcast.
In this study, the team worked to determine the quality antisense ONs (ASOs) attributes, such as purity, potency, and sequence for ONs, which can be challenging. This is due to the relatively large size of oligonucleotides, in addition to their polyanionic nature and large numbers of synthetic modifications. Chromatography technologies are quickly evolving to meet these challenges, the authors wrote, with one notable area of advancement being the use of hydrophilic interaction liquid chromatography (HILIC) for ON separations.
HILIC, a variation of reversed-phase chromatography, allows for strong retention of polar, hydrophilic compounds that are unrelated under conventional reversed-phase conditions, without requiring an ion-pair additive in the mobile phase (2). Few studies address factors affecting separation kinetics, leaving unclear the potential gains from altering flow rate or particle size during method development.
ONs are short single- or double-stranded DNA or RNA molecules used to modulate gene and protein expression, which can accumulate in various tissues after administration (3). ONs, unlike biologic drugs, target errors in the genetic code. This has led to their use for rare and previously untreatable diseases, such as neural conditions (4). ONs allow for the development of therapeutics that affect protein targets that cannot be effectively treated through small-molecule or protein therapeutics. ASOs are a subset of ON therapeutics that are made of single-stranded RNA, which are typically 20–30 nucleotides in length. The longer an ON, the more difficult it is to separate failure sequences of similar lengths. Additionally, stable secondary structures can cause an ON to elute as a broad peak or a series of peaks (5).
The retention of ASOs decreases significantly with increasing pressure, complicating separation. To address this, the team adjusted the mobile phase composition to maintain a nominally constant retention factor across different flow rates. The results showed that plate height increases sharply with rising flow rate, exhibiting a ten-fold increase over the range of 0.1 to 4.0 mL/min using a 4.6 mm i.d. column. Notably, the minimum reduced plate height at the lowest flow rate is around 2, which is low. This dependence of plate height on flow rate leads to improved resolution at lower flow rates, both in conventional one-dimensional separations and in the second dimension of two-dimensional separations.
Despite strong dependence on velocity, the minimum measured reduced plate heights were approximately 2.2. Considering the ON molecules studied held intermediate size and structural complexity, this was impressive, the team wrote.
The dependence of plate height on flow rate observed for a single ON predictably translates to a dependence of resolution on flow rate for paired and closely related ONs. This applies to first- and second-dimensional liquid chromatography (LC) separations. In an example presented by the scientists, decreasing flow rate through the 2D column in a 2D-LC separation of closely related ONs from 0.75 to 0.10 mL/min. improved the resolution of ONs with zero or one phosorothioate modifications from 0.9 to 1.8. Overall, quantifying the dependencies of plate height and resolution on flow rate is important to optimize challenging ON separations, especially when opportunities to improve resolution by changing selectivity are limited.
(1) 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
(2) HILIC Overview. ThermoFisher Scientific 2025. https://www.thermofisher.com/us/en/home/industrial/chromatography/chromatography-learning-center/liquid-chromatography-information/hilic-hplc-uhplc-columns-information/hilic-overview.html (accessed 2025-1-22)
(3) Oligonucleotide. ScienceDirect 2013. https://www.sciencedirect.com/topics/neuroscience/oligonucleotide (accessed 2025-1-22)
(4) Lamb, A. Oligonucleotide Therapeutics: A Biopharmaceutical Basics Overview. ThermoFisher Scientific 2023. https://www.thermofisher.com/blog/analyteguru/oligonucleotide-therapeutics-a-biopharmaceutical-basics-overview/ (accessed 2025-1-22)
(5) Chapter 7: Purification and Characterisation of Oligonucleotides. ATDBio 2025. https://atdbio.com/nucleic-acids-book/Purification-of-oligonucleotides (accessed 2025-1-22)
Assessing Thorium-Peptide Interactions Using Hydrophilic Interaction Liquid Chromatography
February 4th 2025Paris-Saclay University scientists used hydrophilic interaction liquid chromatography (HILIC) coupled to electrospray ionization mass spectrometry (ESI-MS) and inductively coupled plasma mass spectrometry (ICP-MS) to assess thorium’s interaction with peptides.