HPLC/UHPLC Technical Tip
Level: Advanced
Method Development Strategies for Oligonucleotides
The clinical utility of oligonucleotides as a means of controlling gene expression makes them of increasing interest as therapeutics. Oligonucleotides have very different chemical properties to typical small molecule drugs, which makes them an interesting target, but also makes their analysis more complicated than many small molecule therapeutics. They are inherently polar compounds and require quite specific conditions to allow the analysts to fully characterize them and their impurities.
In this two-part series we will look at the challenges scientists face in the analysis of oligonucleotides and some of the method development levers they could pull when working on their analysis. Part 2, on the use of bio-inert hardware in oligonucleotide analysis can be found here.
Traditional oligonucleotide methods employ the use of an ion-pair agent (typically an alkylamine such as triethylamine) to facilitate their retention on a C18 column. C18 phases are still preferred as they offer the highest level of efficiency when the goal is to resolve closely eluting impurities. In this first part, we will look at the effect of the mobile phase, specifically the alkylamine ion pair selected, on resolution and characterization.
In the literature TEA is often listed as a standard alkylamine to be used alongside hexafluoroisopropanol (HFIP) for the analysis of oligonucleotides. However, studies by the Bartlett laboratory would suggest that the different alkylamine types should be evaluated when developing a method for the analysis of oligonucleotides.1
Some general guidelines for selecting alkylamine and alkylamine concentration for use in your method are outlined below. Please be mindful these are guidelines to help method development and should form part of good experimental design.
Higher concentrations of alkylamine leads to increased retention time and higher ionization efficiency (Fig. 1).
Fig. 1
| Column | Biozen™ 2.6 µm Oligo |
| Dimensions | 100 x 2.1 mm |
| Part No. | 00D-4790-AN |
| Mobile Phase A | 12.5 mM HFIP, Hexylamine in Water |
| Mobile Phase B | 12.5 mM HFIP, Hexylamine in MeOH |
| Gradient | 25-75% B in 14 minutes |
| Flow Rate | 0.3 mL/min |
| Detection | TOF-MS |
| Temperature | 65 °C |
| Sample | Nusinersen |
The concentration of alkylamine may be a concern, as ion-pair agents can linger around in your LC system and MS interface if you haven’t implemented a proper wash. In such cases, there are other strategies you can evaluate to reduce the amount of alkylamine in the first place.
The use of a more hydrophobic alkylamine, such as hexylamine (HA), can allow you to reduce the concentration of ion-pairing agent and run a higher proportion of organic solvent in your gradient (Fig. 2) . Greater retention time, a higher proportion of organic solvent in your mobile phase, and lower concentration of ion-pairing agent will all help to facilitate higher ionization efficiency (Fig. 3).
Fig. 2
| Column | Biozen 2.6 µm Oligo |
| Dimensions | 100 x 2.1 mm |
| Mobile Phase | A: 12.5 mM HFIP, HA or TEA (as indicated) in water B: 12.5 mM HFIP, HA or TEA (as indicated) in methanol |
| Flow Rate | 0.3 mL/min |
| Gradient | As shown (in % B) |
| Temperature | 65 °C |
| Detection | UV @ 260 nm |
| Sample | BNA |
Fig. 3
| Column | Biozen 2.6 µm Oligo |
| Dimensions | 100 x 2.1 mm |
| Part No. | 00D-4790-AN |
| Mobile Phase A | 12.5 mM HFIP, 1 mM TEA in Water 12.5 mM HFIP, 4 mM HA in Water |
| Mobile Phase B | 12.5 mM HFIP, 1 mM TEA in Methanol 12.5 mM HFIP, 4 mM HA in Methanol |
| Gradient | 5-75 % B in 14 min |
| Flow Rate | 0.3 mL/min |
| Detection | TOF-MS |
| Temperature | 65 °C |
| Sample | 2’-MOE Gapmer (200 ng) |
One final thing you should consider when choosing an ion-pairing agent is solubility. When there is lower solubility of the ion pair in the mobile phase, you can see some enhancement of ion pairing. However, if we take octylamine as a unique additional example, this additive is very much less soluble in highly aqueous mobile phases and requires more of an organic component. The high levels of organic which are required to solubilize this ion-pair agent make it less applicable in all situations, particularly when analyzing shorter oligonucleotides that are not well retained in highly organic mobile phases.
In summary, there is no magic bullet when it comes to selecting the best ion-pairing agent for the analysis of oligonucleotides, but following a few simple guidelines can significantly improve the characterization data you are able to obtain from your analysis.
1 Basiri, B.; Murph, M. M.; Bartlett, M. G. J Am Soc Mass Spectrom. 2017, 28(8), 1647–1656
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