• Application Note

UPLC Analysis of Phosphorothioate Oligonucleotides: Method Development

UPLC Analysis of Phosphorothioate Oligonucleotides: Method Development
  • Martin Gilar
  • Waters Corporation
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Abstract

In this application note, we illustrate and provide guidelines for fast, high resolution, and efficient analysis of therapeutic phosphorothioate oligonucleotides in a single analysis method.

Benefits

Fast, high-resolution, efficient analysis of therapeutic phosphorothioate oligonucleotides in a single analysis method.

Introduction

Antisense phosphorothioate oligonucleotide therapies are a promising treatment for a number of diseases, including cancer, diabetes, high cholesterol, and AIDS. The inherently unique characteristics of phosphorothioate oligonucleotides, combined with the multiple-step manufacturing process, make analysis of these oligonucleotides challenging. Post-purification analysis is a difficult and time-consuming process, typically requiring multiple orthogonal methods (CGE and SAX HPLC), adding significant costs and burden to a regulated QC laboratory. 

The ACQUITY UltraPerformance LC (UPLC) System with Oligonucleotide Separation Technology (OST) Columns, packed with 1.7 µm sorbent, offer superior analytical performance for phosphorothioate oligonucleotide separations compared to HPLC and fast LC separations. As a result, method development and analysis are accomplished in dramatically shorter time, saving valuable time and analytical resources.

Results and Discussion

The ACQUITY UPLC System and OST Columns are used for fast and efficient separation of oligonucleotides using ion-pairing reversed phase liquid chromatography (IP-RP LC) mode. Figure 1 illustrates the oligonucleotide separation on the mix of 15, 20,  25, 30, and 35 nt oligodeoxythymidines. The minor peaks are by-products of failed synthesis. Baseline n-1 resolution of all species is achieved in less than ten minutes. 

Figure 1. Separation of synthetic oligodeoxythymidines phosphorodiester oligos on a 2.1 x 50 mm, 1.7 µm UPLC OST C18 Column.

Phosphorothioate oligonucleotides are more difficult to analyze than phosphorodiester ones. When replacing an oxygen atom in the oligo backbone for sulfur, multiple diastereomers are created. Partial separation of isomers broadens the peaks in both capillary electrophoresis (CE) and liquid chromatography (LC), and complicates the analysis.

While the traditional triethylammonium acetate (TEAA) ion-pairing system is useful for phosphorodiester oligonucleotides, it fails when applied for separation of phosphorothioate oligonucleotides. Recently, Fountain and Gilar described a novel ion-pairing buffer suitable for efficient analysis of therapeutic phosphorothioate oligonucleotides.1,2 The buffer is comprised of triethylamine (TEA, an ion-pairing agent) and aqueous hexafluoroisopropanol (HFIP, a volatile weak acid used as buffering component to bring the pH to ~8). In addition, this ion-pairing system is compatible with both UV and electrospray MS detection.

The method development for oligonucleotide separation includes an optimization of gradient slope and initial mobile phase elution strength. The method development for analysis of modified oligonucleotides should reflect the fact that these are often more retained in IP-RP LC. An adjustment of initial mobile phase strength may be necessary, especially for 2'O-methylated oligos.

Figure 2 shows the separation of 25 nt phosphorothioate oligonucleotide that was partially hydrolyzed with snake venom phosphodiesterase (3'-exonuclease). The main 25 nt peak was clearly resolved from the N-x 3' truncated species. The identity of the peaks was confirmed by their mass (data are not shown).

The gradient slope used for phosphorothioate separation was 0.2% MeOH per minute. In order to maintain a smooth gradient profile when generating the gradient from 100% aqueous and 100% organic mobile phases, the larger mixer (425 µL) is recom-mended. Figure 2 illustrates that the analysis time can be reduced without sacrificing a resolution. This is achieved by appropriately adjusting the initial gradient strength while keeping the gradient slope constant.

Figure 2. Separation of 25 nt phosphorothioate oligonucleotide from 3' truncated metabolites (3'-exonuclease digested sample). Gradient slope was 0.2% MeOH/min. Gradient started at 19% (A) or at 19.5% MeOH (B).

Figure 3 shows the analysis of a purified synthetic 25 nt phosphorothioate oligonucleotide. Interestingly, the failed synthesis by-products correspond to 3'-truncated parent oligonucleotide fragments. N+x peak (cyanoethyl protection group adduct; EtCN) was resolved from the target compound.

Figure 3. Separation of 25 nt phosphorothioate oligonucleotide from shorter species. (A) 3'exonuclease digested 25 nt, (B) synthetic 25 nt oligo contaminated with a trace amounts of N-x peaks and EtCN 25 nt synthetic by-products.

Conclusion

The ACQUITY UPLC System with Oligonucleotide Separation Technology Columns enable high-resolution, high-throughput analysis of phosphorothioate oligonucleotides in a single method. The development of fast analytical methods for native and modified oligonucleotides can be achieved quickly, increasing the overall operational efficiency of a laboratory. UPLC technology will increase the productivity of any laboratory developing LC and LC-MS methods and performing analysis of oligonucleotides.

References

  1. Fountain KJ, Gilar M, Gebler JC. Analysis of Native and Chemically Modified Oligonucleotides by Tandem Ion-pair Reversed-phase Highperformance Liquid Chromatography/Electrospray Ionization Mass Spectrometry. Rapid Commun Mass Spectrom. 2003. 17 (7): 646–53.
  2. Gilar M, Fountain KJ, Budman Y, Holyoke JL, Davoudi H, Gebler JC. Characterization of Therapeutic Oligonucleotides Using Liquid Chromatography with On-line Mass Spectrometry Detection. Oligonucleotides. 2003; 13 (4): 229–43.
  3. UPLC Separation of Oligonucleotides: Method Development. Waters Application Note. 2007. 720002383EN.
  4. UPLC-MS Separation of Oligonucleotides in Less Than 5 Minutes: Method Development. Waters Application Note. 2007. 720002387EN.

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Analyzing phosphorothioate oligonucleotides holds significant importance across diverse sectors, especially in drug development and nucleic acid therapies, necessitating precise characterization and quality control measures. Ultra-performance liquid chromatography (UPLC ) has emerged as a pivotal tool due to its superior resolution, rapidity, and sensitivity.

It is robust UPLC method tailored specifically for phosphorothioate oligonucleotide analysis. Method development encompasses multiple essential stages focused on optimizing chromatographic conditions for effective separation and detection of oligonucleotide species.

Initially, method development entails carefully selecting suitable chromatographic columns and mobile phase compositions. Columns with high-efficiency packing materials and specific affinity for oligonucleotides are typically preferred to ensure optimal separation. Moreover, meticulous optimization of the mobile phase composition, including buffer pH and organic modifiers, aims to amplify chromatographic resolution and sensitivity.

Subsequently, fine-tuning of chromatographic conditions involves systematic adjustments of parameters like column temperature, flow rate, and gradient profile. These refinements aim to achieve rapid, efficient separation of phosphorothioate oligonucleotides while minimizing issues such as peak broadening and tailing, which could compromise analytical precision and sensitivity.

Furthermore, the selection of a suitable detection method is critical to ensure accurate quantification of oligonucleotide species. While UV detection is commonly utilized due to its sensitivity and compatibility with phosphorothioate oligonucleotides, alternative approaches such as mass spectrometry may be employed for enhanced selectivity and sensitivity in certain scenarios.

Once the UPLC method is meticulously optimized, it undergoes rigorous validation to evaluate its performance characteristics, encompassing linearity, accuracy, precision, and robustness. Method validation is imperative to ascertain that the developed method complies with regulatory standards and can consistently quantify phosphorothioate oligonucleotides across diverse sample matrices.

In summary, UPLC Analysis of Phosphorothioate Oligonucleotides through a single, efficient analytical method offers comprehensive insights into the intricate process of formulating a reliable UPLC method for phosphorothioate oligonucleotide analysis. Through meticulous optimization and validation procedures, the developed method emerges as a valuable asset for researchers and analysts engaged in assessing and ensuring the quality of oligonucleotide-based therapeutic agents.