Analysis & Purification of Therapeutic Oligonucleotides: Method Development Optimization from the Analytical Scale Through Semi-prep and Preparative Purification

Significance of the Topic

Oligonucleotides are emerging therapeutic agents that modulate gene expression to treat a wide range of diseases. Accurate analysis and purification of these short nucleic acid sequences are essential to ensure product integrity, remove closely related sequence impurities, and accelerate the drug development pipeline from discovery through large-scale manufacturing.

Objectives and Study Overview

This work outlines a systematic approach to develop and optimize ion-pair reversed-phase liquid chromatography (IP-RP LC) methods for oligonucleotide analysis at the analytical scale and to transfer these methods to semi-preparative and preparative purification workflows. The goals include:

• Improving resolution and sensitivity of target oligo separations.
• Assessing the impact of ion-pair reagents and temperature on chromatographic performance.
• Demonstrating method scalability across column chemistries and dimensions.

Methodology and Instrumentation

The analytical technique employs ion-pair reversed-phase (IP-RP) separation on C18 stationary phases. Key variables include:

• Ion-pairing agents: triethylamine (TEA), tripropylamine (TPA), hexylamine (HA), dibutylamine (DBA).
• Mobile phase pH and alkylamine concentration to tune retention of anionic oligonucleotide backbones.
• Elevated column temperatures (up to 60 °C) to disrupt secondary structure and reduce mobile phase viscosity.

Instrumentation used: Agilent HPLC systems with column heating and AdvanceBio Oligonucleotide superficially porous particles, designed for stability at high pH and temperature.

Main Results and Discussion

1. Ion-Pairing Effects

• More hydrophobic amines (HA, DBA) increased retention and improved separation of full-length products from truncated impurities.

2. Temperature Influence

• Elevating column temperature from 25 °C to 60 °C sharpened peaks, enhanced resolution of closely eluting species, and lowered backpressure.

3. Scalability and Method Transfer

• Analytical columns (2.1×50 mm, 2.7 μm) were successfully scaled to 4.6×150 mm (4 μm) and semi-prep 10×150 mm (4 μm) formats with proportional flow and injection volume increases.
• Example semi-prep run at 60 °C loaded 3.2 mg of crude antisense oligo in four injections. Fraction yields ranged from 2 % to 33 % with purities up to 99 %.

Benefits and Practical Applications

The described workflow enables:

• Rapid method development at the analytical scale with straightforward scale-up to bench-top semi-prep and preparative purification.
• High resolution sequence confirmation and removal of near-identical impurities.
• Flexible throughput for small-scale research and larger-scale production needs.

Future Trends and Potential Applications

Advancements expected in:

• Novel superficially porous stationary phases and ion-pair chemistries to further enhance resolution.
• Integration of LC-MS and automated fraction collection for real-time impurity profiling.
• Continuous chromatography platforms to increase purification efficiency for large-scale oligonucleotide manufacture.

Conclusion

The Agilent AdvanceBio Oligonucleotide column portfolio delivers robust IP-RP LC solutions from analytical method development to preparative purification. By optimizing ion-pair reagents, temperature, and column chemistry, users can achieve high-resolution separations, simplify scale-up, and ensure high yield and purity of therapeutic oligonucleotides.

Instrumentation Used

• Agilent HPLC systems with column heating module.
• AdvanceBio Oligonucleotide columns: 2.1×50 mm, 2.7 μm; 4.6×150 mm, 4 μm; 10×150 mm, 4 μm.

References

• Superficially Porous Columns for Semi-Preparative Purification of Synthetic Oligonucleotides. Agilent Technologies application note, 5994-7478EN, 2024.
• Fast and Selective Purification of Oligonucleotides Using Preparative HPLC/MS and Software Support. Agilent Technologies application note, 5994-4877EN, 2022.