Analysis & Purification of Therapeutic Oligonucleotides: Method development optimization from the analytical scale through semi-prep and preparative purification

Importance of the Topic

Therapeutic oligonucleotides have emerged as powerful tools for regulating gene expression across diverse disease targets. Their structural diversity and anionic backbone present unique analytical challenges. Reliable liquid chromatography methods are essential for tracking synthesis impurities, confirming sequence integrity, and ensuring product purity throughout the drug development pipeline.

Objectives and Study Overview

This study focused on developing and optimizing ion-pair reversed-phase (IP-RP) HPLC methods for oligonucleotide analysis at the analytical scale and scaling these protocols to semi-preparative and preparative purifications. Key goals included evaluating the effects of temperature, ion-pairing reagents, and column chemistry on resolution and transferability across column formats.

Methodology and Instrumentation

A series of IP-RP experiments were performed using Agilent’s HPLC platform equipped with column heating. Alkylamine ion-pairing agents (triethylamine, tripropylamine, hexylamine, dibutylamine) were screened to modulate retention and selectivity. Temperature studies compared separations at 25 °C and 60 °C to assess resolution gains. Analytical separations employed AdvanceBio Oligonucleotide columns (2.1 × 50 mm, 2.7 µm and 4.0 µm particles). Preparative work used AdvanceBio Oligonucleotide semi-prep columns (10 × 150 mm) for benchtop purifications.

• System: Agilent HPLC with column heater module
• Analytical columns: AdvanceBio Oligonucleotide, 2.7 µm and 4.0 µm, 2.1 × 50 mm
• Semi-prep columns: AdvanceBio Oligonucleotide, 4.0 µm, 10 × 150 mm
• Mobile phases: High pH buffers with selected alkylamine ion-pair reagents

Main Results and Discussion

Increasing column temperature to 60 °C sharpened peaks by reducing intra- and intermolecular interactions, improving crude oligo product confirmation and impurity separation. More hydrophobic ion-pairing agents enhanced retention, enabling baseline resolution of closely eluting sequences. The novel surface chemistry of the AdvanceBio Oligonucleotide support maintained performance under high-pH, high-temperature conditions. Direct method transfer from analytical to semi-prep columns was demonstrated by scaling a 4.6 × 150 mm analytical method to a 10 × 150 mm semi-prep format: a 160 µL injection (3.2 mg total) yielded fractions with purities up to 99.2% and peak yields up to 33.4%.

• Elevated temperature reduces mobile phase viscosity and improves resolution
• Hydrophobic ion-pair reagents increase selectivity for target vs. impurities
• AdvanceBio columns enable seamless scale-up without method re-development

Benefits and Practical Applications

The optimized IP-RP methods streamline method development across scales, supporting high-resolution analytics, accurate sequence confirmation, and efficient benchtop purification. Laboratories can leverage the same column chemistry from discovery through manufacturing, reducing time and cost in therapeutic oligonucleotide workflows.

Future Trends and Application Possibilities

Looking ahead, integration of mass spectrometric detection will further enhance impurity profiling. Advances in column chemistries may allow even larger oligonucleotide separations, including CRISPR guide RNAs and modified backbones. Automation of semi-prep purifications and novel ion-pair reagents are likely to expand throughput and selectivity.

Conclusion

Agilent’s AdvanceBio Oligonucleotide columns deliver robust, high-resolution separations and scalable purification solutions for therapeutic oligonucleotides. Elevated temperatures and optimized ion-pair chemistries enable rapid method transfer from analytical assays to preparative workflows, supporting both research and production needs.

References

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