Oligonucleotide Purification using Anion Exchange Liquid Chromatography

Significance of the Topic

Oligonucleotides have become indispensable tools in molecular biology, diagnostics, and therapeutics. As synthetic methods approach high coupling efficiencies, the removal of closely related impurities remains a critical bottleneck. Scalable, reproducible purification workflows are essential for obtaining high-purity oligonucleotides across research, clinical, and industrial applications.

Objectives and Study Overview

This guide outlines best practices for anion exchange liquid chromatography (AEX-LC) purification of synthetic oligonucleotides using polymer-based strong anion-exchange (PL-SAX) media. Key goals include:

• Defining criteria for selecting column chemistry, pore size, and particle dimensions.
• Presenting method development strategies for analytical to preparative scales.
• Describing scale-up calculations and bulk media approaches.
• Providing practical tips for column conditioning, cleaning, and storage.

Methodology and Instrumentation

The approach employs Agilent PL-SAX columns built on a stable PS-DVB backbone available in 1000 Å (10–200 bases) and 4000 Å (up to mRNA) pore sizes. Common mobile phases include tris or phosphate buffers with NaCl gradients. Method parameters optimized include pH (up to 12), temperature (up to 80 °C), and organic additives (10–15 % ACN). For small-scale analysis, ion-pair reversed-phase can be used, but AEX-LC is preferred for preparative runs due to lower salt consumption and direct UV detection.

Main Results and Discussion

Key findings and recommendations:

• Pore size selection: 1000 Å for 18–200 bases, 4000 Å for large mRNA species.
• Particle size and column length: 5–10 µm analytical up to 30 µm preparative; scale-up column length adjusted to maintain theoretical plates.
• Method optimization illustrated with sgRNA (105 bases): best separation achieved at pH 10 in EDA-HCl with 60 °C and 30–50 % salt gradient.
• Scale-up formula: maintain linear velocity (180–360 cm/hr) and adjust volumetric flow by diameter squared ratio.
• Bulk media operations: Load & Lock columns for process scale with 10 g to 1 kg PL-SAX resin.

Benefits and Practical Applications

The PL-SAX AEX-LC workflow delivers:

• High resolution between full-length product and n-1 to n-x impurities.
• Cost-effective operations due to common buffers and UV detection.
• Flexibility from analytical scouting (0.1 mL/min) to preparative throughput (up to 200 mL/min).
• Compatibility with existing Agilent LC platforms, offering seamless method transfer.

Future Trends and Applications

Emerging directions in oligonucleotide purification include:

• Integration of MS-compatible ion-pair strategies for detailed impurity profiling.
• Automated, high-throughput platforms combining AEX and reversed-phase modules.
• Development of novel stationary phases with improved selectivity for modified oligos.
• Application of machine-learning algorithms for rapid method scouting and optimization.

Conclusion

Anion exchange chromatography on PL-SAX media provides a robust, scalable solution for purifying synthetic oligonucleotides from small research quantities to large-scale production. With optimized pore and particle selection, controlled pH, temperature, and organic modifiers, users can reliably achieve high purity and yield across a range of oligonucleotide lengths.

Instrumentation Used

Systems referenced:

• Agilent 1220/1260/1290 Infinity II Analytical-Scale LC Purification (0.1–10 mL/min).
• Agilent 1260 Infinity II Bio Analytical-Scale LC (1–50 mL/min, biocompatible flow path).
• Agilent 1290 Infinity II Preparative LC (1–200 mL/min, columns up to 50 mm id).

References

1. Agilent Technologies. High Resolution Separations of Oligonucleotides using PL-SAX Strong Anion-Exchange HPLC Columns. Application Note 5990-8297EN.
2. Agilent Technologies. PL-SAX Anion-Exchange Media for Nucleotide and Oligonucleotide Analysis. Technical Overview 5990-8779EN.
3. Agilent Technologies. Purification of Single-Stranded RNA Oligonucleotides Using High-Performance Liquid Chromatography. Application Note 5994-3514EN.
4. Agilent Technologies. Direct Analysis of In-Process Oligonucleotides Without Manual Purification. Application Note 5991-9490EN.
5. Agilent Technologies. Improved Column Lifetime with Thermally Stable Polymer Columns for Oligonucleotide Ion-Pair RP HPLC. Application Note 5990-7764EN.
6. Agilent Technologies. Use Temperature to Enhance Oligonucleotide Mass Transfer and Improve Resolution in Ion-Pair RP HPLC. Application Note 5990-7765EN.
7. Agilent Technologies. Purify Your Way, Agilent Load & Lock Columns. Application Note 5994-3907EN.
8. Agilent Technologies. InfinityLab LC Purification Solutions. Brochure 5991-9153EN.
9. Agilent Technologies. Purify Your Samples with Maximum Flexibility. Brochure 5991-9154EN.