Fast and Selective Purification of Oligonucleotides Using Preparative HPLC/MS and Software Support

Importance of the Topic

Oligonucleotides shorter than 100 nucleotides are central to modern life‐science research and diagnostics, including applications in therapeutics, genome editing, and molecular detection. Reliable purification of these compounds is essential to ensure their structural integrity, biological efficacy, and reproducibility in downstream assays.

Study Objectives and Overview

This application note evaluates a preparative HPLC/MS workflow for fast and selective isolation of synthetic DNA oligonucleotides. Key aims include:

• Replacing costly HFIP ion-pair reagents with dibutylamine (DBA) and TRIS buffer.
• Maintaining mass-selective detection capabilities for precise fraction collection.
• Leveraging automated software to transfer analytical methods to preparative scale without manual reoptimization.

Methodology and Instrumentation

The purification strategy combines ion-pair reversed-phase HPLC with mass‐triggered fraction collection:

• Analytical separation: Agilent InfinityLab Poroshell HPH-C18, 3×100 mm, 2.7 µm, using hexylamine/HFIP mobile phases to profile full‐length product (FLP) and fragments.
• Preparative purification: Agilent Poroshell HPH-C18, 21.2×150 mm, 4 µm, with DBA/TRIS mobile phases compatible with high pH, eliminating the need for HFIP.
• Detection and control: Agilent 1290 Infinity II Autoscale Preparative LC/MSD system, OpenLab CDS ChemStation, and Automated Purification Software to generate focused gradients and trigger collection based on selected m/z of multiply charged ions.
• Fraction collection: Active split to MSD with volatile make-up solvent; peak-based mode combining UV and MS thresholds.

Main Results and Discussion

Analytical HPLC/MS of two DNA oligonucleotides (30–50 bases) distinguished full-length products from truncated fragments. Automated software identified target m/z values and calculated optimized preparative gradients. In preparative runs at 25 mL/min:

• Short ON: Eight fractions collected; seven exceeded 99% purity after reanalysis; early fraction contained minor impurities.
• Long ON: Ten fractions collected; all but the first achieved >99% purity.

Mass-triggered fraction collection narrowed the collection window, reduced impurity carryover, and minimized the number of fractions requiring reanalysis.

Benefits and Practical Applications

This workflow offers:

• Cost savings by replacing HFIP with DBA/TRIS while preserving MS‐triggered collection.
• Rapid scale-up from analytical to preparative separations using matching stationary phases.
• Streamlined method transfer and reproducibility via automated gradient calculation.
• High selectivity and yield through precise MS‐based fraction triggers.

Future Trends and Possibilities

Emerging directions include:

• Integration of real‐time data analytics and machine learning to refine gradient design.
• Extension to longer or chemically modified oligonucleotides (e.g., LNA, morpholino).
• Automated end-to-end purification platforms combining synthesis, purification, and QC.
• Development of new ion-pair reagents and stationary phases for enhanced resolution.

Conclusion

The presented method demonstrates a robust preparative HPLC/MS workflow for oligonucleotide purification, leveraging DBA/TRIS buffers and superficially porous Poroshell columns to achieve high purity, reduced costs, and efficient scale-up. Automated software support ensures rapid method translation and reliable fraction collection based on MS triggers.

Reference

1. Catani M. et al. Oligonucleotides: Current Trends and Innovative Applications in the Synthesis, Characterization, and Purification. Biotechnology Journal 2020, 15(8).
2. Evaluation of Different Ion-Pairing Reagents for LC/UV and LC/MS Analysis of Oligonucleotides. Agilent Technologies application note 5994-2957EN, 2021.
3. Purification of Single-Stranded RNA Oligonucleotides Using High-Performance Liquid Chromatography. Agilent Technologies application note 5994-3514EN, 2021.