Oligonucleotide Purification using Ion Pair Reversed-Phase Liquid Chromatography

Significance of the Topic

The development of reliable purification workflows for synthetic oligonucleotides is essential to support advances in biochemical research, therapeutic development, and quality control. As oligonucleotide technologies expand into RNA interference, antisense applications, guide RNAs for CRISPR, and mRNA vaccines, robust methods are needed to isolate full-length products from closely related impurities across scales from analytical screening to gram-level production.

Objectives and Study Overview

This guide outlines strategies for consumables selection, method development, and scale-up of ion-pair reversed-phase (IP-RP) liquid chromatography using polymeric PLRP-S columns. Key goals are to compare column chemistries, pore and particle sizes, ion-pair reagents, and hardware configurations, along with practical tips for conditioning, operation, and ordering of supplies to achieve high-purity oligonucleotide fractions.

Methodology and Instrumentation

• Chromatographic Technique: IP-RP separation employs alkylamine counterions (e.g., triethylamine, tripropylamine, hexylamine, dibutylamine) to pair with the anionic phosphate backbone, enhancing retention and resolution of oligonucleotide chains.
• MS vs UV Compatibility: Acetate buffers and acetonitrile permit UV detection, while MS-compatible protocols replace acetate with hexafluoroisopropanol combined with methanol to improve ionization and impurity identification.
• Temperature Control: Column temperatures up to ~80 °C reduce secondary interactions, sharpen peaks, and facilitate separation of closely related n-1 and chemical modification impurities.
• Pore and Particle Size Selection: PLRP-S columns are available in 100, 300, 1000, and 4000 Å pores and 3–50 µm particles; 100 Å suits short oligos (15–30 bases), 300 Å for mid-length (75–200 bases), and large-pore media for mRNA constructs (up to thousands of bases).
• Scale-Up Considerations: Linear velocity targets (180–360 cm/hr) guide flow-rate calculations when transitioning from analytical (e.g., 4.6 × 150 mm) to semipreparative and preparative dimensions; particle size changes are compensated by adjusting column length to maintain theoretical plates.
• Consumables and System Configuration: Analytical systems (Agilent 1220/1260/1290 Infinity II) support 0.1–10 mL/min, semipreparative Bio-Analytical units 1–50 mL/min, and preparative LC 4–200 mL/min; compatible filters, fittings, vials, and fraction collectors streamline sample handling.

Main Results and Discussion

The guide demonstrates that selecting the correct pore size and ion-pair reagent concentration dramatically improves resolution of full-length oligonucleotides from failure sequences and modification by-products. Temperature elevation proves critical for high-efficiency separations, while HFIP-based mobile phases enable direct coupling to MS for mass confirmation of target and impurity profiles. Scale-up equations and best practices ensure consistent performance across column formats, and bulk resin plus Load & Lock hardware facilitate larger-scale purifications.

Benefits and Practical Applications

• Enhanced Purity: Optimized IP-RP methods yield >95% removal of n-1, oxidation, and adduct impurities.
• Scalability: A seamless transition from analytical scouting to preparative production reduces development time and resource consumption.
• Versatility: Compatible with UV and MS detection, supporting both routine QC and advanced characterization.
• Workflow Efficiency: Prepacked columns, bulk media, and a defined supply list minimize procurement complexity and downtime.

Future Trends and Opportunities

As mRNA therapeutics and gene-editing reagents gain traction, demand will increase for high-capacity, high-throughput purification platforms. Emerging column chemistries with improved stability at extreme pH and temperature, advancement of greener mobile phases, and integration with automated fraction collection and inline MS detection are poised to streamline workflows further. Continuous processing and single-use consumable systems may also drive rapid adoption in manufacturing settings.

Conclusion

Ion-pair reversed-phase chromatography on polymeric PLRP-S media offers a robust, scalable solution for oligonucleotide purification across a spectrum of chain lengths. By carefully selecting column pore and particle size, ion-pair reagents, temperature, and flow conditions, practitioners can achieve consistent high-purity separations. A defined suite of consumables and Agilent LC configurations ensures streamlined method transfer from research to production.

References

1. Purification of Single-Stranded RNA Oligonucleotides Using High-Performance Liquid Chromatography, Agilent Application Note 5994-3514EN.
2. Direct Analysis of In-Process Oligonucleotides Without Manual Purification, Agilent Application Note 5991-9490EN.
3. Evaluation of Different Ion-Pairing Reagents for LC/UV and LC/MS Analysis of Oligonucleotides, Agilent Application Note 5994-2957EN.
4. Ion-Pair Reversed-Phase Purification of De-Protected Oligonucleotides ‑ Choice of Pore Size, Agilent Application Note 5990-7763EN.
5. Improved Column Lifetime with Thermally Stable Polymer Columns for Oligonucleotide Ion-Pair RP HPLC, Agilent Application Note 5990-7764EN.
6. Agilent PLRP-S HPLC Columns and Media, Agilent Technical Overview 5990-8187EN.
7. Dynamic Binding Capacity of Oligonucleotides on PLRP-S Columns and Stationary Phases, Agilent Application Note 5994-4526EN.
8. Use Temperature to Enhance Oligonucleotide Mass Transfer and Improve Resolution in Ion-Pair RP HPLC, Agilent Application Note 5990-7765EN.
9. Purify Your Way, Agilent Load & Lock Columns, Agilent Technical Overview 5994-3907EN.
10. Agilent InfinityLab LC Purification Solutions, Agilent Brochure 5991-9153EN.
11. Purify Your Samples with Maximum Flexibility, Agilent Infobody 5991-9154EN.