Purification of Single-Stranded RNA Oligonucleotides Using High‑Performance Liquid Chromatography

Significance of the Topic

Purification of single stranded RNA oligonucleotides is a critical process in pharmaceutical production due to the high similarity between desired products and their truncated or otherwise modified impurities. Ion pair reversed phase high performance liquid chromatography (HPLC) offers the resolution and reproducibility required to achieve the high purity standards demanded by research and therapeutic applications.

Objectives and Study Overview

This study demonstrates an end to end workflow for purifying a 22 mer all 2 O methyl synthetic RNA oligonucleotide. The method is first developed at analytical scale, then transferred to preparative scale to produce a product in excess of 99 percent purity with yields above 56 percent.

Methodology

An ion pair reversed phase HPLC method was optimized on an analytical system using PLRP S stationary phase with hexylammonium acetate and acetonitrile in a shallow gradient. To maintain denaturing conditions at preparative scale, urea was added to the aqueous phase. Gradient slopes were kept below one percent per minute to separate full length product from n minus one and n minus two truncations.

Instrumentation Used

• Analytical HPLC Agilent 1260 Infinity II with binary pump vialsampler multicolumn thermostat and diode array detector
• Preparative LC Agilent 1290 Infinity II with preparative binary pump with titanium heads and combined open bed sampler fraction collector
• Columns PLRP S analytical column 4.6 by 150 millimeter 8 micron and preparative column 25 by 150 millimeter 8 micron

Main Results and Discussion

The analytical method at two milligram per milliliter sample concentration resolved the full length peak from shorter impurities. Scaling to preparative conditions used a twenty milligram injection on column. Peak based fraction collection with nine second time slices yielded eleven fractions across the full length peak. Reanalysis at eighty degrees Celsius resolved aggregates and confirmed purity by peak area percentages. Pooling fractions three through seven achieved a product purity of 99.1 percent and a process yield of 56.3 percent. Broader pooling schemes were evaluated to balance purity against maximum recovery.

Benefits and Practical Applications

• Scalable workflow from analytical to preparative scale with reproducible retention profiles
• High purity output exceeding 99 percent suitable for research and pharmaceutical use
• Flexible fraction collection enabling precise control of yield versus purity trade off
• Compatibility with a wide pH range and solvent conditions thanks to titanium pump heads

Future Trends and Opportunities

Advances in oligonucleotide synthesis and purification are expected to focus on higher throughput preparative systems integration with mass spectrometry for real time fraction assessment greener solvent alternatives and implementation of machine learning for automated method optimization. Expanding column chemistries and further improvements in pump precision will further enhance separation of increasingly complex oligonucleotide mixtures.

Conclusion

This application highlights a robust and reproducible ion pair reversed phase preparative HPLC protocol for single stranded RNA oligonucleotide purification. The workflow consistently delivers products above 99 percent purity with yields exceeding 56 percent demonstrating its suitability for demanding pharmaceutical and research applications.

References

1. Roberts TC Langer R Wood MJ Advances in Oligonucleotide Drug Delivery Nat Rev Drug Discov 2020 19 673 694
2. Fueangfung S Yuan Y Fang S Denaturing Reversed Phase HPLC Using a Mobile Phase Containing Urea for Oligodeoxynucleotide Analysis Nucleos Nucleot Nucleic Acids 2014 33 7 481 488
3. Guillarme D Method Transfer for Fast Liquid Chromatography in Pharmaceutical Analysis Application to Short Columns Packed with Small Particles Part II Gradient Experiments Eur J Pharm Biopharm 2008 68 2 430 440