Efficient Reversed-Phase Separation of Oligonucleotides by Using High pH Stable Superficially Porous Columns

Significance

Synthetic oligonucleotides are rapidly becoming key therapeutic agents, but their multi-step synthesis generates truncated impurities that challenge analytical laboratories. High-resolution, rapid and robust separation techniques are essential to ensure product purity and support drug development workflows.

Objectives and Study Overview

This study evaluates a novel superficially porous reversed-phase column designed for stability at high pH. The goals are to assess its performance for RNA and DNA oligonucleotide separations and compare it to conventional totally porous C18 columns in terms of resolution, speed and backpressure.

Methods and Used Instrumentation

The separations employed Agilent AdvanceBio Oligonucleotide superficially porous columns (2.1×50 mm, 2.7 µm). Mobile phases consisted of 100 mM triethylammonium acetate (TEAA) in water (A) and in acetonitrile (B), using a gradient from 10 % to 14 % B over 10 min. An alternative HFIP/TEA mobile phase for LC–MS compatibility was also tested. Chromatographic conditions included 65 °C column temperature, 0.6 mL/min flow rate and UV detection at 260 nm. An Agilent 1290 Infinity Binary pump and autosampler system was used throughout.

Main Results and Discussion

The superficially porous column achieved near-baseline separation of main oligonucleotides from N-1 and N-2 truncations for both RNA and DNA ladders within 10 min. Peak widths were narrower than those on a competitive 1.7 µm totally porous C18, indicating faster mass transfer. At equivalent resolution, backpressure on the 2.7 µm superficially porous column was ~108 bar versus ~292 bar on the 1.7 µm column, enabling compatibility with both 600 bar HPLC and 1200 bar UHPLC systems. Column durability was demonstrated over 300 consecutive injections of a 25-mer DNA oligo under pH > 8, with retention times and peak shapes remaining constant.

Benefits and Practical Applications

• High-throughput, high-resolution profiling of therapeutic oligonucleotides for QC and R&D.
• Compatibility with both UV and MS detection workflows.
• Extended column life under harsh, basic conditions.

Future Trends and Opportunities

Advances may include the integration of superficially porous materials in next-generation UHPLC platforms to further increase throughput. Development of optimized buffer systems for direct MS coupling will streamline biopharma pipelines. Ongoing material science innovations may yield even smaller superficially porous particles with low backpressure and enhanced resolution.

Conclusion

High pH stable superficially porous HPLC columns deliver superior speed, resolution and durability for oligonucleotide separations compared to traditional totally porous columns, making them highly valuable for analytical laboratories in therapeutic development and quality control.

References

None provided.