Evaluation of new high pH stable, superficially-porous particle columns for the reversed-phase separation of oligonucleotides

Importance of the Topic

The development of synthetic oligonucleotides as therapeutic agents necessitates rapid and high-resolution analytical methods to detect and quantify N-1 and N-2 deletion impurities arising from coupling failures. High-pH mobile phases at elevated temperatures offer improved separation but require chemically stable stationary phases.

Aims and Overview of the Study

This study evaluates high-pH stable superficially-porous particle columns (AdvanceBio Oligonucleotide, 2.7 µm) for reversed-phase HPLC separation of RNA and DNA oligonucleotides. Performance metrics such as resolution, backpressure, analysis time, and column stability are compared with conventional totally porous C18 columns and hybrid particle phases. Both UV and LC/MS-compatible mobile phase systems are investigated.

Methodology and Instrumentation Used

• Columns: AdvanceBio Oligonucleotide superficially-porous particles (2.1×50, 2.1×100, 2.1×150, and 4.6×50, 4.6×100, 4.6×150 mm; 2.7 µm), with fast guard cartridges.
• Mobile phases: A) 100 mM triethylammonium acetate (TEAA) in water and 100 mM TEAA in acetonitrile; B) LC/MS-friendly: 400 mM hexafluoroisopropanol (HFIP):15 mM triethylamine (TEA) in water and methanol mix.
• Gradient programs: e.g., 10–14% B in 10 min for RNA, 7–11% B in 5 min then to 80% B for DNA ladder.
• Instrument: Agilent 1290 Infinity Binary (and compatibility with 1260 Infinity Quaternary) HPLC systems; UV detection at 260 nm; column temperature 65 °C; flow rates 0.6 mL/min.

Main Results and Discussion

Superficially-porous columns demonstrated narrower peak widths and shorter mass transfer distances, achieving baseline resolution of N and N-1 oligonucleotide impurities (21-mer RNA, 20-mer RNA) within 11–13 min at pressures around 108 bar versus 292 bar for competitor 1.7 µm C18. DNA ladder separation (15–40 mers) completed in under 10 min, with the 15-mer eluting at ~2 min and mid-length species around 4–5 min. MS-compatible conditions yielded the 25-mer DNA oligo and its impurities in ~3 min with clear resolution.
Column stability tests over 300–400 consecutive injections showed retention time reproducibility and maintained resolution, affirming the long lifetime of these high-pH stable columns.

Benefits and Practical Applications

• Fast, high-resolution separation of RNA and DNA oligonucleotides for therapeutic development and QC.
• Low backpressure enabling use on both 600 bar and 1200 bar HPLC platforms.
• Robust performance under high-pH conditions and elevated temperatures.
• Compatibility with UV and MS detection workflows for method flexibility.

Future Trends and Potential Applications

Further integration with high-throughput LC-MS workflows and automation can streamline oligonucleotide analysis. Miniaturized column formats and AI-driven method optimization may enhance sensitivity and throughput. Expanding to modified nucleic acids and longer sequences will support next-generation oligonucleotide therapeutics.

Conclusion

AdvanceBio Oligonucleotide superficially-porous particle columns offer rapid, high-resolution, and robust separation of RNA and DNA oligonucleotides under high-pH conditions. Their low backpressure, extended lifetime, and compatibility with UV and MS detection make them well suited for research, QA/QC, and therapeutic development applications.

Reference

• Bidlingmeyer BA, Wang X, Luke S, Duong PT. Evaluation of new high pH stable superficially-porous particle columns for the reversed-phase separation of oligonucleotides. Agilent Technologies Application Note PSB-BIOAN-07; HPLC 2015.