Oligonucleotide characterization using μPAC columns in ion-pairing reversed-phase high performance liquid chromatography mode

Importance of the Topic

Single-stranded oligonucleotides, including antisense oligonucleotides (ASOs), are a leading therapeutic platform for gene regulation. Phosphorothioate (PS) backbone modifications improve stability and potency but create chiral centers, yielding complex diastereomer mixtures. Accurate separation and characterization of these isomers are vital for assessing drug purity, safety, and efficacy.

Objectives and Study Overview

This study evaluates Thermo Scientific μPAC HPLC columns in ion-pairing reversed-phase mode to resolve native, modified, and diastereomeric oligonucleotides. The performance of 50 cm and 200 cm μPAC pillar array columns is directly compared with conventional packed bed columns under strong and weak ion-pairing conditions.

Methodology and Instrumentation

Oligonucleotide standards (5–30mer dT ladder; T5 and T15 with defined PS linkages; model therapeutic-like sequences with 3–5 PS bonds) were analyzed by nanoLC at flow rates of 250–1500 nL/min. Two mobile phase systems were applied: a strong ion-pairing buffer (16.3 mM triethylamine, 400 mM HFIP, pH 7.9) to maximize size-based resolution, and a weak buffer (15 mM TEAA, pH 7) to promote diastereomer separation. Columns tested included μPAC micro pillar array columns (C18-functionalized, 18 µm pillars, 50 cm and 200 cm bed length) and two silica-based packed bed columns (75 µm × 25 cm, 75 µm × 15 cm).

Main Findings and Discussion

• In strong ion-pairing mode, μPAC columns achieved peak capacities up to 734 (200 cm) and 348 (50 cm), significantly outperforming packed beds for 5–30mer separations.
• Under weak ion-pairing conditions, μPAC columns resolved 2, 4, and 8 baseline diastereomers for sequences with 1–3 PS bonds, and up to 16 diastereomers for a 19-mer with 4 PS linkages. Packed beds showed severe peak broadening and incomplete resolution.
• High-throughput separations (10 min gradient at 1.5 µL/min) on a 50 cm μPAC column delivered peak capacity of 89—56% higher than packed bed alternatives—enabling a four-fold increase in separation efficiency.
• Resolution gains were most pronounced for central PS modifications and at optimized nanoLC flow rates, attributed to the uniform flow distribution and minimal dispersion in the micro pillar array.

Benefits and Practical Applications

• Enhanced purity profiling of therapeutic oligonucleotides by robust impurity and diastereomer separation.
• Flexible throughput: the 50 cm μPAC column suits routine assays, while the 200 cm column delivers ultra-high resolution for complex mixtures.
• Mass-spectrometry compatibility under both ion-pairing modes streamlines direct LC-MS workflow for detailed structural analysis.

Future Trends and Opportunities

Micro pillar array technology offers potential for integration into multidimensional LC and advanced MS workflows, improving impurity profiling in biopharmaceutical development. Ongoing enhancements in pillar chemistries and microfabrication are expected to boost peak capacity further, reduce analysis times, and enhance column robustness for next-generation oligonucleotide analytics.

Conclusion

Thermo Scientific μPAC micro pillar array columns deliver unparalleled resolution, efficiency, and flexibility for ion-pairing reversed-phase separation of oligonucleotides. Their superior diastereomer selectivity and throughput, combined with MS compatibility, support accelerated development and quality control of oligonucleotide-based therapeutics.

References

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