Reproducible Hydrophilic Interaction Chromatography for Denaturing and Non- Denaturing Analyses of Oligonucleotides Using GTxResolve Premier BEH Amide Columns

Significance of Topic

Hydrophilic interaction chromatography (HILIC) is critical for accurate, ion-pair reagent-free analysis of therapeutic oligonucleotides in both research and QC settings. It offers enhanced retention and resolution of polar biomolecules, facilitating pure and efficient characterization of single- and double-stranded oligonucleotides up to 100 nucleotides.

Objectives and Study Overview

This application note evaluates novel GTxResolve Premier BEH Amide 300 Å 1.7 µm columns to achieve reproducible, high-resolution HILIC separations of a 20 to 100 mer ssDNA ladder and siRNA duplex standards under denaturing and non-denaturing conditions. Key goals include assessing batch-to-batch consistency, peak symmetry, and retention behavior.

Methodology and Instrumentation

Chromatographic conditions were optimized for ssDNA and siRNA using acetonitrile-rich mobile phases containing ammonium acetate and minor methanol additives to stabilize the aqueous layer and suppress residual silanol interactions. Gradient methods were performed on a 2.1 × 150 mm GTxResolve Premier BEH Amide column at flow rates of 0.3 to 0.4 mL/min, with column temperatures varied between 25 °C and 60 °C to switch between non-denaturing and denaturing modes.

Used Instrumentation

• ACQUITY Premier UPLC system with MaxPeak High Performance Surface hardware
• ACQUITY UPLC PDA and tunable UV detectors with a 500 nL flow cell
• Premier flow-through needle autosampler

Main Results and Discussion

GTxResolve Premier BEH Amide columns demonstrated excellent access of oligonucleotides to the 300 Å pores, resulting in sharp, symmetric peaks across 20 to 100 mers and minimal batch variability. Addition of low proportions of methanol improved peak shapes. Temperature modulation allowed clear separation of intact siRNA duplexes from single-stranded RNA impurities under non-denaturing (25 °C) and denaturing (60 °C) conditions without changing mobile phases.

Benefits and Practical Applications

• Ion pairing reagent-free mobile phases reduce instrument contamination and MS suppression
• High resolution and reproducibility support regulated QC and therapeutic development
• Single platform for both denaturing and non-denaturing impurity profiling

Future Trends and Opportunities

Advances may include tailored amide phases for specific oligonucleotide modifications, integration with high-resolution mass spectrometry for in-depth impurity mapping, and automated batch testing for increased throughput in biopharmaceutical workflows.

Conclusion

The GTxResolve Premier BEH Amide 300 Å 1.7 µm column offers robust, reproducible HILIC separations of oligonucleotides with excellent peak quality and temperature-tunable analysis modes, making it well suited for therapeutic oligonucleotide characterization and QA/QC in regulated environments.

Reference

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