Emerging Alternative Analytical Techniques for Oligonucleotide Separation and Purification: HILIC & SEC

Importance of the Topic

As oligonucleotide-based therapeutics and diagnostics continue to expand, accurate separation and characterization of these molecules become critical for ensuring product purity, safety, and efficacy. Traditional methods such as ion-pair reversed-phase (IP-RP) and ion-exchange (IEX) chromatography present challenges related to mass spectrometry compatibility and dedicated instrumentation. Alternative approaches that enable flexible workflows and direct coupling to MS detection are therefore of high practical value.

Study Objectives and Overview

This study evaluates hydrophilic interaction liquid chromatography (HILIC) and size exclusion chromatography (SEC) as emerging analytical techniques for oligonucleotide separation and purification. The main goals are:

• To assess the retention mechanisms and selectivity of various HILIC stationary phases for DNA and RNA oligonucleotides across different pH conditions.
• To characterize SEC column performance for monitoring higher-order structures, including aggregation and conformational differences in oligonucleotides.

Methodology and Instrumentation

The experimental work was divided into HILIC and SEC workflows:

• HILIC separations were performed on five Agilent columns (Poroshell 120 HILIC, HILIC-OH5, HILIC-Z, AdvanceBio Glycan Mapping, AdvanceBio Amide) in a 2.1×150 mm format with 2.7 µm particles. Mobile phases were 10 mM ammonium acetate at pH 4.4 or pH 9.0 mixed with acetonitrile or water in 10:90 ratios.
• SEC analyses employed an AdvanceBio SEC 1000 Å column (2.7 µm, 7.8×300 mm) using either 100 mM Tris acetate with 2.5 mM EDTA or 150 mM phosphate buffer (pH 7.0) at 0.6 mL/min and 40 °C.

Key Results and Discussion

HILIC Screening:

• Twelve key analyte pairs were assessed via the Tanaka test to quantify contributions from hydrophilicity, hydrophobicity, hydrogen bonding, steric selectivity, and ion exchange on HILIC columns.
• The HILIC-Z phase exhibited robust performance at elevated pH, improving peak shape and resolution for both DNA and RNA oligo standards.

SEC Characterization:

• Larger pore size SEC columns resolved single-strand RNA and DNA ladders, revealing that hydrodynamic behavior of oligonucleotides does not strictly correlate with molecular weight due to elongated conformation.
• Comparative elution of Firefly luciferase (FLuc) and β-galactosidase mRNAs highlighted differences in 3D structure, with shorter FLuc mRNA eluting earlier, indicating more extended conformation.

Benefits and Practical Applications

HILIC offers MS-compatible mobile phases and flexible instrumentation use, simplifying workflows for oligonucleotide analysis without dedicated IP-RP systems. SEC provides a gold-standard approach for monitoring aggregation and higher-order structures, essential for quality control of long oligonucleotide therapeutics and mRNA products.

Future Trends and Applications

Continued development of tailored HILIC chemistries and optimized SEC pore architectures will further enhance resolution of sequence variants, impurities, and conformers. Integration with hyphenated MS techniques and automated data analysis pipelines promises more comprehensive characterization of complex oligonucleotide modalities.

Conclusion

This work demonstrates that HILIC and SEC are viable emerging alternatives to IP-RP and IEX for oligonucleotide separation. Their complementary strengths—MS compatibility and aggregation monitoring—address critical quality attributes in the evolving landscape of oligonucleotide therapeutics.