Getting in the right phase: HILIC for oligonucleotide analysis

Significance of the topic

Hydrophilic interaction liquid chromatography (HILIC) has become an important complementary tool for the characterization and impurity profiling of therapeutic oligonucleotides (ONs). Compared with ion-pair reversed phase liquid chromatography (IP-RPLC), HILIC offers orthogonal selectivity that reflects hydrophilicity, charge distribution and base composition rather than the behaviour of ion-pair complexes. HILIC methods using MS‑compatible buffers therefore facilitate LC–MS workflows, help resolve impurities that co-elute in IP‑RPLC, and serve routine QC, impurity characterization and orthogonal method strategies for ON drug development and manufacturing.

Objectives and study overview

This application study evaluated a zwitterionic sulfobetaine HILIC stationary phase (Eurospher II HILIC) for the analysis of a panel of five representative oligonucleotides (20–64 mer; DNA and RNA, various modifications and linkers). The goals were to establish buffer and gradient conditions compatible with LC–MS, optimize peak shape and run time, compare ammonium acetate versus ammonium formate buffers, and assess method transfer and scaling across three column dimensions (50 × 4 mm, 30 × 4 mm and 5 × 4 mm guard). Repeatability and retention behaviour (including whether an IP‑RPLC–type on/off mechanism appears in HILIC) were also investigated.

Methodology

Key method features and general workflow:

• Stationary phase: Zwitterionic sulfobetaine HILIC (Eurospher II 100‑3 HILIC).
• Mobile phases: Acetonitrile as organic component; aqueous buffers evaluated were ammonium acetate (preferred) and ammonium formate. Initial aqueous concentration used for optimization: 50 mM ammonium acetate.
• Temperature: 30 °C; detection UV @ 260 nm.
• Column geometries tested: 50 × 4 mm (analytical), 30 × 4 mm (short analytical) and 5 × 4 mm (guard column).
• Flow rates and gradients were iteratively optimized (initial longer gradients shortened through G1→G3 then scaled for shorter columns G4–G7) to improve peak shape and reduce cycle time.
• Samples: five purified/desalted oligonucleotides: a 20mer DNA semi‑thioate, a 21mer RNA antisense, a 25mer DNA with C12 linker, a 26mer modified RNA (mHH) and a 64mer oxidized DNA.

Used instrumentation

The study was performed on a KNAUER AZURA platform configured as follows:

• Pump: AZURA P 6.1L (stainless steel).
• Autosampler: LH 8.1 Liquid Handler with robotic cooler, injection valve and fast wash station.
• Detector: AZURA DAD 2.1L diode array detector; UV flow cell 10 mm, 2 µL.
• Column models: Eurospher II 100‑3 HILIC columns in 50 × 4 mm, 30 × 4 mm and a 5 × 4 mm guard.
• Software: ClarityChrom 10.1 for control and PDA processing.

Main results and discussion

Retention and selectivity

• All oligonucleotides showed retention on the 50 × 4 mm HILIC column with the initial method; HILIC retention reflected hydrophilic/charge interactions and separated species not always resolved in IP‑RPLC.
• The zwitterionic sulfobetaine phase provided moderated electrostatic interactions compared with bare silica or amino phases, increasing buffer/ionic strength flexibility and MS compatibility.

Buffer choice and chromatographic performance

• Ammonium acetate (50 mM) outperformed ammonium formate in the tested conditions: better peak symmetry and higher peak intensity for the studied samples. Improved performance is attributed to higher effective ionic strength in the water/acetonitrile mixture and possible pH effects in organic‑rich HILIC media.
• Lowering salt concentration (down to 10 mM) degraded resolution; 50 mM ammonium acetate was optimal across samples.

Gradient optimization and runtime reduction

• Initial gradients (G1) were optimized (G2,G3) to shorten runtime while improving peak shape; on the 50 × 4 mm column total analysis time was reduced from 35 to 20 minutes without loss of critical resolution.
• Scaling to a 30 × 4 mm column and increasing flow to 1.0 mL/min further reduced cycle time to ~8 minutes (≈77% reduction versus the original G1), while maintaining acceptable retention and repeatability.

Repeatability and robustness

• Ten consecutive injections of the 20mer under optimized 30 × 4 mm conditions (G4) gave excellent repeatability: RSD 0.16% for retention time and 0.89% for peak area.

Limitations and scale‑down to guard column

• Further scaling to a 5 × 4 mm guard column shortened run times but caused a significant loss of resolution; the 64mer was not retained under guard‑column conditions tested.
• Compared with IP‑RPLC methods, HILIC required longer absolute analysis times—IP‑RPLC methods can separate ONs in under 2 minutes using appropriate ion‑pairing systems—but HILIC delivers orthogonal selectivity and avoids ion‑pair reagents.

Benefits and practical applications

HILIC on a zwitterionic sulfobetaine phase offers several practical advantages for ON analysis:

• Orthogonal selectivity to IP‑RPLC, useful for impurity profiling and resolving shortmers/longmers that co‑elute in IP‑RPLC.
• Compatibility with MS workflows due to ammonium acetate/formate buffers and absence of persisting ion‑pair reagents.
• Method scalability: analytical to shorter columns can substantially shorten cycle times while preserving acceptable performance for many ONs.
• Robust repeatability suitable for QC and routine analyses.

Future trends and opportunities

Potential directions to increase the impact and applicability of HILIC for oligonucleotides include:

• Deeper LC–MS integration using optimized volatile buffers and source conditions to maximize sensitivity for modified ONs and phosphorothioates.
• Stationary phase engineering to improve retention for very long ONs (e.g., 64+ mer) while preserving MS compatibility.
• Miniaturization and UHPLC HILIC formats to recover speed advantages of IP‑RPLC without ion‑pair reagents.
• Systematic comparison and orthogonal method workflows combining IP‑RPLC and HILIC to comprehensively profile impurities and degradation products.
• Regulatory adoption and method harmonization for QC in therapeutic ON production.

Conclusion

Zwitterionic HILIC with ammonium acetate/acetonitrile mobile phases is a reliable, reproducible and MS‑compatible complement to IP‑RPLC for oligonucleotide analysis. The method provided sufficient retention and good peak shapes for a range of ON sizes and modifications, allowed substantial run time reductions through gradient and column scaling (35 → 20 → 8 min), and delivered high repeatability. Limitations remain for very long oligonucleotides under aggressive scale‑down, and IP‑RPLC retains advantages for ultra‑fast separations. HILIC is therefore particularly suitable as a routine QC/characterization tool and as an orthogonal method for impurity profiling.

References

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