Efficient Method Development of Oligonucleotides by Reversed-Phase Ion-Pair Chromatograph

Significance of the topic

Precise separation of synthetic oligonucleotides and related impurities is critical for quality control and safety in nucleic acid drug development. Reversed-phase ion-pair chromatography provides high-resolution analysis but involves many interacting parameters, necessitating efficient method development strategies.

Objectives and Overview of the Study

This work aims to establish a robust and accelerated workflow for developing LC methods for oligonucleotides. Using Shimadzu’s LabSolutions MD software and an AQbD approach, the authors performed systematic screening and design-space evaluation to identify optimal mobile-phase and instrument conditions.

Methodology and Instrumentation

The study consisted of two stages:

• Initial screening: Mobile-phase variables (HFIP 100–200 mmol/L, TEA 5–20 mmol/L, ACN/MeOH ratios 0–100 %) were tested, and an Evaluation Value (number of peaks × sum of resolution factors) was calculated to rank conditions.
• Design-space optimization: Using the best initial condition (100 mmol/L HFIP, 10 mmol/L TEA, ACN 50 %), further experiments varied acetonitrile ratio (40–60 %), column temperature (55–65 °C), and starting gradient concentration (6–8 %). Peak resolution was visualized to define robust operating ranges.

Used Instrumentation:

• LC: Nexera XS inert Method Scouting System with Shim-pack Scepter Claris column (100 × 2.1 mm, 3 µm).
• Detector: Photodiode array at 260 nm and LCMS-2050 single-quadrupole mass spectrometer (ESI/APCI negative mode).

Main Results and Discussion

The initial screening identified 100 mM HFIP, 10 mM TEA, and 50 % acetonitrile as yielding the highest Evaluation Value. Design-space plots showed improved resolution at higher temperatures and allowed simultaneous fulfillment of criteria: resolution of n-1(5’)/n-1(3’), resolution of n-1(3’)/PO > 0.7, and final peak retention time < 16 min. The optimized condition (ACN 54 %, oven 65 °C, initial gradient 8 %) achieved n-1(3’)/PO resolution > 0.7 and final retention around 15 min, though baseline separation of n-1(5’) and n-1(3’) remained challenging.

Benefits and Practical Applications

This automated, equation-driven approach significantly reduces manual workload in method scouting, enhances data quality, and accelerates nucleic acid drug analysis workflows. It provides a reproducible framework for early-phase method development under AQbD principles.

Future Trends and Applications

Emerging directions include integration of machine-learning algorithms for predictive separation, expansion to multi-attribute methods combining MS/MS data, and increased automation of gradient and reagent optimization. The methodology can be extended to diverse oligonucleotide modifications and complex biological matrices.

Conclusion

A systematic, software-assisted strategy for RP-IP LC method development for oligonucleotides has been demonstrated. By combining initial screening, design-space evaluation, and automated peak tracking, the study achieved robust separation conditions efficiently, fostering streamlined drug development.