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

Significance of the Topic

Nucleic acid therapeutics such as antisense oligonucleotides require robust chromatographic methods to separate target compounds from synthesis-related impurities. Achieving optimal resolution is critical for accurate quantification, regulatory compliance, and ensuring patient safety. Analytical Quality by Design software streamlines this process by integrating experimental design, automated data processing, and design space visualization, reducing development time and human error.

Objectives and Study Overview

This study demonstrates an efficient workflow for developing reversed-phase ion-pair (RP-IP) chromatography methods for oligonucleotides using LabSolutions MD. Key objectives include:

• Screening mobile phase compositions (HFIP and TEA concentrations, acetonitrile/methanol ratios) to identify initial separation conditions.
• Optimizing gradient, column temperature, and organic solvent ratio to maximize resolution of a full-length product (FLP) and five related impurities.
• Implementing automated peak tracking via LCMS data to ensure accurate compound identification.
• Visualizing resolution design spaces and overlaying retention time constraints to select the final optimal method.

Methodology and Instrumentation

Initial screening evaluated 24 mobile phase combinations: HFIP at 100 or 200 mmol/L, TEA at 5, 10, 15 or 20 mmol/L, and acetonitrile ratios of 0, 50 or 100% (balance methanol). LabSolutions MD automatically generated analysis schedules, prepared mobile phases, and calculated an Evaluation Value for each chromatogram based on the number of peaks and their resolution (Eq. 1). The top-ranking condition (100 mmol/L HFIP, 10 mmol/L TEA, 50% acetonitrile) was selected for further optimization. Optimization varied acetonitrile ratio (40–60%), oven temperature (55–65 °C), and gradient start (6–8%). Automated peak tracking used UV data and LCMS deconvoluted molecular weights to resolve peaks with similar UV spectra. Design spaces plotted resolution versus acetonitrile ratio and temperature, and overlay with retention time limits identified the final optimum.

Used Instrumentation

• Nexera XS inert UHPLC system with inert flow path.
• Shim-pack Scepter™ Claris column (100 × 2.1 mm I.D., 3 µm, metal-free inert coating).
• SPD-M40 UV detector at 260 nm with inert flow cell.
• LCMS-2050 single-quadrupole mass spectrometer (ESI/APCI DUIS, negative mode, m/z 500–2000).
• LabSolutions MD software for method development, automated scheduling, evaluation and design space analysis.

Main Results and Discussion

Initial screening identified 100 mmol/L HFIP, 10 mmol/L TEA, and 50% acetonitrile as optimal among 24 conditions. High HFIP with 100% acetonitrile caused baseline fluctuations, affecting quantification. During optimization, higher acetonitrile ratio, increased oven temperature, and elevated gradient start improved resolution. LCMS-based peak tracking provided accurate identification (mass errors <2 Da) even when UV spectra were nearly identical. Design space visualization revealed that resolution increases with temperature, while optimal acetonitrile ratio varies by impurity pair. Overlay of resolution and retention time constraints yielded a final method: 100 mmol/L HFIP, 10 mmol/L TEA, 54% acetonitrile, 46% methanol, column oven at 65 °C, gradient start 8%, achieving resolution >0.7 for critical pairs and total run time <16 min.

Benefits and Practical Applications

• Automated mobile phase blending and schedule generation reduces manual workload and errors.
• Quantitative ranking and design space tools accelerate decision-making and enhance method robustness.
• LCMS-based peak tracking ensures reliable identification of closely eluting or spectrally similar species.
• Approach is adaptable to various oligonucleotide sequences and modifications, benefiting research, QA/QC, and production labs.

Future Trends and Potential Applications

The integration of AQbD software with advanced detectors and AI-driven experimental design will further accelerate chromatographic method development. Combining high-resolution mass spectrometry with predictive modeling may enable real-time optimization and adaptive control in regulated environments. Wider adoption of automation and digital workflows will support high-throughput screening of complex biologics and personalized nucleic acid therapeutics.

Conclusion

By leveraging LabSolutions MD alongside Nexera XS inert UHPLC and LCMS-2050, this workflow automates screening, optimization, peak tracking, and design space evaluation for oligonucleotide separations. The result is a robust, high-throughput method that meets resolution and run-time criteria, reducing development timelines and ensuring reliable impurity profiling.

Reference

• Fujisaki S, Suzuki R. LabSolutions MD: Software for Efficient Method Development based on Analytical Quality by Design. Shimadzu Application News, First Edition, Sep 2024.