Superficially Porous Columns for Semi-Preparative Purification of Synthetic Oligonucleotides

Importance of the Topic

Synthetic oligonucleotides are essential tools in modern biopharma, but their manufacture yields mixtures containing deletion sequences and by-products. Efficient purification at milligram scale without switching to large preparative equipment reduces time and cost. Superficially porous columns offer high resolution and loading capacity at moderate pressures, making them attractive for semi-preparative workflows using standard analytical LC systems.

Objectives and Study Overview

This study aimed to develop and optimize an ion-pair reversed-phase LC method for crude 22-mer RNA purification, and to demonstrate seamless scale-up from analytical to semi-preparative formats on a single Agilent 1290 Infinity II system. Key goals included identifying optimal pH and temperature conditions, evaluating superficially porous stationary phases, and validating fraction collection strategies.

Used Instrumentation

• Agilent 1290 Infinity II high-speed pump (G7120A)
• Agilent 1290 Infinity II multisampler with sample thermostat (G7167B)
• Agilent 1290 Infinity II multicolumn thermostat (G7116B)
• Agilent 1290 Infinity II diode array detector with 10 mm max-light cell (G7117C, G7117-60020)
• Agilent 1260 Infinity bio-inert analytical fraction collector (G5664A)

Applied Methodology

Crude all-2'-O-methylated 22-mer RNA was diluted in mobile phase A containing 0.1 M TEAA. A 1 M TEAA stock was prepared by dissolving acetic acid and triethylamine, then adjusting pH to 7.0 or 8.65. Analytical separations used AdvanceBio Oligonucleotide superficially porous columns (2.7 µm or 4 µm, 4.6 × 150 mm) with a 5–35 % ACN gradient over 30 min. Temperature was varied between 25 °C and 60 °C. Scale-up employed 10 × 50 mm and 10 × 150 mm semi-preparative columns operated at matched linear velocities (0.42 mL/min vs. 2.0 mL/min) and the same gradient profile. Sample volumes up to 40 µL were injected using an enlarged loop, and 0.2-min time-based fractions were collected.

Main Results and Discussion

Optimization showed pH 8.65 and 60 °C delivered the sharpest peaks and best impurity resolution. Superficially porous phases maintained high efficiency at lower pressures. Scale-up chromatograms were virtually identical to analytical runs when flow rates were adjusted for linear velocity, confirming predictable performance. Fraction analysis of the main peak across six 0.2-min slices demonstrated that pooling fractions F08–F10 achieved >98 % purity and ~60 % yield, while extending the pool to F08–F11 raised overall yield to ~89 % with 96 % purity.

Benefits and Practical Applications of the Method

• High-resolution purification of synthetic oligonucleotides with minimal instrument changes
• Reduced system downtime by using the same analytical LC setup for scale-up and fraction analysis
• Lower operating pressures due to superficially porous particles, enabling robust semi-preparative runs
• Scalable workflow from milligram to larger batch purifications without revalidation of method conditions

Future Trends and Potential Applications

Emerging directions include integrating automated fraction collection with real-time purity monitoring, extending superficially porous technology to longer or chemically modified oligonucleotides, and applying machine learning to predict optimal method parameters. Further scale-out to pilot or production scale using larger column diameters and continuous purification formats may streamline oligonucleotide manufacturing.

Conclusion

The developed ion-pair reversed-phase LC method on superficially porous columns provides a robust, reproducible route for semi-preparative RNA purification. Seamless scale-up on the same analytical instrument simplifies transition from method development to production, ensuring high purity and yield with minimal reconfiguration.

References

1. Goyon A, Fan Y, Zhang K. Chapter 10 - Analysis of Oligonucleotides by Liquid Chromatography. In: Fanali S, et al., editors. Handbooks in Separation Science, Liquid Chromatography (Third Edition). Volume 2; 2023. p. 357–380.