Dynamic Binding Capacity and High‑Resolution Oligonucleotide Analysis with Agilent Bio SAX Columns

Significance of the Topic

The increasing development of oligonucleotide therapeutics and diagnostics requires analytical techniques capable of delivering high resolution, reproducibility and minimal nonspecific interactions. Nonporous anion‐exchange (AEX) chromatography addresses these needs by offering robust LC-UV separations without reliance on volatile ion-pair reagents.

Study Objectives and Overview

This work evaluates dynamic binding capacity and analytical performance of Agilent Bio SAX columns packed with nonporous polystyrene divinylbenzene particles grafted with a hydrophilic layer. The study compares three particle sizes (1.7, 5 and 10 µm) for oligonucleotides ranging from 25 to 100 bases, with emphasis on binding efficiency and impurity resolution.

Methodology

Oligonucleotide samples (25, 50, 75 and 100 mer) were dissolved in 10 mM Tris pH 8 mobile phase and quantified spectrophotometrically. Chromatographic conditions included:

• Mobile phase A: 10 mM Tris, pH 8.0
• Mobile phase B: 2 M NaCl in mobile phase A
• Flow rate: 0.5 mL/min
• Column temperature: 25 C for binding capacity; 80 C for analytical separations
• Gradient profiles for cleanup and analytical runs optimized per oligo length

Instrumentation

• Agilent 1290 Infinity II high-speed pump (G7132A)
• Agilent 1290 Infinity II Bio multisampler with sample thermostat (G7137A, option 101)
• Agilent 1290 Infinity II multicolumn thermostat with biocompatible heat exchanger (G7116B)
• Agilent 1290 Infinity II diode array detector with bio-inert flow cell (G7115A, option 28)

Main Results and Discussion

Dynamic binding studies revealed that the 1.7 µm Bio SAX column exhibited the highest capacity (~8.49 mg/mL), followed by 5 µm (~6.52 mg/mL) and 10 µm (~3.22 mg/mL), independent of oligo length. Analytical separations using the 5 µm column achieved baseline resolution of crude 25, 50, 75 and 100 mer samples and even resolved n – 1 species in RNA standards, demonstrating the platform's high resolving power.

Benefits and Practical Applications of the Method

• High resolution impurity profiling of therapeutic oligonucleotides
• Avoids use of volatile ion-pair reagents, simplifying method development and validation
• Robust dynamic binding suitable for preparative and analytical workflows
• Minimal nonspecific adsorption due to hydrophilic surface grafting

Future Trends and Potential Applications

Further work may focus on coupling AEX separations with mass spectrometry for detailed structural characterization, scaling methods for preparative purification, and exploring novel stationary phases to boost throughput and selectivity. Automation and real-time monitoring of oligonucleotide synthesis may also benefit from these advances.

Conclusion

Agilent Bio SAX nonporous columns deliver exceptional binding capacity and high-resolution LC-UV separations for oligonucleotides across a broad length range. Their performance in impurity profiling makes them a valuable tool for analytical and preparative applications in oligonucleotide research and quality control, although individual method validation remains essential.

References

1. Cook K; Thayer J. Advantages of Ion-Exchange Chromatography for Oligonucleotide Analysis. Bioanalysis. 2011;3(10):1109–1120.
2. Hsiao J; Apffel A; Turner M. Optimizing Separation of Oligonucleotides with Anion-Exchange Chromatography. Agilent Technologies Application Note 5994-4753EN, 2022.