Large scale purification – of oligonucleotides with ion exchange chromatography

Significance of the Topic

Oligonucleotide-based therapeutics have emerged as a transformative class of drugs, offering precise modulation of gene expression and favorable safety profiles. The rapid expansion of research and clinical applications has driven the demand for large-scale, high-purity oligonucleotides. Efficient purification strategies are essential to meet stringent quality requirements for research, development and clinical-grade materials.

Objectives and Study Overview

This work presents a case study by BianoGMP GmbH on the development and scale-up of ion exchange chromatography (IEX) for the purification of an oligo(dT) sample. The aim was to achieve at least 95 % purity and maintain a high yield, demonstrating the suitability of preparative IEX for gram-scale production under GMP-relevant conditions.

Methodology

The crude oligonucleotide was obtained by solid-phase synthesis, followed by cleavage, deprotection and neutralization steps to reduce conductivity below 30 mS/cm. Purity and concentration were evaluated by analytical HPLC and UV photometry. The purification protocol involved a preparative gradient elution with phosphate and sodium bromide buffers under non-denaturing conditions. High loading was employed to maximize throughput, and manual fractionation was performed based on UV detection.

Instrumentation Used

• AZURA Prep System with ternary pump (0–220 mL/min) and single UV detector at 260 nm
• YMC Pilot glass preparative column (100 mm ID × 500 mm, strong anion exchanger, SOURCE 15 Q resin)
• ClarityChrom® software with fraction collection control

Main Results and Discussion

Analytical HPLC of the crude sample showed 89.1 % initial purity. After gradient IEX purification of a 1 L sample, pooling of fractions yielded the target oligo(dT) at 95 % purity with a 90 % recovery. Chromatograms demonstrated sharp, well-resolved peaks and consistent UV saturation at high loading, confirming the method’s robustness.

Benefits and Practical Applications of the Method

• High binding capacity enables processing of large sample volumes with minimal column size.
• Non-denaturing aqueous buffers improve safety and sustainability compared to organic solvents.
• Scalability from analytical to preparative formats facilitates seamless technology transfer.
• Simplified resin regeneration and column reuse support cost-effective manufacturing.

Future Trends and Applications

Advancements in resin chemistry and column design are expected to further enhance resolution and throughput. Automation of loading and fractionation steps will reduce manual intervention and improve reproducibility. Integration of continuous IEX processes and in-line analytical monitoring could accelerate production timelines and support real-time quality control in GMP settings.

Conclusion

Preparative ion exchange chromatography proved to be an efficient and scalable solution for large-scale purification of oligonucleotides. The demonstrated protocol achieved the required purity and yield while using environmentally friendly conditions, making it well suited for research, development and clinical manufacturing.

Reference

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2. Minkner R, Boonyakida J, Park EY, Wätzig H. Oligonucleotide separation techniques for purification and analysis: What can we learn for today's tasks? Electrophoresis. 2022;43(23-24):2402–2427.