Method Development for Preparative Purification of Long Oligonucleotides

Importance of the topic

The purification of long oligonucleotides is critical for ensuring the quality and safety of therapeutic agents such as antisense oligonucleotides and siRNAs. Impurities arising from incomplete synthesis, degradation, or side reactions can compromise efficacy, regulatory compliance, and downstream analyses.

Study objectives and overview

This work aimed to develop a robust preparative ion-pair reversed-phase liquid chromatography (IP-RPLC) method for a 60-mer 2′-O-methyl and 2′-hydroxyl modified oligonucleotide. The study systematically screened multiple stationary phases and ion-pairing reagents to optimize resolution of the target sequence from closely related process impurities and to enable seamless scale-up from analytical to preparative purification.

Instrumentation

• Thermo Scientific UltiMate 3000 LC System with diode array detection at 260 nm for method development.
• Waters LC Prep AutoPurification System with 2489 UV/Vis detector at 280 nm for semi-preparative purification.
• XBridge Premier Oligonucleotide BEH C18 columns (300 Å) in analytical format (2.5 µm, 4.6 × 100 mm) and preparative format (2.5 µm and 5 µm, 10 × 100 mm).

Results and discussion

Analytical screening compared triethylammonium acetate (TEAA) and hexylammonium acetate (HAA) as ion-pairing reagents. HAA demonstrated stronger hydrophobic interactions, improving separation of the full-length oligonucleotide from truncated fragments. On the 2.5 µm analytical column, HAA provided superior resolution guiding its selection for preparative scale.

In preparative trials, the 2.5 µm, 10 × 100 mm column at 4 mL/min achieved 70 % purity with a 4 mg yield at ~3000 psi, while the 5 µm format at 8 mL/min delivered a 62 % purity with a 5 mg yield under similar backpressure. The 5 µm column offers compatibility with lower-pressure systems, illustrating a practical trade-off between purity and hardware flexibility.

Benefits and practical applications

• Batch-tested XBridge Premier columns deliver predictable, low secondary interaction separations.
• MaxPeak HPS technology minimizes nonspecific adsorption, improving recovery of early-eluting components.
• Wide 300 Å pore size enhances mass transfer for long oligonucleotides, reducing peak broadening.
• Scalable method design ensures direct translation from analytical evaluation to preparative purification.

Future trends and perspectives

Next-generation chromatographic materials and novel ion-pairing chemistries may further enhance resolution of increasingly longer or heavily modified oligonucleotides. Automated gradient optimization and greener solvent systems will streamline method development. Additionally, integration with real-time detection and process analytical technologies could accelerate purification workflows.

Conclusion

A systematic approach combining MaxPeak HPS-protected hardware, optimized ion-pairing with HAA, and a wide-pore BEH C18 stationary phase enabled high-resolution preparative purification of a 60-mer oligonucleotide. The study underscores the importance of informed reagent and column selection to balance purity, yield, and system compatibility.

References

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