Increasing the Productivity of Oligonucleotide Purification through Column Scaling and Method Optimization

Importance of the topic

A reliable, high-throughput purification strategy for synthetic oligonucleotides is critical to support research, discovery, and late-stage development of genetic therapeutics. Efficient, scalable methods reduce cost and time, minimize exposure to hazardous solvents, and supply sufficient material for biological assays and quality control.

Objectives and overview of the study

This work aimed to develop a cost-effective, HFIP-free ion-pair reversed-phase purification workflow that scales seamlessly from analytical UPLC to preparative HPLC. Key goals included mobile phase optimization, column diameter scaling, method parameter tuning, and evaluation of purity, recovery, and productivity for a 20-mer oligonucleotide.

Methodology and instrumentation used

Sample: Crude 20-mer (5’-GCCTCAGTCTGCTTCCACCT-3’) reconstituted in 10 mM NH4OAc.
Analytical separation: Waters ACQUITY UPLC Oligonucleotide BEH C18, 1.7 µm, 2.1×100 mm at 60 °C using 8.6 mM triethylamine/100 mM HFIP–water (A) and MeOH (B); alternative method with 100 mM TEAA pH 7.0–water (A) and ACN (B) on XBridge Oligonucleotide BEH C18, 2.5 µm, 4.6×50 mm at 25 °C.
Preparative purification: XBridge Oligonucleotide BEH C18 OBD Prep, 2.5 µm, 30×50 mm; 100 mM TEAA pH 7.0–ACN gradient; flow rate 25 mL/min; fraction collection via Waters 3767 Sample Manager, Autopurification System (SFO, PDA/TUV, SQD2 MS) and ACQUITY ISM pump.

Main results and discussion

The TEA/HFIP analytical method produced ~90% purity at 60 °C. Switching to a TEAA–ACN system at ambient temperature simplified scale-up and eliminated HFIP hazards while preserving resolution of n-1 impurities. Scaling to the 30 mm preparative column at a 410 µg injection achieved 96.3% pooled purity, 85% recovery, and ~1 mg/hour throughput. Increasing load to 25 mg broadened peaks but improved productivity; optimizing gradient focus and increasing flow rates during clean-up boosted throughput to ~75–100 mg/hour.

Benefits and practical applications of the method

• Eliminates costly and hazardous HFIP, enhancing lab safety and lowering consumable expense.
• Maintains high chromatographic performance when scaling from analytical to preparative columns.
• Delivers >95% purity, >85% recovery, and up to 100 mg/hour productivity with simple gradient and flow adjustments.

Future trends and potential uses

Adoption of wider bore columns (e.g., 50 mm ID) can further raise load capacity and throughput. Offline column regeneration and MS-triggered fraction collection promise additional efficiency gains. Continued development of ion-pair reagents and high-strength packing materials will support emerging modalities in nucleic acid therapeutics.

Conclusion

A robust, HFIP-free TEAA ion-pair reversed-phase method was successfully transferred from UPLC to widebore HPLC, achieving high purity, recovery, and productivity. The approach provides a practical path for lab-scale oligonucleotide manufacturing and can be further enhanced by hardware and method refinements.

References

1. McDonald PD et al., Optimum Bed Density columns: Enabling Technology for Laboratory-Scale Isolation and Purification, Waters Corporation.
2. Gilar M et al., Best Practices for Oligonucleotide Analysis Using Ion-Pair Reversed-Phase Liquid Chromatography, Waters Corporation.
3. Gilar M et al., HPLC and UPLC Columns for Analysis of Oligonucleotides, Waters Corporation.
4. Lefebvre P et al., Evaluating the Tools for Improving Purification Productivity, Waters Application Note, 2007.