Semi-preparative reversed-phase liquid chromatographic purification of oligonucleotides

Significance of the topic

Oligonucleotides have become essential tools and therapeutics in modern biotechnology and diagnostics, with applications ranging from qPCR assays to forensic human identification and gene transfer studies. High-purity oligonucleotides are critical for reliable analytical results and therapeutic safety. Reversed-phase high-performance liquid chromatography (RP-HPLC) remains the preferred method for purifying synthetic oligonucleotides, eliminating truncated sequences and other synthesis byproducts.

Goals and overview of the study

This application note outlines a semi-preparative RP-LC purification workflow for three dual-labeled 25-mer oligonucleotides using the Thermo Scientific™ Vanquish™ Analytical Purification LC system. The objectives are to demonstrate:

• Integration of the Vanquish LC platform, including the Vanquish Fraction Collector, into a high-throughput production environment.
• Efficient peak-based fraction collection and re-analysis of purified fractions on the same LC system.
• Flexibility of the Vanquish UHPLC and HPLC systems to perform both analytical and semi-preparative purifications.

Methodology

Synthetic oligonucleotides ([CY5]-BHQ2, [AT647]-BHQ2, [FAM]-BHQ1, each 1 µmol) were dissolved to 400 µM in deionized water. A semi-preparative C18 column (130 Å, 5 µm, 10 mm × 100 mm) was employed with a binary gradient of 50 mM triethylammonium acetate (TEAA), pH 7, and acetonitrile over 40 minutes at 5.0 mL/min and 60 °C. Fractions were collected based on UV detection at 260 nm using individualized peak detection thresholds and slopes to ensure precise cutting of the main product peaks, including combined treatment of AT647 isomers as a single product.

Instrumentation used

• Vanquish Flex Analytical Purification LC system (System Base Horizon/Flex, Binary Pump F, Split Sampler FT, Column Compartment H with pre-heater, Diode Array Detector FG)
• Integral Vanquish Fraction Collector with delay capillary and flush buffer loop
• Thermo Scientific Chromeleon™ Chromatography Data System version 7.3.1

Main results and discussion

Initial purities of the three oligonucleotides ranged from 89% to 93%, with multiple baseline impurities observed. After semi-preparative RP-LC fractionation and re-analysis:

• All UV‐detectable impurities were effectively removed, yielding 100% relative purity in the collected fractions.
• The Vanquish fluidics minimized band broadening, enabling high-resolution separation and accurate fraction cuts.
  
• Automated delay volume determination and precise needle positioning (30 mm height, 2 mm puncture offset) ensured reproducible, low-dispersion collection in deep-well plates.
• External needle wash and internal rinse prevented carry-over between runs, facilitating unattended multi-sample purification.

Benefits and practical applications

• High-throughput purification of custom oligonucleotides with minimal manual intervention.
• Dual use of the Vanquish system for both analytical QC and semi-preparative production.
• Robust fraction collection enabling isolation of closely eluting species, including dye isomers when required.
• Enhanced lab productivity and reproducibility in pharmaceutical and diagnostic oligonucleotide manufacturing.

Future trends and possibilities

Advances in oligonucleotide purification may include:

• Integration of real-time mass spectrometric detection for fraction guidance.
• Automated method development using machine learning to optimize gradients and collection windows.
• Scale-up to preparative workloads and continuous-flow purification systems.
• Application to longer RNA therapeutics and CRISPR guide RNAs requiring stringent purity.

Conclusion

The Thermo Scientific™ Vanquish™ Analytical Purification LC system with integrated fraction collection delivers a robust, high-resolution semi-preparative RP-LC workflow for oligonucleotide purification. The seamless analytical-to-preparative transition, precise fluidics, and customizable fraction collection parameters ensure high-purity products suitable for demanding pharmaceutical and diagnostic applications.

References

1. Roberts T, Langer R, Wood M. Advances in oligonucleotide drug delivery. Nat Rev Drug Discov. 2020;19:673–694.
2. Zhang Q, Lv H, W. Recent Methods for Purification and Structure Determination of Oligonucleotides. Int J Mol Sci. 2016;17(12):2134.
3. Catani M, et al. Oligonucleotides: Current Trends and Innovative Applications in the Synthesis, Characterization, and Purification. Biotechnol J. 2020;15(8):1900226.
4. Kanwal F, et al. Large-Scale in Vitro Transcription, RNA Purification and Chemical Probing Analysis. Cell Physiol Biochem. 2018:1915–1927.
5. Cramer H, Finn K, Herzberg E. Purity analysis and impurities determination by RP-HPLC. CRC Press; 2011.
6. Thermo Fisher Scientific. Technical Note 72940: Principles of fraction collection using the Vanquish HPLC and UHPLC systems; 2022.