Method transfer for the purification of synthetic oligonucleotides

Importance of the Topic

Effective purification of synthetic oligonucleotides is essential for applications such as qPCR, forensic DNA analysis and gene therapy development.
Reliable method transfer between HPLC platforms streamlines workflow development, reduces validation time, and ensures consistent product purity.

Study Aims and Overview

This work aimed to transfer a complete purification and quality control workflow for dual-labelled 15mer oligonucleotides from a competitor HPLC system to a Thermo Fisher Scientific solution.
The study covered peak-based RP-HPLC purification, LC-UV QC and high-resolution MS confirmation under Thermo Scientific Chromeleon CDS control.

Methodology and Instrumentation

The purification employed TEAA-based reversed-phase gradients, isolating target oligonucleotide peaks.
Quality control used LC-UV on a Vanquish Flex UHPLC system and exact mass identification via Orbitrap Exploris 480.

Used Instrumentation

• Agilent 1260 Infinity II Analytical-Scale LC Purification System
• Thermo Scientific Vanquish Analytical and Semi-Preparatory LC Systems
• Vanquish Flex UHPLC for LC-UV QC
• Orbitrap Exploris 480 Mass Spectrometer
• Hypersil GOLD C18 Prep (P/N 25005-159070A) and Analytical (P/N 25002-052130) Columns

Key Results and Discussion

• Hypersil GOLD prep column matched Daisogel performance in purity and yield while reducing fraction volumes.
• Hypersil GOLD analytical column produced LC-UV profiles comparable to Waters ACQUITY BEH C18 benchmarks.
• Vanquish purification yields and LC-UV QC metrics were equivalent to those obtained on the Agilent 1260 system.
• Mass spectra confirmed the expected monoisotopic masses (±0.2 Da) with no detectable critical impurities.

Benefits and Practical Applications of the Method

• Seamless instrument and column vendor switching without extensive revalidation.
• Reduced solvent consumption and faster downstream processing due to optimized fraction volumes.
• High-throughput compatibility supports diagnostic assay development and quality assurance processes.

Future Trends and Applications

Enhanced automation and integration with chromatography data systems are expected to further accelerate method development.
Miniaturized and microflow purification platforms coupled with real-time mass detection may enable in-line monitoring and rapid decision making.
Expansion to longer and chemically modified oligonucleotides will broaden the method’s relevance in therapeutic oligo production.

Conclusion

The successful transfer of oligonucleotide purification and QC workflows from a non-Thermo Fisher platform to Thermo Fisher instrumentation demonstrates robust method versatility and performance equivalence, facilitating flexible deployment in diverse analytical laboratories.

References

1. Roberts et al. Advances in Oligonucleotide Drug Delivery. Nature Reviews Drug Discovery, 19, 673–694 (2020)
2. Zhang et al. Recent Methods for Purification and Structure Determination of Oligonucleotides. International Journal of Molecular Sciences, 17(12), 2134 (2016)