Method transfer case study for instrument and LC column migration of the purification and analysis workflow for synthetic oligonucleotides

Význam tématu

The purification of synthetic oligonucleotides is critical for research, diagnostic assays and therapeutics. Impurities arising during synthesis, including truncated sequences and degradation byproducts, can compromise performance and safety. Achieving high purity is essential to meet stringent quality requirements in pharmaceutical and diagnostic applications.

Cíle a přehled studie / článku

This case study aimed to transfer a semi-preparative reverse-phase purification and analytical workflow for dual-labeled 15mer oligonucleotides from a third-party liquid chromatography system onto the Thermo Scientific Vanquish LC platform. The objectives included demonstrating equivalence in purity, yield and identity confirmation between existing Agilent and Waters workflows and the new Thermo Fisher Scientific solution.

Použitá metodika a instrumentace

• Preparative separation performed on a Daisogel semi-preparative column and a Hypersil GOLD C18 prep column under identical gradient and flow conditions.
• Analytical quality control via reversed-phase UHPLC using Waters ACQUITY UPLC BEH C18 and Thermo Scientific Hypersil GOLD C18 RP columns.
• Fraction collection controlled by Chromeleon CDS 7.3.2 with predefined peak detection and collection windows.
• Identity confirmation by LC-UV and high-resolution accurate mass spectrometry on the Vanquish Flex system.

Hlavní výsledky a diskuse

The transferred prep method achieved 99.7 ± 0.1 % purity for ABY-MGB and 99.9 ± 0.1 % for JUN-MGB, matching or exceeding results from the original columns. The Hypersil GOLD prep column provided sharper peak separation and reduced fraction volumes. Analytical QC demonstrated comparable purity and yield between Waters and Hypersil GOLD analytical columns, with both systems yielding over 99 % purity post-purification.

Přínosy a praktické využití metody

• Seamless migration to a unified vendor platform reduces maintenance and training burden.
• High-resolution separation enhances removal of close-eluting impurities.
• Integration with Chromeleon CDS provides streamlined data handling and reporting.
• Robust method supports consistent quality control for therapeutic oligonucleotides.

Budoucí trendy a možnosti využití

As oligonucleotide therapeutics advance, demand for higher throughput and automation will grow. Future developments may include online coupling of purification and mass spectrometry detection, use of novel stationary phases to resolve complex secondary structures, and integration with solid-phase synthesis modules for real-time process monitoring.

Závěr

The method transfer to the Thermo Scientific Vanquish LC platform demonstrated equivalent or improved purification performance, yielding high-purity oligonucleotides with robust reproducibility. Adoption of this workflow can simplify operations and support quality requirements across research and manufacturing environments.

Reference

1. Roberts P et al Advances in oligonucleotide drug delivery Nature Reviews Drug Discovery 2020;19:673–694
2. Zhang X et al Recent methods for purification and structure determination of oligonucleotides International Journal of Molecular Sciences 2016;17(12):2134
3. Catani M et al Oligonucleotides current trends and innovative applications in synthesis characterization and purification Biotechnology Journal 2020;15(8):1900226
4. Kanwal R et al Large-scale in vitro transcription RNA purification and chemical probing analysis Cellular Physiology and Biochemistry 2018;51:1915–1927