Instrument and LC column migration for the purification and analysis of synthetic oligonucleotides

Significance of the Topic

The purification and analysis of synthetic oligonucleotides are essential for applications in molecular diagnostics, therapeutics, and biochemical research. Impurities arising during multi-step solid-phase synthesis, such as truncated sequences and dye degradation, necessitate high-resolution purification and rigorous purity assessment to ensure performance in downstream assays.

Aim and Study Overview

This study demonstrates the complete transfer of a semi-preparative reversed-phase HPLC purification and analytical HPLC-UV QC workflow from a third-party system (Agilent 1260 Infinity II with Daisogel columns) to the Thermo Scientific Vanquish LC platform using Hypersil GOLD columns. The goal includes method consistency under Chromeleon CDS, comparable purity, yield, and identity confirmation by LC-HRAM-MS.

Methodology and Instrumentation

Sample preparation involved dissolving dual-labeled oligonucleotides in TEAA buffer (Solvent A) and MeCN/MeOH (Solvent B), followed by semi-preparative purification. The purification gradient on both Daisogel SP-100 ODS-P and Hypersil GOLD C18 prep columns employed a stepped aqueous–organic profile at 4.5 mL/min and 50 °C. Collected fractions were analyzed by LC-UV in two formats—Waters ACQUITY UPLC BEH C18 and Hypersil GOLD analytical columns—and quantified by UV-Vis. Identity confirmation used Vanquish HPLC-HRAM Orbitrap MS with Xtract deconvolution.

Used Instrumentation

• Thermo Scientific Vanquish Analytical Purification LC System (Binary Pump, Split Sampler, Column Compartment, Diode Array Detector, Fraction Collector)
• Thermo Scientific Hypersil GOLD Prep and Analytical C18 Columns
• Agilent 1260 Infinity II Preparative LC System with Daisogel SP-100 ODS-P column
• Vanquish Flex UHPLC–Orbitrap High-Resolution Accurate-Mass MS
• Savant SpeedVac SPD1010 Concentrator
• SpectraMax 384+ UV-Vis Spectrophotometer

Main Results and Discussion

Instrument-to-instrument transfer yielded comparable purities (98.4–99.9%) and yields (45–48 nmol) for ABY-MGB and JUN-MGB oligonucleotides. Hypersil GOLD prep columns provided sharper peaks, reduced co-eluting shoulders, and smaller fraction volumes versus Daisogel. Analytical QC on Hypersil GOLD matched performance of Waters ACQUITY, confirming equivalent purity assessment. Solvent evaporation for shipping led to minor degradation, highlighting the value of direct integration.

Benefits and Practical Applications

• Seamless migration of oligonucleotide workflows to a single LC–MS platform
• Improved resolution and reduced solvent consumption with Hypersil GOLD columns
• Consistent purity and yield across instruments and columns
• Centralized data management via Chromeleon CDS

Future Trends and Possibilities for Use

Advancements may include online fraction evaporation, real-time LC–MS QC integration, automated method optimization, and expansion to longer or modified oligonucleotides. Novel stationary phases and microfluidic LC promise higher throughput and lower sample requirements.

Conclusion

The Thermo Fisher Vanquish LC platform, combined with Hypersil GOLD columns and Chromeleon CDS, enables robust method transfer for semi-preparative purification and analytical QC of synthetic oligonucleotides. Comparable purity, yield, and identity confirmation validate this unified workflow.

Reference

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5. Cramer J. et al. Purity analysis by RP-HPLC. In Handbook of Analysis of Oligonucleotides; CRC Press; 2011:1–46.