AdvanceBio Your Oligonucleotide Workflow

Significance of the Topic

Synthetic oligonucleotides—including aptamers, guide RNAs, siRNAs, and antisense oligos—are increasingly central to life-science research and therapeutic development. However, effective monitoring, identification, and removal of impurities from incomplete coupling, side reactions, and post-synthesis processing remain major challenges. Robust analytical and purification workflows are critical to ensure high quality and yield of target sequences.

Objectives and Study Overview

This document presents the Agilent AdvanceBio Oligonucleotide workflow, a fully integrated solution for analytical characterization and preparative purification of DNA and RNA oligonucleotides. It covers scalable column chemistries, mobile-phase strategies, software integration, and instrumentation enabling efficient method transfer and reduced downtime.

Methods and Instrumentation

• Instrumentation
  
  • Agilent 1290 Infinity II UHPLC System
  • Agilent 6530 LC/Q-TOF Mass Spectrometer
  • Agilent MassHunter BioConfirm 12.0 software for automated sequence confirmation
• Stationary Phase Details
  
  • AdvanceBio Oligonucleotide superficially porous columns
  • Particle sizes: 2.7 µm (analytical) and 4 µm (scalar/preparative)
  • pH stability: 3–11; temperature up to 65 °C; pore size 120 Å; pressure tolerance 600 bar
  • Internal diameters available: 2.1 mm, 4.6 mm, and 21.2 mm
• Mobile-Phase Composition
  
  • Analytical: 15 mM triethylamine (TEA)/400 mM hexafluoroisopropanol (HFIP) in water with methanol gradient (10–40% B over 10 min)
  • Preparative: Dibutylamine (DBA)/TRIS alternative to reduce HFIP usage, compatible with mass-directed fraction collection

Main Results and Discussion

• Sub-two-micron performance achieved high separation efficiency at reduced backpressure compared to fully porous particles.
• High-resolution LC/MS and UV separation of DNA ladder (15–40 mer) and RNA standards (14–21 mer) demonstrated clear baseline resolution.
• MassHunter BioConfirm 12.0 enabled automated target-peptide-ion (TPI) data processing, reducing analysis time and improving reproducibility.
• Preparative purification on 21.2 mm columns with DBA/TRIS mobile phase delivered rapid fraction collection, enhanced purity, and high recovery of target oligonucleotides.

Benefits and Practical Applications

• Seamless method scale-up from analytical to preparative workflows with minimal reoptimization.
• Dual compatibility with LC/MS and UV detection for comprehensive impurity profiling and purification.
• Reduced reliance on expensive HFIP through optimized ion-pairing reagent alternatives.
• Batch-qualified stationary phases ensure consistent performance across lots.

Future Trends and Opportunities

Ongoing and future advances may include:

• Further optimization of alternative ion-pairing reagents to lower costs and environmental impact.
• Integration of AI-driven method development and real-time data analysis for higher throughput.
• Expansion of column chemistries to accommodate longer, modified oligonucleotides and complex conjugates.
• Implementation of process analytical technology (PAT) for in-line monitoring in oligonucleotide manufacturing.

Conclusion

The Agilent AdvanceBio Oligonucleotide workflow combines superficially porous column technology, optimized mobile-phase strategies, and integrated software to deliver high-resolution, scalable solutions for oligonucleotide analysis and purification. This platform addresses key impurity challenges, streamlines method transfer, and enhances cost efficiency, supporting the rapid development of nucleic acid therapeutics.

References

• Vanhoenacker G, Lecluyse C, Debyser G, Sandra P, Sandra K. Evaluation of Different Ion-Pairing Reagents for LC/UV and LC/MS Analysis of Oligonucleotides. Agilent Technologies application note.
• Schipperges S, Schneider S, Huber U. Simplified Oligonucleotide Analysis and Characterization with AdvanceBio Oligonucleotide Columns and MassHunter BioConfirm 12.0. Agilent Technologies application note.
• Rieck F. Fast and Selective Purification of Oligonucleotides Using Preparative HPLC/MS and Software Support. Agilent Technologies application note.