End-To-End Spectroscopy-Based Workflow Solutions for Oligonucleotide Analysis

Significance of the Topic

Synthetic oligonucleotides, including siRNA, antisense oligonucleotides, aptamers and CRISPR guides, play a growing role in research and genetic testing. Reliable characterization of their mass, sequence, purity and impurity levels is essential for both discovery research and production quality control. Compliance with regulatory guidelines for raw materials, residual solvents and elemental impurities further underscores the need for robust analytical workflows.

Study Objectives and Overview

This guide presents end-to-end spectroscopy-based workflows for oligonucleotide analysis, covering raw material identification, final product confirmation, trace elemental impurity profiling and residual solvent determination. By integrating Raman, FTIR, HPLC/UV-Vis, ICP-MS and GC/GC-MS techniques, the proposed solutions support research, development and cGMP production environments.

Methodology and Instrumentation

The workflows are structured as follows:

• Workflow 1: Raw material identification using Agilent Vaya handheld Raman in warehouse settings or Agilent Cary 630 FTIR and 1290 Infinity II Bio LC with OpenLab ChemStation in the lab.
• Workflow 2: Finished product confirmation via UV-Vis using the Cary 3500 Multizone spectrophotometer and Cary UV Workstation software for concentration and melting temperature analysis.
• Workflow 3: Trace elemental impurity analysis by ICP-MS employing I-AS/SPS4 autosamplers, USP/ICH Q3D kits, the 7850 or 7900 ICP-MS systems and MassHunter software.
• Workflow 4: Residual solvent analysis by GC or GC/MS using the 8890 GC with headspace 8697 sampler or Intuvo 9000 system and 5977C MSD, controlled by OpenLab CDS.

Main Results and Discussion

• Raman and FTIR methods enable rapid, non-destructive identification of raw oligonucleotide starting materials even through transparent and opaque containers.
• HPLC/UV-Vis provides sensitive separation and quantification of impurities and raw material confirmation with automated data handling.
• UV-Vis thermal melt analysis accurately determines nucleic acid concentration and melting temperature, facilitating base composition assessment.
• ICP-MS workflows achieve low detection limits and high robustness for multiple elemental impurities, meeting USP <232>/<233> and ICH Q3D requirements.
• GC and GC/MS residual solvent analysis complies with USP <467>, offering routine, high-throughput or unknown screening options with reliable separation and detection.

Benefits and Practical Applications of the Method

• Comprehensive coverage of all stages from raw material verification to final product QC.
• Modular workflows suitable for warehouse screening, research laboratories and cGMP manufacturing.
• Secure data integrity, user management and compliance with regulatory guidances.
• High throughput and automation reduce time to result and minimize manual intervention.

Future Trends and Opportunities for Use

Analytical demands for oligonucleotides will continue to evolve with new modalities and stricter regulatory requirements. Future trends include integration of machine learning for spectral interpretation, real-time monitoring in manufacturing, miniaturized and portable devices, and expanded multi-attribute methods combining mass spectrometry and chromatography. These advances will further enhance speed, sensitivity and data-driven decision making.

Conclusion

The spectroscopy-based workflows detailed in this guide deliver robust, end-to-end solutions for oligonucleotide analysis. By combining complementary detection techniques with secure data handling, these methods support reliable material identification, impurity profiling and regulatory compliance across research and production settings.

References

• Neo A, Welsby C. Rapid Testing of Solvents Through Amber Bottles Using an Agilent Vaya Handheld Raman Spectrometer. Agilent Technologies application note 5994-5929EN, 2023.
• Alwan W, Zieschang F. The Agilent Cary 630 FTIR Spectrometer for Material Identification Applications. Agilent Technologies application note 5994-4992EN, 2022.
• Best Practice for Nucleic Acid Thermal Stability Measurements Using the Cary 3500 UV-Vis Spectrophotometer. Agilent Technologies white paper 5994-4028EN, 2022.
• Sanderson J. Residual Solvent Analysis in Hemp Consumer Products Using Headspace Gas Chromatography and Mass Spectrometry. Agilent Technologies application note 5994-5237EN, 2022.
• Analysis of Artificial Tear Eye Drops for Elemental Impurities. Agilent Technologies application note 5994-1561EN, 2022.
• Zhang Y, Na S. Analysis of USP <467> Residual Solvents of Class 1, 2 and 3 Using the Agilent 8890 GC/FID/5977B MSD System. Agilent Technologies application note 5994-1488EN, 2019.