Driving the Field of Oligonucleotide Therapeutics

Importance of the Topic

Oligonucleotide therapeutics represent a breakthrough modality for precise gene regulation, offering potential treatments for rare and common diseases across multiple therapeutic areas. Their development relies on robust analytical and preparative methods to ensure sequence fidelity, purity, and safety for downstream modifications, delivery, and clinical application.

Objectives and Overview

This text reviews the challenges and solutions in oligonucleotide development, covering raw material assessment, synthesis impurity control, sequence confirmation, purity analysis, and scale-up from analytical characterization to preparative manufacturing under cGMP conditions.

Methodology and Instrumentation

The eBook describes a multi-platform analytical pipeline:

• Raw material identification via Raman and FTIR spectroscopy, and HPLC/UHPLC.
• Purity and product-related impurity analysis by ion-pair reversed-phase and anion-exchange LC coupled with UV and MS detection.
• Sequence and ID confirmation using high-resolution LC-Q-TOF/MS, LC/MS/MS deconvolution software, and UV/Vis melting point analysis.
• Process-related impurity testing: residual solvents by GC/GC-MS and elemental impurities by ICP-MS.
• Orthogonal methods, including capillary electrophoresis, to resolve closely related species.
• Preparative LC scale-up guided by column scaling calculators and matched column chemistries.

Instrumentation Used

• Handheld and bench FTIR/Raman spectrometers for non-destructive raw material ID.
• Agilent 1290 Infinity II Bio UHPLC and 6230B TOF LC/MS for purity and impurity profiling.
• 6545XT AdvanceBio LC/Q-TOF for high-resolution sequence confirmation.
• Cary 3500 UV/Vis spectrophotometer for melting temperature analysis.
• 8890/Intuvo GC with headspace sampling and 5977 GC/MSD for residual solvent testing.
• Agilent 7850 and 7900 ICP-MS instruments for trace elemental impurity quantification.
• Analytical to preparative LC systems (1260 Infinity II, 1290 Infinity II Bio, 1290 InfinityLab LC/MSD XT) and specialized columns (AdvanceBio Oligonucleotide, PLRP-S, PL-SAX, Bio-SAX).

Main Results and Discussion

Integrated workflows successfully detect and quantify synthesis by-products (shortmers, longmers, depurinated species), confirm full-length sequences, and ensure compatibility of analytical methods with preparative scale-up. Implementation of orthogonal techniques enhances impurity detection and sequence verification.

Benefits and Practical Applications

These solutions streamline method development, reduce manual calculations, and ensure compliance with emerging regulatory frameworks (ICH Q3C, Q3D) by providing end-to-end analytical and preparative capabilities. They support QC/QA for clinical and commercial oligonucleotide API production.

Future Trends and Applications

Continued growth in oligo therapeutics—including siRNA, ASO, mRNA, and CRISPR-based modalities—will drive demand for higher-throughput, more sensitive analytical platforms and scalable purification methods. Advances in lipid nanoparticle and ligand conjugation will require specialized characterization workflows, while evolving regulatory guidance will standardize impurity thresholds.

Conclusion

A comprehensive suite of Agilent technologies addresses key challenges in oligonucleotide analytics and manufacturing, enabling reliable raw material assessment, rigorous purity and sequence confirmation, and efficient scale-up to cGMP API production. These integrated workflows accelerate therapeutic discovery and support quality control across the pipeline.

References

1. Roberts TC, Langer R, Wood MJA. Advances in oligonucleotide drug delivery. Nat Rev Drug Discov. 2020;19:673–694.
2. Crooke ST. Molecular mechanisms of antisense oligonucleotides. Nucleic Acid Ther. 2017;27(2):70–77.
3. Egli M, Manoharan M. Chemistry, structure, and function of approved oligonucleotide therapeutics. Nucleic Acids Res. 2023;19(6):2529–2573.
4. ICH Q3C(R6) Guidelines for Residual Solvents. 2016.
5. ICH Q3D(R1) Guideline for Elemental Impurities. 2019.