Analysis of Impurities in Oligonucleotide Therapeutics

Significance of the Topic

Oligonucleotide therapeutics have emerged as versatile agents for modulating gene expression and treating genetic disorders. Ensuring the purity of these compounds is critical due to potential safety concerns related to sequence-specific and truncated impurities. Robust analytical methods are required for quality control and characterization during drug development and production.

Objectives and Study Overview

This application note describes a high-performance liquid chromatography–mass spectrometry (LC–MS) workflow for the analysis of impurities in therapeutic oligonucleotides. The study aims to demonstrate reliable separation and identification of full-length products and shorter fragment impurities using a bioinert reversed-phase column combined with sensitive MS detection.

Methodology

The method employs ion-pair reversed-phase chromatography with a gradient of methanol in 100 mM hexafluoroisopropanol (HFIP) and 10 mM triethylamine (TEA). A flow rate of 0.3 mL/min and column temperature of 60 °C ensure reproducible retention and peak shape. Ultraviolet detection between 190 nm and 400 nm is used to monitor elution profiles, while online MS analysis in negative mode provides mass-based confirmation of analytes.

Used Instrumentation

• Liquid chromatograph: Nexera XS inert system
• Column: Shim-pack Scepter Claris C18-300, 100 mm × 2.1 mm I.D., 1.9 µm particle size
• Mobile phases: A = 100 mM HFIP and 10 mM TEA in water; B = methanol; gradient from 10% to 90% B over 22 minutes, re-equilibration to 10% B by 26 minutes
• Detection: Photodiode array (PDA) at 190–400 nm
• Mass spectrometer: LCMS-2050 with ESI/APCI in negative ion mode; scan range m/z 550–2000; nebulizing gas 3.0 L/min (N₂); drying gas 5.0 L/min (N₂); heating gas 7.0 L/min (N₂); DL temperature 200 °C; desolvation temperature 400 °C; interface voltage –3.0 kV

Main Results and Discussion

The bioinert C18 column effectively minimized nonspecific adsorption of oligonucleotides, yielding sharp and symmetrical peaks. The ion-pairing mobile phase facilitated efficient retention and elution of both the full-length product (FLP) and truncated impurities (n–1, n–3, n–10). MS data enabled direct confirmation of molecular weights, allowing unambiguous identification of impurities present at low levels. The combined UV and MS approach provided a comprehensive impurity profile critical for purity assessment.

Benefits and Practical Applications

• High sensitivity and selectivity for detecting sequence variants and truncated fragments
• Bioinert column chemistry improves reproducibility by reducing analyte adsorption
• Integrated UV and MS detection supports both quantitative and qualitative analysis
• Suitable for routine quality control in oligonucleotide drug development and manufacturing

Future Trends and Potential Applications

Advancements in bioinert stationary phases and tailored ion-pair reagents are expected to further enhance separation efficiency and MS compatibility. Emerging high-resolution mass spectrometers and novel fragmentation techniques will improve structural characterization of complex oligonucleotides. Automation and miniaturization of LC–MS workflows may accelerate high-throughput screening and in-process monitoring.

Conclusion

The described LC–MS method using a Shim-pack Scepter Claris C18-300 column demonstrates robust performance for impurity profiling in oligonucleotide therapeutics. The optimized chromatographic conditions and sensitive MS detection provide a reliable platform for quality control and regulatory compliance.