ANALYSIS OF OLIGONUCLEOTIDE IMPURITIES ON THE BIOACCORD LC-MS SYSTEM WITH ACQUITY PREMIER

Significance of the Topic

The growing prominence of oligonucleotide therapeutics necessitates highly selective and sensitive analytical methods for impurity profiling and mass confirmation. Conventional reversed-phase LC–MS methods often suffer from analyte losses and poor reproducibility due to interactions between the negatively charged phosphate backbone and metal surfaces in the fluidic path. Mitigating these surface interactions is essential to achieve reliable quantitation and accurate mass measurements of modified oligonucleotides and their low-level impurities.

Objectives and Study Overview

This study evaluates a novel bio-inert chromatographic platform combining MaxPeak High Performance Surface (HPS) technology across the entire fluidic path with an Optimally Surface Treated (OST) column (ACQUITY Premier OST). A heavily modified 21-mer oligonucleotide and its impurities were analyzed to demonstrate:

• The recovery and resolution of low-abundance impurities.
• Linearity and sensitivity across a broad concentration range.
• Comparative performance versus a standard OST column.

Methodology

Ion-pair reversed-phase LC–MS was performed under the following conditions:

• Mobile phases: A = 7 mM triethylamine (TEA)/40 mM HFIP in water; B = 3.5 mM TEA/20 mM HFIP in 50% methanol.
• Gradient: 25–35% B over 25 min, total run time 40 min; flow rate 300 µL/min; column temperature 60 °C.
• Columns: 2.1 × 100 mm Premier OST (P/N 186009485) versus regular OST (P/N 186003950), both packed with 1.7 µm BEH C18.
• MS detection: negative-mode ESI, m/z 400–5000, 2 Hz scan rate, capillary voltage 0.8 kV, cone voltage 40 V, source 120 °C, desolvation 400 °C.
• Data processing: waters_connect software with BayesSpray deconvolution for intact mass determination.

Instrumentation

The following components were employed:

• Waters BioAccord LC–MS system.
• ACQUITY Premier UPLC with HPS-treated fluidics.
• ACQUITY Premier OST and standard OST C18 columns (2.1 × 100 mm, 1.7 µm, 130 Å).

Main Results and Discussion

The bio-inert Premier platform demonstrated superior performance:

• All 14 oligonucleotide impurities were resolved using the Premier OST column versus only seven impurities on the regular OST column, attributed to reduced adsorption at inlet/outlet frits.
• Calibration over 0.5 to 1000 nM exhibited excellent linearity (R2 = 0.9999) and detection down to 0.5 nM (5 fmol on-column).
• Carryover was negligible, with no detectable signal in blank runs following high-load injections.
• Mass accuracy for all components was better than 15 ppm, allowing confident intact mass confirmation of both major and trace impurities.
• UV peak area RSDs remained below 15% across replicates, ensuring reproducible quantitation.

Benefits and Practical Applications

This advanced LC–MS workflow offers:

• Enhanced impurity recovery and chromatographic peak shape due to minimized metal surface interactions.
• High sensitivity and broad dynamic range for low-level impurity detection.
• Robust mass confirmation for modified oligonucleotides in regulated and non-regulated environments.
• Automated data processing for streamlined QC and compliance readiness.

Future Trends and Opportunities

Anticipated developments include:

• Further integration of high-performance surface treatments throughout analytical systems.
• Automation enhancements enabling real-time impurity monitoring in manufacturing.
• Expansion to longer and more complex oligonucleotides and conjugated modalities.
• Advanced informatics pipelines leveraging AI for rapid spectral deconvolution and impurity identification.

Conclusion

The BioAccord LC–MS platform with ACQUITY Premier UPLC and Premier OST column provides a bio-inert system that overcomes traditional metal interaction issues. It delivers comprehensive impurity profiling, high sensitivity, reproducible quantitation, and accurate mass confirmation of modified oligonucleotides, supporting rigorous quality control in therapeutic development.

References

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3. Sharma VK, Watts JK. Future Med Chem. 2015;7(16):2221–2242.