Expediting Method Development for Oligonucleotide Impurity Analysis Using the ACQUITY™ QDa™ II Mass Detector

Importance of the Topic

Therapeutic oligonucleotides have become crucial in modern biopharma, requiring precise and rapid impurity profiling to ensure patient safety and drug efficacy. Traditional LC-UV detection often lacks the mass specificity needed to resolve closely related sequence variants, such as n-1 and n-2 species. Integrating compact mass spectrometry and advanced solvent blending markedly accelerates method development while enhancing confidence in impurity identification.

Objectives and Study Overview

This study demonstrates an expedited workflow for impurity analysis of 25-mer oligonucleotides by combining the Waters ACQUITY Premier QSM LC system with quaternary solvent blending and the ACQUITY QDa II Mass Detector. Two full-length products—one with a phosphodiester backbone (FLP-PO) and one with a phosphorothioate backbone (FLP-PS)—were assessed alongside their n-1 shortmer impurities.

Methodology

• Sample preparation: FLP-PO and FLP-PS at 0.1 mg/mL in water, spiked with 10% (w/w) n-1 shortmers.
• Chromatography: Ion-pair reversed-phase LC using alkylamine/HFIP mobile phases, leveraging quaternary blending of water, organic solvent (ACN/MeOH 50/50), and concentrated ion-pairing stocks.
• Gradient screening: Organic and ion-pairing concentrations were varied by adjusting reservoir ratios to optimize resolution and MS response.
• Detection: UV at 260 nm and negative electrospray MS, full scan (350–1500 m/z) plus selected ion recording (SIR) targeting lower charge states (e.g., [M-6H]⁻⁶).

Used Instrumentation

• Waters ACQUITY Premier QSM LC System with quaternary solvent blending
• ACQUITY Premier Oligonucleotide BEH C18 Column (130 Å, 2.5 µm, 4.6×150 mm)
• ACQUITY QDa II Mass Detector
• ACQUITY UPLC TUV Detector (260 nm)
• Empower 3.8.1 chromatography software

Main Results and Discussion

• FLP-PO Optimization: Screening DBA/HFIP concentrations from 1 to 10 mM revealed 2.5 mM DBA as optimal, achieving baseline resolution (USP > 1.5) while preserving MS signal.
• Mass Load Robustness: Peak areas scaled linearly with injected mass, and resolution remained stable across increased loads, confirming method reproducibility.
• Impurity Profiling (FLP-PO): SIR quantitation measured 4.10% total n-1 impurities, closely matching UV integration (4.19%). Full-scan MS identified the n-C shortmer as the predominant impurity.
• FLP-PS Optimization: TEA/HFIP at 15 mM provided an optimal balance of PO and PS impurity separation and MS sensitivity. An 8–11% organic gradient yielded efficient analysis time and peak resolution.
• Impurity Profiling (FLP-PS): UV suggested 14.56% impurity; SIR deconvolution revealed 11.15% oxidized PO impurity and 2.55% n-C shortmer, highlighting co-elution masked by UV alone.

Benefits and Practical Application of the Method

• Seamless UV-MS integration accelerates method development by providing real-time mass confirmation of chromatographic peaks.
• Quaternary solvent blending reduces downtime and simplifies ion-pairing optimization.
• The compact QDa II detector delivers sufficient mass range and sensitivity for routine QC without the complexity of full-scale MS systems.

Future Trends and Opportunities

Analytical demands for larger and more complex oligonucleotides will continue to rise. Future advancements may include automated real-time feedback loops for ion-pair optimization, expanded mass range detectors, and advanced data analytics to further reduce development timelines and enhance impurity characterization.

Conclusion

The integrated workflow of quaternary solvent blending on an ACQUITY Premier system combined with inline mass detection via the QDa II detector significantly streamlines oligonucleotide impurity method development. It delivers robust, reproducible separations and mass-based confirmation of closely related species, effectively meeting stringent QC and regulatory requirements.

References

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