High-throughput Screening of Oligonucleotides for Identity and Purity Assessment Using the ACQUITY QDa Detector and ProMass for MassLynx

Significance of the Topic

The development of therapeutic oligonucleotides has created a growing need for rapid, reliable assays to confirm product identity and purity. Mass spectrometry detection added to ion-pairing reversed-phase UPLC workflows offers orthogonal confirmation of molecular weight, critical for quality control in synthetic oligonucleotide production.

Aims and Scope of the Study

This study evaluates the combination of the ACQUITY QDa detector and ProMass software within the MassLynx environment to provide automated, high-throughput screening of synthetic oligonucleotides. The work demonstrates mass accuracy, deconvolution performance, and batch processing capability for identity and purity assessments.

Methodology and Instrumentation

• Chromatography: ACQUITY UPLC H-Class system with BEH C18 column (1.7 μm, 2.1×50 mm), TEA/HFIP buffered mobile phases, gradient methods for polyT standards and high-throughput screening.
• Detection: ACQUITY UPLC TUV (260 nm) and ACQUITY QDa detector operating in negative‐ESI mode (mass range 410–1250 Da).
• Software: MassLynx SCN 9.25 with MaxEnt1 for deconvolution and ProMass HR (ZNova algorithm) for automated batch processing; ProMassBridge interface for raw data integration.
• Samples: PolyT standards (15–35 nt) and a 21-mer siRNA sequence with N-1 and N+1 variants, prepared at 10 pmol/μL, 5 μL injections.

Main Results and Discussion

Deconvolution of polyT standards yielded mass accuracy between –0.1 Da and +0.5 Da across a triplicate. A high-resolution separation of the siRNA strand resolved the N-1 and N+1 species in under ten minutes, with ProMass reporting mass errors of +0.1 Da to +0.4 Da. Batch processing of a 48-well plate using ProMassBridge enabled automated identification of target sequence wells, with interactive web-based summaries showing color-coded pass/fail results, spectra, and chromatograms.

Benefits and Practical Applications

• Automated deconvolution and reporting reduce manual data processing time and increase throughput.
• Integration of mass detection into established LC workflows enhances confidence in oligonucleotide identity and purity.
• Interactive browser-based results facilitate rapid review of large sample sets.

Future Trends and Applications

Advances may include broader application to diverse biotherapeutic modalities, enhanced deconvolution algorithms, and integration with real-time process analytics. Machine learning-driven data interpretation could further accelerate screening and decision-making in oligonucleotide development and manufacturing.

Conclusion

The ACQUITY QDa detector, combined with ProMass software, delivers a cost-effective, high-throughput solution for identity and purity assessment of synthetic oligonucleotides. This workflow streamlines QC processes, offering reliable mass confirmation and automated data analysis within existing UPLC platforms.

References

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