Purity, Assay, and Impurity Profiling of Single-Stranded Oligonucleotides Using Agilent Oligo Analysis Accelerator for OpenLab CDS

Importance of the Topic

Quality control of synthetic oligonucleotides is vital in biopharmaceutical development to ensure safety, efficacy, and regulatory compliance. Routine release testing demands methods that combine sensitivity, specificity, and robustness while maintaining data integrity and minimizing manual intervention.

Objectives and Overview

This article evaluates the Agilent Oligo Analysis Accelerator (OAA) for OpenLab CDS in combination with Agilent InfinityLab LC/MSD XT single quadrupole LC-MS. It aims to streamline purity, assay, and impurity profiling of single-stranded therapeutic oligonucleotides under quality control conditions.

Methodology

• Chromatography: Ion-pair reversed-phase LC using tributylammonium acetate and acetonitrile gradient on an AdvanceBio Oligonucleotide column (2.1 x 150 mm) at 50 °C.
• Mass Spectrometry: Negative-mode electrospray source with dual source settings – standard (soft) to preserve the four-charge envelope and harsh to confirm adduct identity.
• Calibration: Four-point UV and MS calibration; UV uses linear regression, MS uses a quadratic fit combining full-length product (FLP) and single-oxidation FLP peaks.
• Data Analysis Workflow in OAA: Project setup, data selection, system suitability, ion classification, peak integration, and result summary, all within a compliant web interface linked to OpenLab CDS.

Instrumentation Used

• Agilent 1260 Infinity II or 1290 Infinity II LC system
• Agilent AdvanceBio Oligonucleotide column, 2.1 x 150 mm
• Agilent InfinityLab LC/MSD XT single quadrupole mass detector (G6135C)
• OpenLab Control Panel, OpenLab CDS, and Oligo Analysis Accelerator software add-on

Main Results and Discussion

The OAA platform automates the extraction of ion chromatograms (m/z ± 150 around FLP) and the integration of UV and MS peaks using early- and late-eluting impurity boundaries. Predefined impurity thresholds (0.2% for the ≤ -4 charge state; 0.3% for higher charge states) enable reliable impurity classification. The dual-source approach discriminates adducts from true impurities, and automated EEI/LEI peak finding reduces manual error. The method achieves reproducible impurity quantitation down to 0.2% in a routine QC environment.

Benefits and Practical Applications of the Method

• Compliance with 21 CFR Part 11 via integrated audit trails and user-role controls
• Reduced analyst time through automated integration and impurity classification
• Minimized transcription errors by eliminating manual data transfers
• High-throughput QC release testing for synthetic oligonucleotide therapeutics

Future Trends and Possibilities for Use

• Extension of automated workflows to larger oligonucleotides and duplex RNA formats
• Integration of machine learning algorithms for real-time anomaly detection
• Expansion toward hybrid or higher mass range MS platforms
• Enhanced cloud-based data sharing and collaborative analysis

Conclusion

The combination of Agilent InfinityLab LC/MSD XT with the Oligo Analysis Accelerator streamlines and secures the analytical workflow for oligonucleotide QC. By automating data analysis and ensuring compliance, this solution addresses key challenges in purity, assay, and impurity profiling of therapeutic oligos.

References

• Rentel C.; Gaus H.; Bradley K.; Luu N.; Kolkey K.; Mai B.; Madsen M.; Pearce M.; Bock B.; Capaldi D. Assay, purity, and impurity profiling of phosphorothioate oligonucleotide therapeutics by ion pair–HPLC–MS. Nucleic Acid Therapeutics 2022, 32(3):206–220. DOI: 10.1089/nat.2021.0056