Automated LC/MS Quality Control of Anti-Sense Oligonucleotides

Significance of the topic

Antisense oligonucleotides are an advanced class of therapeutic nucleic acids requiring precise and high sensitivity quality control to ensure purity and compliance in pharmaceutical production. Liquid chromatography mass spectrometry remains the gold standard for routine impurity profiling but can involve time consuming manual data processing steps.

Objectives and study overview

This study demonstrates a streamlined LC/MS workflow for assessing antisense oligonucleotide purity by integrating an extended mass range single quadrupole detector with specialized software to automate data analysis tasks and accelerate method transfer into compliant QC environments.

Methodology and instrumentation

Sample preparation involved a 20 mer antisense oligonucleotide at 0.1 mg/mL. Instrumentation comprised an Agilent InfinityLab Pro iQ Plus LC/MS system with electrospray ionization in negative polarity, coupled to an AdvanceBio Oligonucleotide column and diode array detection. Two MS source conditions (standard and harsh) were used to discriminate adduct formation. Data acquisition and analysis were carried out in Agilent OpenLab CDS with the Oligo Analysis Accelerator module.

Main results and discussion

The Oligo Analysis Accelerator software automated extraction and integration of ion chromatograms for all identified m/z species, determined early and late eluting impurity boundaries for UV peak integration, and classified ions by comparing spectra under standard and harsh conditions to identify adducts. The workflow yielded an MS purity of 86.9% and UV purity of 98.0% for the antisense oligonucleotide. Unknown late eluting species and potential long mers were detected, emphasizing the need for high selectivity and sensitivity.

Benefits and practical applications

• Significant reduction of manual data processing tasks
• Improved accuracy and traceability in impurity profiling
• Simplified method transfer into regulated QC labs
• Enhanced detection of closely related impurities and adducts

Future trends and potential applications

Emerging chromatographic approaches and high resolution detectors may further improve separation and quantitation of complex oligonucleotide impurities. Integration with cloud based data management and machine learning algorithms promises advanced automated identification and classification of novel process related species.

Conclusion

The combination of extended mass range single quadrupole detection and purpose built software enables a robust and efficient LC/MS quality control workflow for antisense oligonucleotides while ensuring compliance and facilitating rapid throughput in routine QC environments.

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 profile of phosphorothioate oligonucleotide therapeutics by ion pair HPLC MS. Nucleic Acid Therapeutics. 2022;32(3):206 220.