Detailed study into ASO impurity analysis, lessons learned, and myths dispelled while moving to compliant platform methods

Importance of the topic

Oligonucleotide impurity analysis is critical for ensuring the safety, efficacy and regulatory compliance of antisense oligonucleotide (ASO) therapeutics. As more synthetic RNA modalities enter clinical trials, robust methods are required to accurately characterize low-level impurities generated during manufacturing and to support quality control and product registration.

Objectives and study overview

This study addresses common misconceptions in ion-pair reversed-phase LC-MS of ASOs and demonstrates a compliant, automated workflow for impurity profiling. Key aims include:

• Dispelling myths about adduct formation and in-source fragmentation
• Optimizing source and chromatographic conditions for high-resolution MS
• Comparing deconvolution and extracted ion chromatogram (XIC) approaches for quantitation
• Implementing automated annotation and reporting with Chromeleon software

Methodology and Instrumentation

Sample preparation involved synthetic antisense oligonucleotides spiked with low-level impurities. Chromatographic separation used a Thermo Scientific DNAPac RP column (2.1×100 mm, 4 μm) on a Vanquish Flex UHPLC with pentylamine/HFIP ion-pair reagents. MS detection employed a Thermo Scientific Orbitrap Exploris system controlled by Chromeleon 7.3.2 software. Data processing included high-resolution deconvolution, targeted XIC extraction and automatic annotation of full-length products (FLP) and impurities. Reporting integrated UV, MS and quantitation results in a GLP-compliant format.

Main results and discussion

Optimized source conditions effectively removed metal adducts while minimizing in-source fragmentation up to 70 eV. Multiple charge states were evaluated, showing that selecting appropriate states improves quantitation precision. Deconvolution and targeted XIC methods yielded comparable impurity profiles, with automated transfer of m/z and abundance values into component tables. Key findings:

• Adduct removal without artifact generation enhances signal clarity
• Charge-state profiling enables robust identification of true impurities
• Deconvolution and XIC quantitation agree within analytical variability
• Automated annotation accelerates data review and supports new peak detection

Benefits and practical applications

The presented workflow offers several advantages for ASO impurity analysis:

• Fully automated, GLP-compliant reporting saves time and reduces manual errors
• High-resolution MS combined with intelligent XIC selection increases confidence in impurity assignments
• Compatibility with existing UHPLC platforms allows rapid implementation in QC laboratories

Future trends and opportunities

Advancements in oligonucleotide analytics will focus on:

• Integration of advanced data-processing algorithms and AI-driven peak classification
• Higher-throughput platforms enabling multiplexed oligonucleotide profiling
• Expansion to novel modalities such as siRNA, mRNA and conjugated oligonucleotides
• Enhanced compliance through standardized software workflows and electronic records

Conclusion

Ion-pair reversed-phase LC-HRAM MS, when combined with optimized source conditions and intelligent data processing, provides a powerful, reproducible method for ASO impurity analysis. Automated deconvolution, targeted XIC quantitation and comprehensive reporting within Chromeleon software deliver a streamlined, compliant solution for QC and research laboratories.