Compliance-ready workflow for antisense oligonucleotide impurity analysis without ion pairing agents

In recent years, ion pair-free analytical approaches have gained increasing attention with the aim of mitigating the limitations and drawbacks of ion pair reversed phase chromatography for the characterization of therapeutic oligonucleotides and adherence to green chemistry principles. There is a growing interest in developing ion pair-free strategies in alignment with EU REACH regulation, which prioritize reducing toxic chemical usage, such as volatile or hazardous additives in pharmaceutical or biopharmaceutical analyses. In the case of oligonucleotides, there is a need for sustainable, non-toxic alternatives to conventional reagents like alkylamines and fluorinated alcohols. Therefore, the focus of the present work is to establish an alternative ion pair-free strategy with a special focus on high-throughput analysis and applicability for QC environments using compliance-ready software for data acquisition, data processing and reporting. Method development was performed with pharmaceutically relevant RNA-based single stranded antisense oligonucleotides with different modifications and lengths, that had previously been quantified using an ion pairing method. Chromatographic gradient and MS parameters were optimized for rapid analysis in less than 12 min while maintaining adequate chromatographic resolution where possible, of the full-length product and major impurities. The use of a low ammonium acetate concentration at pH > 9.5 showed high sensitivity with very clean spectra containing minimal metal adduction. Two different quantitation approaches were assessed in the present study for sequence verification and quantitation purposes; one based on untargeted isotopic deconvolution over a wide range of charge states and a new targeted approach using selected extracted ion chromatogram signals for only two most abundant charge states. Although some differences were observed in terms of fractional abundances, resulting values were comparable. Sensitivity of the ion pair-free method showed lower values compared to IPRP-HRMS method, but still within acceptable LODs/LOQs to monitor main impurities during high throughput analysis.