A Software Workflow Using Wide Mass Range Single Quadrupole Mass Spectrometry Data Stream Applied to Oligonucleotides Confirmation

Importance of the Topic

Oligonucleotide therapeutics such as antisense oligonucleotides and small interfering RNAs require accurate molecular weight confirmation and impurity profiling to ensure product integrity, efficacy, and safety. Emerging backbone chemistries and sequence modifications increase analytical complexity, necessitating robust workflows for comprehensive characterization.

Goals and Study Overview

This work evaluates a production prototype single quadrupole LC-MS system with an extended mass range (m/z 3000) coupled with LabSolutions Insight Biologics software. The study focuses on confirming full-length products (FLPs) and optimizing quantitation workflows for a 20-mer antisense oligonucleotide (mipomersen) and a synthetic 30-mer polyT sequence.

Methodology and Instrumentation

• Liquid chromatography performed on a Shim-pack Scepter™ Claris column with ion-pair mobile phases: A (10% ACN, 5 mM TBuAA, 1 μM EDTA) and B (80% ACN, 5 mM TBuAA, 1 μM EDTA) using a 6-minute gradient.
• Mass spectrometry using a modified Shimadzu LCMS-2050 single quadrupole, extended to m/z 3000 via reduced RF frequency (1.0 MHz), negative ESI (–3.0 kV), desolvation at 450 °C, and 1 scan/s acquisition.
• Data processing in profile mode with LabSolutions Insight Biologics, employing full-length sequence input for charge deconvolution and reporting.

Results and Discussion

• Mipomersen FLP confirmation yielded a deconvoluted mass of 7176 Da across three principal charge states (z=–4 to –6) with excellent linearity (R² ≈ 0.9999).
• PolyT30 quantitation demonstrated that summing all charge state signals improved precision and extended the lower limit of quantitation compared to monitoring a single charge state.
• Full-range acquisition (m/z 600–3000) provided unbiased detection of all charge states, reducing the need for iterative method optimization and ensuring consistent identification across varying injection levels.
• Mobile phase selection influenced charge state distribution: TBuAA/EDTA produced higher signal intensities on dominant charge states, while HFIP/TEA generated broader charge distributions albeit at lower individual intensities.

Benefits and Practical Applications

• Streamlined workflow for oligonucleotide confirmation without the complexity of high-resolution instruments.
• Robust quantitation achieved by summing multiple charge states, mitigating variability of low-intensity ions.
• Extended mass range broadens applicability to larger oligonucleotides and impurity profiling tasks.
• User-friendly software accelerates data analysis, reporting, and compliance in research and QA/QC settings.

Future Trends and Potential Applications

• Application to larger oligonucleotide classes such as single guide RNAs (sgRNAs) and siRNAs for both qualitative screening and quantitative assays.
• Integration of advanced algorithms for automated deconvolution, impurity annotation, and batch processing.
• Refinement of mobile phase chemistries to tailor charge state distributions for specific sequence chemistries and lengths.
• Adoption in high-throughput and regulated laboratories, paving the way for validated methods in biopharmaceutical development.

Conclusion

The extended-range single quadrupole LC-MS platform, combined with dedicated data processing software, offers a versatile and efficient solution for oligonucleotide molecular weight confirmation and quantitation. Full-range data acquisition and charge state summation deliver high precision, reduced method development time, and flexible adaptation to diverse oligonucleotide classes.