Efficient Method Development for Separation of Capped mRNA Fragments

Significance of the Topic

Rapid and reliable characterization of 5 prime capped mRNA fragments is critical for quality control in mRNA therapeutics, including COVID-19 vaccines. The cap structure (Cap-0 and Cap-1) influences mRNA stability, translational efficiency, and immunogenicity. Accurate separation and identification of capped fragments and related impurities support regulatory compliance and ensure product consistency.

Objectives and Study Overview

This study demonstrates an efficient workflow for method development using LabSolutions MD software combined with LCMS-2050 and LCMS-9050 systems. Key goals include:

• Screening mobile‐phase additives and columns to achieve baseline separation of Cap-0, Cap-1, and impurities.
• Optimizing gradient and temperature parameters via design space evaluation.
• Confirming molecular weights of separated components with single‐quad and high‐resolution MS.

Methodology and Instrumentation

Model samples comprised 36-base mRNA fragments with Cap-0 (m7GpppR-) and Cap-1 (m7GpppRm-), plus impurities (pppR-, ppR-, GpppR-). Reversed‐phase ion‐pair chromatography employed 100 mm × 2.1 mm Shim-pack Scepter C18 columns (120 and 300 Å) with 1.9 µm particles. Aqueous phases contained 100 mmol/L HFIP and 10–20 mmol/L ion-pair reagents (DIPEA, TEA, DBA, HA). Organic solvents were acetonitrile or methanol. LabSolutions MD automated schedule generation, mobile-phase blending, ranking of chromatograms by resolution, and design space visualization. Molecular weight confirmation used:

• LCMS-9050 Q-TOF MS for high‐accuracy mass determination.
• LCMS-2050 single‐quadrupole MS for routine QC checks.

Main Results and Discussion

First‐phase screening revealed that DIPEA and TEA caused coelution of fragments, while DBA and HA produced distinct peaks. Second‐phase screening identified optimal separation with a 300 Å column, 20 mmol/L HA, and 75 % acetonitrile. Optimization experiments showed that a 30-minute gradient, 20 % initial organic concentration, and 40 °C oven temperature maximized resolution (peak-to-valley >7.7; Rs ≈1.8). Design space plots confirmed these trends. LCMS-9050 component spectra distinctly resolved 14 Da differences among GpppR-, ppR-, and pppR-. Final conditions with LCMS-2050 provided clear UV and mass chromatograms, enabling unambiguous identification of Cap-0, Cap-1, and impurities.

Benefits and Practical Applications

By integrating LabSolutions MD with Shimadzu LC/MS platforms, laboratories can:

• Reduce manual labor and human error through automated method scouting and mobile‐phase preparation.
• Rapidly evaluate multiple parameters and visualize design space to ensure robust separations.
• Confirm fragment identities with complementary MS approaches, streamlining QC workflows.

Future Trends and Opportunities

Advances may include machine-learning–guided parameter selection, real-time feedback loops for dynamic method adjustment, and extension to other oligonucleotide modalities such as siRNA or antisense. Further automation of sample handling and data interpretation will accelerate analytical development and support high-throughput quality control in biopharmaceutical manufacturing.

Conclusion

This work illustrates an efficient, systematic approach to developing ion-pair reversed-phase LC methods for capped mRNA fragments. LabSolutions MD software streamlines parameter screening and optimization, while Shimadzu LCMS-2050 and LCMS-9050 instruments offer complementary capabilities for molecular weight confirmation. The combined workflow enhances method robustness and accelerates analytical readiness for mRNA therapeutics.

Instrumentation Used

• Nexera XS inert UHPLC with Shim-pack Scepter Claris C18-120 and C18-300 columns
• LCMS-9050 quadrupole time-of-flight mass spectrometer (ESI negative, m/z 600–2000)
• LCMS-2050 single-quadrupole mass spectrometer with DUIS negative ion source (SCAN m/z 550–2000)

Reference

1. A. Ramanathan, G. B. Robb, S. H. Chan, mRNA capping: biological functions and applications, Nucleic Acids Res. 2016, 44, 7511–7526.