Characterization of mRNA Therapeutics Using a Novel LC-MS Based Workflow

Significance of the Topic

Messenger RNA (mRNA) therapeutics have emerged as a transformative class of biopharmaceuticals, exemplified by recent mRNA-based vaccines. Robust analytical methods are essential to ensure sequence integrity, detect modifications, and meet regulatory quality standards for research, development, and manufacturing.

Objectives and Study Overview

This work presents a novel liquid chromatography–mass spectrometry (LC-MS) workflow to characterize mRNA molecules. The aim was to establish a controlled partial digestion strategy using RNase T1 on magnetic beads, coupled to high-resolution LC-MS/MS, to generate unique oligonucleotide fragments and achieve comprehensive sequence mapping.

Methodology

A 3,500-base mRNA sample was digested with immobilized RNase T1 beads at 37 °C for incubation times of 5, 10, 15, and 30 minutes. After magnetic separation, formic acid was added to quench the reaction. The digest was analyzed on a Thermo Scientific Vanquish UHPLC system with a DNAPac RP column (50 °C, 300 µL/min) using a 40-minute gradient from 5 % to 20 % methanol (both solvents containing TEA and HFIP). Mass spectrometry employed an Orbitrap Exploris 240, full scan (m/z 450–4000 at 120 000 resolution) and data-dependent MS/MS (m/z 150–2000 at 30 000 resolution, stepped HCD 25/28/31 V). Data were processed in Biopharma Finder software with automated oligonucleotide identification, confidence scoring, and sequence coverage mapping.

Instrumentation Used

• Thermo Scientific Vanquish UHPLC system
• DNAPac RP oligonucleotide column
• Thermo Scientific Orbitrap Exploris 240 mass spectrometer
• RNase T1 immobilized on magnetic beads
• KingFisher Duo Prime (optional automated bead handling)

Main Results and Discussion

Optimization identified 15 minutes as the optimal digestion time to produce fragments of 5–43 nucleotides with one to eleven missed cleavages. Chromatographic retention correlated with fragment molecular weight, enabling separation of isomeric oligonucleotides. MS/MS annotation provided sequence confirmation and modification site information, with a confidence threshold >85 %. Applying these criteria delivered 89 % unique sequence coverage and 91 % total coverage including non-unique fragments.

Benefits and Practical Applications

• Controlled partial digestion yields longer, unique fragments for accurate sequence mapping.
• High-resolution LC separation resolves isomeric sequences, reducing ambiguity.
• Automated MS/MS confidence scoring streamlines data analysis and quality control.
• Applicable to mRNA therapeutic development, batch release testing, and stability studies.

Future Trends and Applications

Advancements may include integration of additional nucleases to enhance sequence coverage, implementation of automated sample preparation platforms, real-time data analytics powered by machine learning for rapid QC decisions, and standardized workflows for regulatory submissions. Expansion to modified mRNA constructs and complex formulations will drive further method development.

Conclusion

The described LC-MS workflow combining controlled RNase T1 digestion, high-resolution chromatography, and MS/MS fragment confirmation enables comprehensive mRNA sequence mapping. This approach delivers high sequence coverage and robust analytical performance, supporting the growing demands of mRNA therapeutic characterization.