Analysis of mRNA 5’ Cap Structure Using a Quadrupole Time-of-Flight Mass Spectrometer

Significance of the Topic

Messenger RNA is a key modality in modern vaccine and therapeutic development, notably for COVID-19 vaccines. The 5 end cap structure influences recognition, translation efficiency and stability of mRNA in cells. Precise analysis of cap modifications and impurities is essential for quality control and regulatory compliance in biologics manufacture.

Objectives and Study Overview

This study demonstrates characterization of the Cap 1 structure and uncapped impurity of 36 base mRNA fragments using a high resolution quadrupole time of flight mass spectrometer coupled to UHPLC and specialized bioinformatics software. It aims to confirm fragment coverage, detect process related impurities and quantify their levels in defined mixtures.

Methodology and Instrumentation

• Sample preparation included in vitro transcription of plasmid DNA templates to generate 5 end modified mRNA fragments and unreacted RNA impurities followed by enzymatic fragmentation.
• Chromatographic separation was achieved on a C18 column using hexafluoroisopropanol and diisopropylethylamine buffer gradient in aqueous organic solvent at 60°C.
• Mass analysis was performed in negative electrospray DDA mode covering MS m/z 550 to 2500 and MS/MS m/z 100 to 2500.

Used Instrumentation

• Shimadzu Nexera XS inert UHPLC system
• Shim-pack Scepter Claris C18 column, 150 mm × 2.1 mm, 1.9 μm
• Shimadzu LCMS 9050 quadrupole time of flight mass spectrometer
• LabSolutions Insight Biologics software for sequence mapping and modification analysis

Main Results and Discussion

Component chromatograms distinguished Cap 1 fragments from uncapped RNA based on retention time and MS1 profiles. Deconvolution of multivalent ions provided accurate mass confirmation. Side products with an extra cytidine residue (N1 groups) were detected at 6.6 to 7.9 percent relative abundance, indicating secondary transcription events. MS/MS enabled mapping of fragment ions across the backbone for both uncapped and N1 variants, highlighting modification sites and small structural changes. Quantitative spiking experiments from zero to ten percent uncapped impurity showed linear response with R2 greater than 0.99.

Benefits and Practical Applications

• High resolution mass data enable unambiguous identification of cap structures and low level impurities.
• Software guided sequence coverage simplifies detection of base modifications, strand length variants and adducts.
• Quantitative capability supports robust quality control of mRNA therapeutics.

Future Trends and Applications

Integration of advanced fragmentation methods such as ultraviolet photodissociation or ion mobility could further enhance sequence coverage. Automated workflows and cloud based data analysis are expected to streamline regulatory submissions. The approach will likely become a standard component in GMP compliant mRNA production, expanding to longer constructs and diverse modification chemistries.

Conclusion

The combination of a quadrupole time of flight LCMS and dedicated biologics software provides a comprehensive platform for structural and quantitative analysis of mRNA cap modifications and impurities. The demonstrated sensitivity, accuracy and sequence resolution support its adoption for quality control in mRNA based drug development.

Reference

• Application News No 01 00595A EN An Oligonucleotide Impurity Analysis Workflow Using LabSolutions Insight Biologics Software
• Application News No 01 00656 EN Simple Analysis of Impurities in Oligonucleotide Therapeutics Using a Single Quadrupole Mass Spectrometer