Analytical solutions for mRNA vaccines and therapeutics

Significance of the topic

Messenger RNA (mRNA) vaccines and therapeutics represent a transformative approach to disease prevention and treatment, relying on the body’s own machinery to produce target proteins. Analytical characterization of mRNA molecules and their lipid nanoparticle (LNP) carriers is critical to ensure product identity, purity, safety and efficacy. Robust, high-throughput methods are needed to support development, quality control and regulatory compliance for these complex biopharmaceuticals.

Objectives and study overview

This work presents an integrated suite of analytical solutions designed to:

• Confirm full-length mRNA sequence directly by LC-HRAM MS without DNA conversion or primers.
• Determine critical quality attributes (CQAs) including capping efficiency, poly(A) tail length and impurity profiles.
• Characterize LNP composition, lipid purity, encapsulation efficiency and degradation products.
• Streamline method development and automate sample preparation and data analysis for high reproducibility.

Methodology

A three-step LC-HRAM MS workflow enables direct mRNA sequence confirmation: controlled partial RNase T1 digestion on magnetic beads, high-pH reversed-phase separation on DNAPac RP columns, and high-resolution Orbitrap MS/MS for oligonucleotide mapping. Impurity scouting uses dual-LC column screening (RP and ion-exchange) on the Vanquish Duo system. Capping and poly(A) tail analysis are performed via targeted cleavage, bead-based cleanup and LC-HRAM MS quantitation. LNP component separation employs UHPLC-CAD for universal detection and LC-MS/MS for sensitive identification of lipids and metabolites.

Used instrumentation

• Thermo Scientific Vanquish UHPLC systems (Flex, Horizon, Duo)
• Thermo Scientific Orbitrap Exploris mass spectrometers (120, 240, 480)
• Thermo Scientific DNAPac RP and PA200RS oligonucleotide HPLC columns
• Thermo Scientific Accucore C30 UHPLC columns
• Thermo Scientific Charged Aerosol Detector (CAD)
• KingFisher Duo Prime magnetic bead purification system
• Thermo Scientific BioPharma Finder and Chromeleon CDS software

Main results and discussion

Direct LC-HRAM MS mapping achieved >85% sequence coverage within two hours, eliminating library prep and multi-day workflows. Partial RNase digests on beads produced large, unique fragments with high reproducibility across replicates. High-pH RP separation resolved sequence isomers and enabled confident MS/MS identification. Capping and poly(A) assays delivered accurate efficiency and length distribution measurements, with mass errors <1 ppm. Dual-column scouting accelerated impurity method optimization. LNP analysis by UHPLC-CAD provided baseline separation of four key lipids and trace impurities in single runs. LC-MS/MS detected lipid degradants at sub-0.1% levels and quantified novel ionizable lipids in complex biological extracts with 2 ppm accuracy.

Benefits and practical applications

• Rapid, end-to-end workflows reduce turnaround from days to hours.
• Direct mRNA sequencing avoids biases and complexities of indirect methods.
• Automated sample preparation and data processing ensure high reproducibility.
• Comprehensive multi-attribute analysis in single runs streamlines QC testing.
• Universal CAD detection simplifies formulation profiling, while HRAM MS supports in vivo metabolite studies.

Future trends and possibilities

Integration of standardized, automated platforms with advanced software and machine learning will further accelerate mRNA and LNP analytics. Emerging technologies such as ion mobility, real-time reaction monitoring, and multiplexed detection promise deeper insight into molecular heterogeneity. Continued innovation in mass spectrometry sensitivity and data processing will support next-generation RNA modalities and personalized therapeutics.

Conclusion

The described analytical solutions combine direct sequence mapping, impurity profiling, and LNP characterization into cohesive, high-throughput workflows. By leveraging LC-HRAM MS, UHPLC-CAD and automation, these methods address the unique challenges of mRNA therapeutics development and manufacturing, ensuring reliable, comprehensive quality control.