Characterization of mRNA vaccine 5’ and 3’ end products using BioPharma Finder 5.0

Importance of the topic

mRNA vaccines require precise end capping and polyadenylation to ensure stability, translation efficiency, and resistance to nucleases. Reliable characterization of 5’ and 3’ ends is critical for quality control throughout development and manufacturing of mRNA therapeutics.

Objectives and study overview

This study presents an automated analytical workflow using Thermo Scientific BioPharma Finder 5.0 to detect, characterize, and quantify the 5’ cap and 3’ poly(A) tail of in vitro transcribed mRNA. Enzymatic digestion followed by high resolution accurate mass LC-MS/MS was applied to identify end modifications and assess their distribution.

Methodology and Instrumentation

Sample preparation for 5’ cap analysis involved annealing a biotinylated DNA–RNA probe to the mRNA 5’ end, RNase H cleavage, magnetic bead cleanup, and UHPLC–MS/MS analysis. For poly(A) characterization, RNase T1 digestion of the poly(A) tail was performed followed by oligo(dT)25 bead isolation.

• UHPLC: Thermo Scientific Vanquish Horizon system with DNAPac RP column
• Mass spectrometry: Thermo Scientific Orbitrap Exploris 240 operated in negative polarity, peptide and intact protein modes
• Software: BioPharma Finder 5.0 with Oligonucleotide Sequencing and Intact Mass Analysis workflows

Main Results and Discussion

Automated deconvolution and annotation enabled clear identification of capped and uncapped species. Relative quantitation yielded a cap efficiency of 24%, within 4% of theoretical. MS/MS fragmentation maps provided sequence confirmation and site-specific modification data. Intact mass analysis of poly(A) tails revealed a length distribution centered between 121 and 126 adenosines, consistent with the expected 140-mer template after accounting for truncated species.

Benefits and Practical Applications

Implementation of BioPharma Finder 5.0 streamlines data processing for mRNA end characterization, reducing manual intervention and increasing confidence in QC workflows. This approach supports regulatory requirements for mRNA vaccine and therapeutic development by delivering accurate, reproducible measurements of end-cap and tail structures.

Future Trends and Potential Applications

Advances in software algorithms and instrument sensitivity will further enhance the throughput and resolution of mRNA end analysis. Integration with automated sample preparation platforms and expanded modification libraries promises broader application to novel RNA modalities and personalized therapies.

Conclusion

Thermo Scientific BioPharma Finder 5.0 provides a robust, automated solution for comprehensive analysis of mRNA 5’ caps and 3’ poly(A) tails, supporting critical quality assessments in vaccine and therapeutic research.

References

Ross RL, Qi Q, Murphy K, Du M. Characterization of mRNA vaccine 5’ and 3’ end products using BioPharma Finder 5.0. Thermo Fisher Scientific.