Characterization of mRNA 5’ capping products using an LC-HRAM-MS/MS analytical platform and Thermo Scientific BioPharma Finder software solution

Significance of the topic

Production of non-natural mRNA for vaccines and therapeutics demands precise characterization of the 5’ cap structure, a critical quality attribute that enhances transcript stability, nuclear export, translation initiation, and evasion of host exonucleases and innate immune sensors.

Objectives and study overview

This study aimed to develop a sensitive and robust LC-HRAM-MS/MS workflow using an Orbitrap Exploris 240 instrument coupled to a Vanquish Horizon UHPLC system and Thermo Scientific BioPharma Finder 5.0 software. The goal was confident identification, sequence confirmation, and relative quantitation of the 5’ cap on in vitro transcription (IVT) mRNA.

Methodology and instrumentation

• Sample preparation: IVT mRNA was decapped with RNA 5’ pyrophosphohydrolase (RppH) to generate a reference standard and mixed with capped mRNA in a defined ratio. A biotinylated 25-mer DNA–RNA chimera probe annealed to the 5’ end of capped mRNA and was digested with RNase H. Cleavage products were captured on streptavidin magnetic beads, washed with reduced sodium concentration to limit adduct formation, eluted in 75% ethanol/water, dried, and reconstituted for analysis.
• Chromatography: Ion-pair reversed-phase LC on a Thermo Scientific DNAPac RP column (2.1×100 mm, 4 μm) at 70°C. Mobile phases were 50 mM HFIP/15 mM TEA in water (A) and methanol (B). Gradient: 2% B to 40% B over 16 min at 400 μl/min.
• Mass spectrometry: Orbitrap Exploris 240 operated in negative-ion mode with data-dependent MS2. Full scan: 120 000 resolution (m/z 550–2500), AGC target custom, max injection 50 ms. MS/MS: 30 000 resolution, isolation window 1.6 m/z, HCD energy 20 V.
• Data analysis: BioPharma Finder 5.0 was used for oligomer sequence mapping. Custom 5’ terminal modifications (m7G cap) were defined, and a “nonspecific” nuclease cleavage setting was applied. MS1 and MS2 matching employed 5 ppm tolerance and minimum confidence of 0.80.

Main results and discussion

• Extracted ion chromatograms based on the top five isotopic peaks of the most abundant charge states (capped m/z 893.7495, z=8; decapped m/z 958.8535, z=7) yielded a measured capped-to-decapped ratio within 4% of the theoretical 4:1 input.
• Reducing Na+ concentration in wash buffer to 100 mM minimized sodium adduction and improved sensitivity in negative-ion detection.
• BioPharma Finder mapping identified the capped and decapped 5’ termini coeluting at 3.24 min and annotated additional probe-related failure fragments throughout the chromatogram.
• MS/MS fragmentation maps provided high ladder coverage and confirmed the location and identity of the m7G cap modification.

Benefits and practical applications

• The platform offers a streamlined, label-free workflow for comprehensive 5’ cap analysis, reducing manual interpretation and accelerating mRNA quality control and process development.
• High-resolution accurate-mass and MS2 confirmation deliver robust identification of cap analogs and truncated probe sequences in complex samples.

Future trends and potential applications

• Automation and high-throughput magnetic bead–based sample prep for large-scale mRNA analytics.
• Multi-attribute MS approaches to profile additional RNA modifications (e.g., 2′-O-methylation) and poly(A) tail heterogeneity.
• AI-driven data analysis for rapid discovery of novel cap structures and modified nucleotides.
• Adoption of LC-HRAM-MS/MS cap characterization in regulated environments to support lot release and stability testing of mRNA therapeutics.

Conclusion

The combined LC-HRAM-MS/MS platform and BioPharma Finder software deliver a sensitive, accurate, and efficient method for mapping and quantitating mRNA 5’ cap structures, supporting critical quality attribute assessment during mRNA product development and quality control.

Instrumentation Used

• Thermo Scientific Vanquish Horizon UHPLC system (Binary Pump H, Split Sampler HT, Column Compartment H)
• Thermo Scientific Orbitrap Exploris 240 mass spectrometer
• Thermo Scientific DNAPac RP column (2.1×100 mm, 4 μm)
• Thermo Scientific BioPharma Finder 5.0 software

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