Analysis of mRNA Cap Impurity Profiles and Capping Efficiency Using RapiZyme™ MC1 Ribonuclease

Significance of the Topic

Messenger RNA (mRNA) has emerged as a transformative modality in gene therapy and cancer treatment. The 5′ cap structure is critical for efficient translation and molecular stability, making its accurate profiling a key requirement for mRNA therapeutic development and regulatory compliance.

Goals and Study Overview

This work presents an integrated method to characterize 5′ cap species and quantify capping efficiency within a single oligonucleotide mapping workflow. By employing RapiZyme MC1 ribonuclease together with ion-pair reversed-phase liquid chromatography–mass spectrometry (IP-RP-LC-MS), the approach eliminates specialized sample-preparation steps and enables simultaneous sequence confirmation and cap analysis.

Materials and Methods

Two mRNA samples—a proprietary sequence (mRNA 1) and a commercial GFP mRNA—were digested with either RNase T1 or RapiZyme MC1 in ammonium acetate buffer. Digestion mixtures were directly subjected to IP-RP-LC-MS without additional enrichment. Data were processed using waters_connect software (mRNA Cleaver, CONFIRM Sequence, INTACT Mass) with in silico digestion and high-accuracy fragment scoring.

Instrumentation Used

• ACQUITY Premier System with binary solvent manager
• Xevo G3 QTof or Vion IMS QTof mass spectrometer
• ACQUITY Premier Oligonucleotide BEH C18 FIT Column (130 Å, 1.7 μm, 2.1 × 150 mm)
• Dipropylethylamine (DPA)/HFIP ion-pair mobile phases

Main Results and Discussion

• RNase T1 digests detected the cap1 oligonucleotide (m7GpppAmGp) and its unmethylated analog but failed to resolve uncapped species due to excessively short fragments.
• RapiZyme MC1 generated longer uridine-specific digestion products preserving cap structures: fully methylated, unmethylated, and uncapped forms were differentiated by distinct m/z values and retention times.
• Quantitative profiles for mRNA 1 showed ~70% cap1, ~20% unmethylated cap, and ~10% uncapped. GFP mRNA exhibited ~96.3% cap1 and minor uncapped species (~2.4%, ~1.3%).

Benefits and Practical Applications

• All-in-one workflow combining sequence mapping and cap profiling.
• No requirement for sequence-specific probes or RNase H/DNAzyme cleavage steps.
• Rapid, reproducible data analysis using waters_connect accelerates QC testing.
• Enables vendor-specific cap impurity profiling to inform process optimization.

Future Trends and Opportunities

Further automation, integration with AI-driven data analytics, and expansion to additional RNA modifications are expected to enhance throughput and deepen quality insights, supporting next-generation mRNA therapeutic pipelines.

Conclusion

The RapiZyme MC1–IP-RP-LC-MS platform delivers a robust, efficient solution for comprehensive 5′ cap profiling and capping efficiency assessment in mRNA therapeutics, streamlining workflows and expediting development and QC processes.

Reference

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