MRNA CRITICAL QUALITY ATTRIBUTE ANALYSIS USING A UPLC-TOF-MS SYSTEM AND CUSTOMIZED SOFTWARE

Significance of the Topic

RNA-based therapeutics, led by recent COVID-19 mRNA vaccines, demand robust analytical controls to ensure product consistency, safety and efficacy. Critical Quality Attributes (CQA) of mRNA—such as 5′ capping efficiency, poly(A) tail length distribution and primary sequence integrity—directly impact stability, translational efficiency and immunogenicity. Developing streamlined, high-throughput workflows for CQA monitoring is therefore essential for biopharmaceutical process development and quality assurance.

Objectives and Overview of the Study

This work outlines an end-to-end analytical strategy for three key mRNA CQAs using a benchtop UPLC-TOF MS platform combined with custom data-processing software. The study aimed to:

• Quantify 5′ capping efficiency and identify cap-related impurities.
• Profile heterogeneity of the poly(A) tail region.
• Map primary sequence after enzymatic digestion to assess coverage and detect modifications.

Methodology and Instrumentation

Sample types included synthetic single guide RNA (sgRNA), luciferase mRNA constructs and model cap oligonucleotides. Key steps:

• RNase T1 digestion for sequence mapping and poly(A) profiling.
• Ion-pair reversed-phase UPLC separation on ACQUITY Premier OST columns with a VanGuard FIT pre-column.
• Binary gradient of HFIP/DIPEA aqueous buffer (pH 8.8) versus ethanol-based organic phase at 300 μL/min.
• Detection via UPLC-TOF MS (BioAccord system) with MaxEnt deconvolution.
• Data processing in INTACT Mass App for capping quantitation, poly(A) tail deconvolution and sequence mapping; supplemental use of mRNA Cleaver App and Sequence Viewer for coverage analysis.

Key Results and Discussion

5′ capping analysis demonstrated high sensitivity, accurately quantifying cap-1 versus precursor species at ratios down to 99:1, with measured cap-1 purity of ~97.3%. Poly(A) tail profiling yielded a weighted average tail length of ~126.5 nucleotides, revealing the distribution of truncated and full-length species. Sequence mapping of a 100-mer sgRNA achieved ~75% coverage at the MS1 level, with elevated-energy fragmentation improving assignment confidence and highlighting ambiguous regions resolved via comparison of candidate sequences.

Benefits and Practical Applications

The integrated workflow delivers:

• Rapid, orthogonal assessment of mRNA quality attributes on a single LC-MS platform.
• Automated data processing to reduce manual interpretation time and potential for error.
• Quantitative insights into cap structure, poly(A) dynamics and sequence integrity, supporting process control and comparability studies.

Future Trends and Applications

Anticipated advancements include:

• Integration of MS1 and MS2 data to enhance sequence coverage and confidence.
• Further automation of data workflows to minimize ambiguous assignments.
• Extension of the platform to complex formulations and larger mRNA constructs.

Conclusion

This study establishes a versatile UPLC-TOF MS-based approach for comprehensive CQA analysis of mRNA therapeutics. Combining efficient separation, sensitive detection and tailored informatics enables precise monitoring of critical attributes, accelerating development and ensuring product quality.

References

1. Waters Application Note 720008130 “RNA CQA Analysis Using the BioAccord LC-MS System and INTACT Mass Waters_Connect Application”
2. Waters Application Note 720007329 “Rapid Analysis of Synthetic mRNA Cap Structure Using Ion-Pairing RPLC with the BioAccord LC-MS System”
3. Waters Application Note 720007925 “Ion Pairing Reversed Phase LC-MS Analysis of Poly(A) Tail Heterogeneity Using the BioAccord LC-MS System”