TIDES: ANALYSIS OF mRNA CRITICAL QUALITY ATTRIBUTES (CQAs) USING THE BIOACCORD UPLC-TOF MS SYSTEM AND INTACT Mass SOFTWARE

Significance of the Topic

The rapid advancement and regulatory approval of mRNA-based COVID-19 vaccines have elevated RNA therapeutics to a central position in biopharmaceutical development. Ensuring consistent quality and efficacy of these molecules depends on robust analytical methods to monitor key Critical Quality Attributes (CQAs). Reliable assessment of 5’ capping, poly(A) tail heterogeneity and sequence integrity is essential for process control, batch release and regulatory compliance in RNA drug manufacturing.

Study Objectives and Overview

This work outlines three complementary UPLC-TOF MS workflows implemented on the BioAccord system in combination with the INTACT Mass App. The goals were to:

• Quantify 5’ cap structures and their precursor impurities.
• Characterize poly(A) tail length distribution and heterogeneity.
• Map primary sequence coverage and resolve isobaric fragments.

By integrating specialized chromatography, high-resolution mass spectrometry and tailored software tools, the study demonstrates a streamlined approach to mRNA CQA analysis.

Methodology and Instrumentation

The analytical workflows share a common platform: RNase T1 digestion of RNA samples followed by ion-pairing reversed-phase UPLC separation and TOF MS detection. Key steps include enzymatic cleavage, optimized chromatographic focusing of heterogeneous fragments, and MaxEnt deconvolution to derive accurate mass series. Sequence mapping employs data-dependent MS/MS and a dedicated Sequence Viewer to calculate coverage percentages and differentiate isobaric candidates.

Used Instrumentation

• Waters BioAccord UPLC-TOF MS system with Max Peak High Performance Surfaces.
• ACQUITY Premier OST column, 1.7µm, 130 Å, 2.1×150 mm.
• INTACT Mass App for cap analysis, tail profiling and sequence mapping.
• Enzyme Cleavage Tool, Cleaver App and Sequence Viewer modules for data processing.

Main Results and Discussion

• 5’ Capping: Cap-1 and precursor impurities were mixed at defined ratios (100:1, 99:1, 9:1) and quantified with high accuracy using extracted ion chromatograms, enabling precise determination of capping efficiency.
• Poly(A) Tail Heterogeneity: Ion-pairing UPLC achieved sharp separation of tail fragments; MaxEnt deconvolution revealed a weighted average length of ~126.5 nucleotides and defined the full distribution profile.
• Sequence Mapping: RNase T1 digest of sgRNA and Fluc mRNA generated MS/MS spectra with >95% coverage. Ambiguous isobaric sequences were distinguished through elevated-energy fragmentation and sequence viewer analysis.

Benefits and Practical Applications

These integrated workflows provide rapid and reliable monitoring of mRNA CQAs in a single platform. Automated data processing reduces manual interpretation time, improves confidence in result assignments, and supports high-throughput process development and quality control in mRNA therapeutic manufacturing.

Future Trends and Opportunities

Advancements are planned to enhance sequence coverage by combining MS1 and MS2 data streams and to fully automate the data analysis pipeline. Further applications may extend these workflows to novel RNA modifications, longer transcript libraries and real-time release testing in continuous manufacturing environments.

Conclusion

The BioAccord UPLC-TOF MS system coupled with INTACT Mass App and supporting informatics offers a powerful, unified solution for comprehensive mRNA CQA assessment. The demonstrated methods deliver precise quantitation of cap structures, poly(A) tail profiling and sequence integrity verification, addressing critical needs in RNA therapeutic development and quality assurance.

References

1. D’Esposito R, Doneanu C, Gastall H, Berger SJ, Yu YQ. RNA CQA Analysis Using the BioAccord UPLC-TOF MS System and INTACT Mass Application. Waters Application Note 720008130; 2024.
2. Waters Corporation. Rapid Analysis of Synthetic mRNA Cap Structure Using Ion-Pairing RPLC with the BioAccord LC-MS System. Application Note 720007329; 2023.
3. Waters Corporation. Ion-Pairing Reversed-Phase LC-MS Analysis of Poly(A) Tail Heterogeneity Using the BioAccord LC-MS System. Application Note 720007925; 2023.