Blueprint for QC-ready mRNA mapping with Thermo Fisher Scientific and the University of Sheffield

Significance of the topic

mRNA therapeutics require unambiguous identity confirmation and impurity profiling to support development, manufacturing and regulatory release. Mass spectrometry-based sequence mapping after enzymatic digestion is emerging as a core analytical strategy because it provides direct, MS/MS-backed evidence of sequence identity and related critical quality attributes (CQAs) such as 5 capping and poly(A) tail length. This case study presents a QC-ready, automated LCMS/MS blueprint developed through collaboration between Thermo Fisher Scientific and the University of Sheffield that addresses key analytical challenges for large, structured RNA molecules.

Objectives and overview of the study

The objective was to develop a reproducible, high-throughput workflow for comprehensive mRNA sequence mapping and multi-attribute monitoring that is suitable for transition from R&D to QC. The approach focuses on controlled partial RNase digestions to generate longer, unique oligoribonucleotide fragments, automated sample processing and high-resolution accurate mass (HRAM) LCMS/MS acquisition, supported by bespoke data visualization tools to accelerate and standardize interpretation.

Methodology

The strategy addresses the analytical challenge posed by RNAonly composition (four bases) and frequent isobaric digestion products. Complete digestion with high-frequency RNases (e.g., RNase T1) tends to produce many overlapping, isobaric fragments; therefore, controlled partial digestion was chosen to yield longer sequence-specific fragments that are more informative in MS/MS.

Two automated partial-digestion formats were developed:

• Offline automated digests using immobilized enzyme beads: immobilized RNase on magnetic beads enables rapid, reproducible stoppage of digestion by bead removal, which is well suited to robotic platforms for routine processing.
• Online 2D-LC partial digestion: immobilized RNase columns integrated into a two-dimensional LC setup permit precise control of digest stringency via column temperature and flow rate, enabling fully automated online digestion and transfer into HRAM analysis.

Both approaches rely on generating overlapping oligoribonucleotide fragments that collectively provide high-confidence sequence coverage and can be combined with complementary enzyme digests to improve coverage across structured regions.

Poue instrumentace

The workflow uses a single-vendor instrument and consumable set to simplify method transfer and QC implementation. Key components used in the case study include:

• Thermo Scientific KingFisher Duo Prime Purification System for automated handling of magnetic-bead immobilized RNase digestions.
• SMART Digest RNase Kits and SMART Digest RNase Columns for controlled partial digestion (offline bead format and online column format, respectively).
• Thermo Scientific Vanquish high-throughput LC system coupled with a DNAPac RP analytical column for oligoribonucleotide separation.
• Thermo Scientific Orbitrap Exploris 240 and 480 mass spectrometers for high-resolution accurate mass MS and MS/MS acquisition.
• Thermo Scientific BioPharma Finder software for peptide/oligo identification and downstream data export.

Main results and discussion

Partial digestion markedly improved unique fragment generation compared with complete digestion. The offline bead-based automated digests provided simple, repeatable control over digestion endpoints because enzyme removal halts reaction instantaneously. The online 2D-LC approach achieved highly reproducible partial digestions across a ~4.2 kb mRNA and enabled an end-to-end automated run that also quantified 5 capping efficiency and poly(A) tail length/heterogeneity within the same LCMS/MS session.

Using oligo-focused fragmentation and HRAM MS/MS allowed confident assignment of long oligoribonucleotides; combining overlapping fragments and complementary enzyme digests reached ~98% unique-fragment sequence coverage in under 60 minutes per run for tested constructs. Reproducibility overlays demonstrated tight retention time and response consistency across multiple injections, supporting routine QC use.

To facilitate rapid, standardized interpretation, the Sheffield team developed bespoke visualization tools that ingest BioPharma Finder outputs and produce linear and spiral sequence maps. These visualizations condense complex MS/MS evidence into clear maps, allow merging results from complementary digests, and can combine under- and over-digest injections to resolve difficult regions. What previously required a day of manual curation was reduced to minutes per visualization.

Method robustness was supported by recommended QC parameters (for example: ppm mass windows, confidence thresholds, acceptable signal ratios, MS2-only identifications and use of random-sequence decoys) that enable SOPs and method transfer between labs.

Benefits and practical applications

The combined approach offers several practical advantages for labs working with mRNA therapeutics:

• QC readiness: high sequence coverage, reproducibility and built-in MS/MS confirmation support identity testing and batch release workflows.
• Multi-attribute capability: concurrent measurement of identity, 5 capping, and poly(A) length/heterogeneity in the same analytical session reduces assay burden.
• Automation-friendly: bead-based and online digestion formats integrate with automated platforms for high throughput and consistent operation.
• Standardization and transferability: single-vendor hardware/software plus defined acceptance criteria facilitate SOP development and inter-lab method transfers.
• Efficient data interpretation: bespoke visualization tools dramatically lower analyst time and increase clarity for QA review.

Future trends and applications

As mRNA therapeutics scale, the need for high-throughput, robust, and auditable sequence-mapping workflows will grow. Expected developments include:

• Broader adoption of online 2D-LC partial digestion for routine QC to minimize manual handling and increase throughput.
• Further integration of visualization and automated decision rules to support regulatory reporting and batch release documentation.
• Extension of complementary enzyme panels and tailored digestion protocols to cover highly structured or chemically modified regions.
• Application of standardized decoy and scoring strategies to harmonize specificity metrics across laboratories and platforms.
• Potential coupling with orthogonal techniques (e.g., nanopore sequencing, capillary electrophoresis) for enhanced impurity profiling and full-length molecule characterization.

Conclusion

The Thermo FisherUniversity of Sheffield collaboration demonstrates a practical, QC-ready LCMS/MS blueprint for mRNA sequence mapping that combines controlled partial digestion, automated sample handling, HRAM MS/MS and bespoke visualization tools. The approach resolves common analytical challenges for large RNAs by producing longer, unique fragments, achieving near-complete sequence coverage rapidly and reproducibly, and enabling multi-attribute monitoring in a single workflow. These attributes make the workflow attractive for adoption in QC labs supporting scaled mRNA production.

References

1. Vanhinsbergh CJ, Criscuolo A, Sutton JN, Murphy K, Williamson AJK, Cook K, Dickman MJ. Characterization and Sequence Mapping of Large RNA and mRNA Therapeutics Using Mass Spectrometry. Analytical Chemistry. 2022;94(20):73397349. doi: 10.1021/acs.analchem.2c00765.
2. Welbourne EN, Copley RJ, Owen GR, Evans CA, Isoko K, Cook K, Cordiner J, Kis Z, Moghadam PZ, Dickman MJ. Mass spectrometry-based mRNA sequence mapping via complementary RNase digests and bespoke visualisation tools. Analyst. 2025;150(5):10121021. doi: 10.1039/d5an00033e.