Accurate and consistent analysis of poly(A) tails of mRNA therapeutics on a UHPLC-HRAM-MS platform

Significance of the Topic

Messenger RNA therapeutics rely on poly(A) tails to enhance stability and translation efficiency. Precise characterization of poly(A) tail length and distribution is critical for understanding the quality, potency, and consistency of in vitro transcribed (IVT) mRNA products. Traditional methods such as chromatography, PCR, or sequencing either lack resolution or require extensive preparation, underscoring the need for a robust, streamlined analytical approach.

Study Goals and Overview

This study aimed to establish an automated, compliance-ready workflow for accurate and reproducible analysis of poly(A) tails in IVT Cas9 mRNA. The method employs ion-pairing reversed-phase UHPLC coupled to high-resolution accurate-mass (HRAM) mass spectrometry. The developed eWorkflow in Chromeleon CDS was tested on three different LC-MS systems—an Orbitrap Exploris 240 and two Orbitrap Exploris MX instruments—to verify seamless method transfer and data consistency across platforms.

Methodology and Instrumentation

Sample Preparation:

• In vitro transcription of Cas9 mRNA followed by RNase T1 digestion.
• Selective extraction of poly(A) tails using Dynabeads Oligo(dT)25 magnetic beads.
• Dry down and reconstitution in 95:5 solvent A:B (15 mM DBA/25 mM HFIP in water : acetonitrile).

Instrumentation and Chromatography:

• UHPLC: Thermo Scientific Vanquish Horizon system with DNAPac RP column (2.1 × 100 mm, 4 μm).
• Mass Spectrometry: Orbitrap Exploris 240 and Exploris MX (BioPharma Option) operating in intact protein mode at 180,000 resolution (m/z 200), negative ion mode.
• Mobile phases: Solvent A—15 mM dibutylamine (DBA) and 25 mM hexafluoroisopropanol (HFIP) in water; Solvent B—acetonitrile; gradient 15–90% B over 15 min.

Data Processing and Reporting:

• Automated eWorkflow in Chromeleon CDS 7.3.2, including full MS acquisition, intact mass deconvolution (Xtract), and fit-for-purpose report template.
• Automatic calculation of monoisotopic mass accuracy (±20 ppm threshold) and median tail length.

Main Results and Discussion

The workflow achieved single-nucleotide resolution of poly(A) tail lengths ranging from 117 to 132 adenosines with mass errors below 5 ppm. Median tail lengths for the three replicate digests were consistently around 124.5–125. Reproducibility was confirmed across all three LC-MS systems, demonstrating the robustness of the eWorkflow for seamless method transfer. Automated reporting eliminated manual data handling, facilitating rapid assessment of poly(A) distributions and key quality attributes.

Benefits and Practical Applications

• High-resolution mass spectrometric analysis provides definitive poly(A) tail length characterization without extensive library preparation.
• Automated, compliance-ready reporting supports quality control in mRNA therapeutic development and manufacturing.
• Seamless eWorkflow transfer across instruments reduces implementation time and variability between laboratories.

Future Trends and Opportunities

As mRNA-based therapies expand beyond infectious disease to oncology and personalized medicine, demand for rapid, precise, and high-throughput analytics will increase. Integration of automated LC-MS workflows with digital lab notebooks and real-time data analytics could further streamline process development and regulatory submissions. Advances in MS instrumentation and software algorithms may enable even higher throughput and deeper profiling of RNA modifications.

Conclusion

The described UHPLC-HRAM-MS eWorkflow enables accurate, reproducible, and automated analysis of IVT mRNA poly(A) tails across multiple mass spectrometry platforms. Its high resolution and ease of transfer make it an attractive solution for routine QC and method validation in the growing field of mRNA therapeutics.

References

1. Clifford T. H. et al. mRNA-LNP expressing PfCSP and Pfs25 vaccine candidates targeting infection and transmission of Plasmodium falciparum. NPJ Vaccines. 2022;7(1). DOI:10.1038/s41541-022-00577-8
2. Neuzil KM. An mRNA Influenza Vaccine—Could It Deliver? N Engl J Med. 2023. DOI:10.1056/NEJMcibr2215281
3. Qin S. et al. mRNA-based therapeutics: powerful and versatile tools to combat diseases. Signal Transduct Target Ther. 2022;7:10.1038/s41392-022-01007-w
4. Beverley M. et al. Poly A tail length analysis of in vitro transcribed mRNA by LC-MS. Anal Bioanal Chem. 2018;410:1667–1677.
5. Thermo Fisher Scientific. Characterization of in vitro-transcribed (IVT) mRNA poly(A) tail by LC-HRAM-MS and BioPharma Finder 5.0 software. Application Note AN001183.