Determining mRNA Capping with HILIC-MS on a Low-Adsorption Flow Path

Importance of the Topic

The success of mRNA vaccines against COVID-19 has driven widespread adoption of mRNA therapeutics. A critical quality attribute (CQA) for in vitro-transcribed (IVT) mRNA is the capping efficiency at the 5' terminus, as the cap structure enhances stability, cellular delivery, and translation efficiency. Reliable measurement of this attribute by liquid chromatography–mass spectrometry (LC-MS) is challenged by adsorption of negatively charged phosphate groups to metal surfaces in conventional flow paths.

Objectives and Overview of the Study

This application note evaluates the use of a low-adsorption hydrophilic interaction liquid chromatography (HILIC) system to determine mRNA capping efficiency. Key goals include:

• Comparing traditional stainless-steel versus PEEK-lined HILIC columns.
• Assessing stainless-steel LC versus biocompatible (metal-free) flow paths on Agilent 1290 Infinity II platforms.
• Characterizing 5'-terminal fragments generated by RNase H and RNase A digestion.

Methodology and Used Instrumentation

mRNA capping was analyzed using two probe-guided digestion workflows:

• RNase H method: Hybridization of a complementary DNA probe near the 5' end, followed by thermostable RNase H cleavage at 50 °C for 45 min, yielding a nonphosphorylated 3' terminus.
• RNase A method: A biotinylated probe protects the 5' region, enabling selective RNase A digestion at 37 °C for 60 min and streptavidin bead enrichment of 5' fragments.

Chromatographic separation employed a diol HILIC column (2.1×100 mm, 1.9 μm) in stainless-steel or PEEK-lined configuration, using a 20 mM ammonium acetate/acetonitrile gradient. Detection was performed on an Agilent 6545 LC/Q-TOF in negative electrospray mode. Two Agilent 1290 Infinity II systems (standard and Bio biocompatible) were evaluated.

Main Results and Discussion

Comparison of column hardware revealed that the PEEK-lined HILIC column dramatically improved peak intensity, shape, and reproducibility for short oligonucleotide standards, even at high dilutions, with no conditioning required. Stainless-steel columns exhibited progressive signal loss and tailing due to phosphate–metal interactions. Similarly, the biocompatible LC system enhanced signal stability compared with the standard stainless-steel system.
In application to IVT mRNA 5'-termini, the PEEK-lined/biocompatible configuration enabled clear detection of capped (m7GpppAm) and uncapped fragments, which were undetectable on conventional hardware. Mass spectra of capped fragments displayed complex charge and adduct distributions, emphasizing the need for maximum sensitivity.

Benefits and Practical Applications

Implementation of a low-adsorption HILIC-MS workflow allows:

• High-sensitivity quantification of mRNA capping efficiency without extensive column conditioning.
• Robust detection of 5'-terminal fragments for CQA monitoring in mRNA therapeutics.
• Reduced carryover and improved peak shape in routine QC laboratories.

Future Trends and Possibilities

Emerging directions include:

• Integration of HILIC-MS/MS sequencing for detailed mapping of cap variants and modifications.
• High-throughput automation of probe digestions and magnetic bead enrichments.
• Development of further inert flow path materials for expanded analysis of modified RNAs and oligonucleotides.

Conclusion

The combination of a PEEK-lined diol HILIC column and a metal-free Agilent 1290 Infinity II Bio LC system overcomes phosphate-metal adsorption issues, delivering reliable, high-sensitivity measurement of mRNA capping efficiency. This workflow enhances quality control in the manufacture of mRNA-based therapeutics.

References

1. Callaway E, Naddaf M. Pioneers of mRNA COVID Vaccines Win Medicine Nobel. Nature 2023;622:228–229.
2. Beverly M, Dell A, Parmar P, Houghton L. Label-Free Analysis of mRNA Capping Efficiency Using RNase H Probes and LC-MS. Anal Bioanal Chem 2016;408:5021–5030.
3. Liau B. Rapid Analysis of mRNA 5′ Capping with High Resolution LC/MS. Agilent Technologies application note 5994-3984EN;2021.
4. Morreel K, t’Kindt R, Debyser G, et al. Diving into the Structural Details of In Vitro Transcribed mRNA Using LC-MS-Based Oligonucleotide Profiling. LCGC Europe 2022;35:220–236.
5. Nwokeoji AO, Chou T, Nwokeoji EA. Low Resource Integrated Platform for Production and Analysis of Capped mRNA. ACS Synth Biol 2023;12:329–339.
6. Wolf EJ, Dai N, Chan SH, Corrêa IR Jr. Selective Characterization of mRNA 5′ End-Capping by DNA-Probe Directed Enrichment with Site-Specific Endoribonucleases. ACS Pharmacol Transl Sci 2023;6:1692–1702.
7. Guimaraes GJ, Kim J, Bartlett MG. Characterization of mRNA Therapeutics. Mass Spectrom Rev 2023;doi:10.1002/mas.21856.
8. Ramanathan A, Robb GB, Chan SH. mRNA Capping: Biological Functions and Applications. Nucleic Acids Res 2016;44:7511–7526.
9. Daniel S, Kis Z, Kontoravdi C, Shah N. Quality by Design for Enabling RNA Platform Production Processes. Trends Biotechnol 2022;40:1213–1228.
10. USP. Analytical Procedures for mRNA Vaccine Quality. USP guideline EA966W_2023-04, draft second edition.
11. Huang M, Xu X, Qiu H, Li N. Analytical Characterization of DNA and RNA Oligonucleotides by Hydrophilic Interaction Liquid Chromatography-Tandem Mass Spectrometry. J Chromatogr A 2021;1648:462184.
12. Goyon A, Nguyen D, Boulanouar S, Yehl P, Zhang K. Characterization of Impurities in Therapeutic RNAs at the Single Nucleotide Level. Anal Chem 2022;94:16960–16966.
13. Li G, Rye P. MS/MS Oligonucleotide Sequencing Using LC/Q-TOF with HILIC Chromatography. Agilent Technologies application note 5994-5632EN;2023.
14. Lardeux H, Goyon A, Zhang K, et al. The Impact of Low Adsorption Surfaces for the Analysis of DNA and RNA Oligonucleotides. J Chromatogr A 2022;1677:463324.
15. Schneider S. Analysis of Phosphate Compounds with the Agilent 1260 Infinity Bio-Inert Quaternary LC System. Agilent application note 5991-0025EN;2012.
16. Sandra K, Vandenbussche J, Vanhoenacker G, et al. Analysis of Nucleotides Using a Fully Inert Flowpath. Agilent Technologies application note 5994-0680EN;2019.
17. Schneider S. Comparability Studies for the Analysis of Nucleotides on Four Different LC Systems. Agilent Technologies application note 5994-4392EN;2021.
18. Vanhoenacker G, Sandra P, Sandra K, Schneider S, Huber U. Improving Peak Shape and Recovery in LC Studies of Phosphated Vitamin B2. Agilent application note 5994-5830EN;2023.