Characterization of Intact mRNA Using Ion Pair-Reversed Phase-Time of Flight-MS, Size Exclusion Chromatography-Multi Angle Light Scattering, and Charge Detection Mass Spectrometry

Significance of the Topic

Recent advances in mRNA based vaccines highlight the need for robust characterization of intact mRNA molecules to ensure quality and consistency in therapeutic products

Objectives and Study Overview

This study investigates three complementary analytical techniques for determining intact mass of three mRNA constructs including human erythropoietin mRNA, firefly luciferase mRNA and Cas9 mRNA with the goal of comparing ion pair reversed phase time of flight mass spectrometry, size exclusion chromatography with multi angle light scattering and charge detection mass spectrometry for accuracy range and workflow efficiency

Methodology and Instrumentation

• Ion pair reversed phase TOF MS using HFIP and DIPEA mobile phases on an ACQUITY HSS T3 C18 column with TUV detection at 260 nm and Synapt XS QTOF in negative mode
• Size exclusion chromatography with multi angle light scattering using a Waters BEH450 SEC column in TMAC buffer with TUV and HELEOS MALS detection
• Charge detection mass spectrometry on a prototype instrument with buffer exchange into ammonium acetate and static nano electrospray using a Triversa Nanomate with optimized trapping and quadrupole settings for high mass detection

Key Results and Discussion

• EPO mRNA mass measured at approximately 245 kDa by IP RP TOF MS, 283 kDa by SEC MALS and 285 kDa by CDMS with variations attributed to fragmentation and detector saturation
• FLuc mRNA gave 625 kDa by IP RP TOF MS, 616 kDa by SEC MALS and 634 kDa by CDMS suggesting possible adducts or modifications
• Cas9 mRNA exceeded the TOF mass range but was measured at 1490 kDa by SEC MALS and 1480 kDa by CDMS, with evidence of a dimer form in SEC MALS data

The three methods demonstrated consistent trend in intact mass determination. Chromatographic MS revealed structural variants, SEC MALS provided reliable aggregate profiling and CDMS enabled direct analysis of very large biomolecules without separation

Benefits and Practical Applications of the Method

• Ion pair reversed phase MS detects low abundance fragments and chemical modifications but may require method optimization and is limited in upper mass capability
• SEC MALS delivers accurate size and aggregate distribution in a single run with minimal development effort
• CDMS offers direct measurement of large biomolecules with simple sample preparation and no chromatography reducing analysis time

Future Trends and Applications

Advances in high mass mass spectrometry instrumentation and ionization strategies will extend range and sensitivity for intact mRNA analysis. Integration of CDMS with automated sample handling and hybrid separation approaches will improve throughput. Novel chromatographic phases and ion pairing chemistries will enhance resolution of sequence variants and modified nucleotides, supporting quality control of mRNA based therapeutics and next generation RNA based modalities

Conclusion

The comparison of IP RP TOF MS, SEC MALS and CDMS illustrates complementary strengths for intact mRNA characterization. SEC MALS and CDMS provide accurate mass across a broad size range while chromatographic MS adds structural insight enabling a comprehensive workflow for ensuring integrity and consistency of mRNA based products