Session: Parallel session 9 - Top-down and Structural analysis

High Mass Analysis of Intact mRNAs by Mass Spectrometry and Mass Photometry: The Good, the Bad, and the Ugly

Evolène DESLIGNIÈRE¹, Lauren BARNES², Thomas POWERS², Olga FRIESE², Albert HECK¹

¹Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, Netherlands
²BioTherapeutics Pharmaceutical Sciences, Pfizer Inc, Chesterfield, MO, United States

The success of mRNA vaccines to combat COVID-19 has encouraged biopharmaceutical companies to invest in broader applications for various diseases. Analytical approaches must keep pace to support the development of mRNA therapies. Intact mass analysis of mid-to-large mRNAs (>1000 nucleotides) poses significant analytical challenges due to their size, heterogeneity, and instability. Here, we show how single-ion Orbitrap-based charge detection MS (CDMS) and mass photometry (MP) can rapidly and accurately measure the mass of intact high mass capped mRNAs, up to 9400 nucleotides (~3 MDa).

mRNAs were purchased from TriLink Biotechnologies and supplied by Pfizer. MS experiments were performed on a UHMR Orbitrap (Thermo Scientific) by static direct infusion. For CDMS, parameters were tuned to maximize ion survival (transient 64–512 ms). Thousands of single ions were recorded for ~30 minutes. Mass photometry measurements were performed on a SamuxMP instrument (Refeyn) using APTES-coated glass slides.

In ensemble MS experiments, broad or even unresolved charge distributions were obtained due to the presence of polyA tail variants. While approximate masses could be extracted for mRNAs <2000 nucleotides, ensemble MS failed to provide information for larger samples.

Drawbacks of ensemble MS could be avoided by recording single ions. Unstable ion behavior made CDMS measurements in native conditions difficult. Working instead in denaturing conditions was particularly beneficial for CDMS as high charges directly translate into better S/N and reduced charge uncertainty, with drastically improved mass accuracy, especially for large mRNAs.

Lastly, in-solution MP allowed to measure large mRNAs with high accuracy (only 0.5% off from theory). MP revealed low amounts of mRNA fragments and dimers that can be overlooked in CDMS.

Overall, CDMS and MP are innovative complementary approaches enabling the study of even large heterogeneous mRNA without requiring prior digestion or online separation.