Characterization of intact mRNA-based therapeutics by charge detection mass spectrometry and mass photometry
The impressive success of mRNA-based vaccines to combat COVID-19 has encouraged biopharmaceutical companies to invest in broader applications of alike vaccines for various diseases. Analytical approaches must keep pace to support this surge in the development of mRNA-based therapies. Intact mass ana...
Saved in:
| Main Authors: | , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2025-06-01
|
| Series: | Molecular Therapy: Methods & Clinical Development |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S232905012500049X |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1850262416687890432 |
|---|---|
| author | Evolène Deslignière Lauren F. Barnes Thomas W. Powers Olga V. Friese Albert J.R. Heck |
| author_facet | Evolène Deslignière Lauren F. Barnes Thomas W. Powers Olga V. Friese Albert J.R. Heck |
| author_sort | Evolène Deslignière |
| collection | DOAJ |
| description | The impressive success of mRNA-based vaccines to combat COVID-19 has encouraged biopharmaceutical companies to invest in broader applications of alike vaccines for various diseases. Analytical approaches must keep pace to support this surge in the development of mRNA-based therapies. Intact mass analysis of mid- to large mRNA molecules (>1,000 nt) poses significant analytical challenges due to mRNA size, heterogeneity, and instability. Here, we demonstrate how single-particle Orbitrap-based charge detection mass spectrometry (CDMS) and mass photometry (MP) approaches can rapidly measure the mass of various intact high-mass capped mRNAs, up to 9,400 nt (∼3 MDa) in size. While ensemble MS yielded approximate masses for mRNAs <2,000 nt, it failed to provide information on samples of longer sequences. The drawbacks of ensemble MS could be avoided by recording individual ions. Low-charge mRNA components showed unstable ion behavior, hampering initial CDMS measurements, whereas high-charge populations offered better signal-to-noise and reduced charge uncertainty, with drastically improved mass accuracy. Lastly, in-solution MP enabled the measurement of mRNAs with high accuracy, while revealing low amounts of mRNA fragments and dimers that are sometimes overlooked in CDMS. Overall, CDMS and MP provide complementary methods that enable the study of large heterogeneous mRNA without requiring prior digestion or online separation. |
| format | Article |
| id | doaj-art-25d5df6171dd404eba510a54a1b6daeb |
| institution | OA Journals |
| issn | 2329-0501 |
| language | English |
| publishDate | 2025-06-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Molecular Therapy: Methods & Clinical Development |
| spelling | doaj-art-25d5df6171dd404eba510a54a1b6daeb2025-08-20T01:55:11ZengElsevierMolecular Therapy: Methods & Clinical Development2329-05012025-06-0133210145410.1016/j.omtm.2025.101454Characterization of intact mRNA-based therapeutics by charge detection mass spectrometry and mass photometryEvolène Deslignière0Lauren F. Barnes1Thomas W. Powers2Olga V. Friese3Albert J.R. Heck4Biomolecular Mass Spectrometry and Proteomics, Bijvoet Centre for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, 3584 CH Utrecht, the Netherlands; Netherlands Proteomics Center, 3584 CH Utrecht, the NetherlandsBioTherapeutics Pharmaceutical Sciences, Pfizer Inc, Chesterfield, MO 63017, USABioTherapeutics Pharmaceutical Sciences, Pfizer Inc, Chesterfield, MO 63017, USABioTherapeutics Pharmaceutical Sciences, Pfizer Inc, Chesterfield, MO 63017, USABiomolecular Mass Spectrometry and Proteomics, Bijvoet Centre for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, 3584 CH Utrecht, the Netherlands; Netherlands Proteomics Center, 3584 CH Utrecht, the Netherlands; Corresponding author: Albert J.R. Heck, Biomolecular Mass Spectrometry and Proteomics, Bijvoet Centre for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, 3584 CH Utrecht, the Netherlands.The impressive success of mRNA-based vaccines to combat COVID-19 has encouraged biopharmaceutical companies to invest in broader applications of alike vaccines for various diseases. Analytical approaches must keep pace to support this surge in the development of mRNA-based therapies. Intact mass analysis of mid- to large mRNA molecules (>1,000 nt) poses significant analytical challenges due to mRNA size, heterogeneity, and instability. Here, we demonstrate how single-particle Orbitrap-based charge detection mass spectrometry (CDMS) and mass photometry (MP) approaches can rapidly measure the mass of various intact high-mass capped mRNAs, up to 9,400 nt (∼3 MDa) in size. While ensemble MS yielded approximate masses for mRNAs <2,000 nt, it failed to provide information on samples of longer sequences. The drawbacks of ensemble MS could be avoided by recording individual ions. Low-charge mRNA components showed unstable ion behavior, hampering initial CDMS measurements, whereas high-charge populations offered better signal-to-noise and reduced charge uncertainty, with drastically improved mass accuracy. Lastly, in-solution MP enabled the measurement of mRNAs with high accuracy, while revealing low amounts of mRNA fragments and dimers that are sometimes overlooked in CDMS. Overall, CDMS and MP provide complementary methods that enable the study of large heterogeneous mRNA without requiring prior digestion or online separation.http://www.sciencedirect.com/science/article/pii/S232905012500049XmRNAintact massnative mass spectrometrycharge detection mass spectrometrymass photometrysingle particle |
| spellingShingle | Evolène Deslignière Lauren F. Barnes Thomas W. Powers Olga V. Friese Albert J.R. Heck Characterization of intact mRNA-based therapeutics by charge detection mass spectrometry and mass photometry Molecular Therapy: Methods & Clinical Development mRNA intact mass native mass spectrometry charge detection mass spectrometry mass photometry single particle |
| title | Characterization of intact mRNA-based therapeutics by charge detection mass spectrometry and mass photometry |
| title_full | Characterization of intact mRNA-based therapeutics by charge detection mass spectrometry and mass photometry |
| title_fullStr | Characterization of intact mRNA-based therapeutics by charge detection mass spectrometry and mass photometry |
| title_full_unstemmed | Characterization of intact mRNA-based therapeutics by charge detection mass spectrometry and mass photometry |
| title_short | Characterization of intact mRNA-based therapeutics by charge detection mass spectrometry and mass photometry |
| title_sort | characterization of intact mrna based therapeutics by charge detection mass spectrometry and mass photometry |
| topic | mRNA intact mass native mass spectrometry charge detection mass spectrometry mass photometry single particle |
| url | http://www.sciencedirect.com/science/article/pii/S232905012500049X |
| work_keys_str_mv | AT evolenedesligniere characterizationofintactmrnabasedtherapeuticsbychargedetectionmassspectrometryandmassphotometry AT laurenfbarnes characterizationofintactmrnabasedtherapeuticsbychargedetectionmassspectrometryandmassphotometry AT thomaswpowers characterizationofintactmrnabasedtherapeuticsbychargedetectionmassspectrometryandmassphotometry AT olgavfriese characterizationofintactmrnabasedtherapeuticsbychargedetectionmassspectrometryandmassphotometry AT albertjrheck characterizationofintactmrnabasedtherapeuticsbychargedetectionmassspectrometryandmassphotometry |