Detection of residual T7 RNA polymerase used in mRNA in vitro transcription by Simple Western.
Simple Western
T7 RNA polymerase
capillary western
mRNA vaccine
purification
Journal
Electrophoresis
ISSN: 1522-2683
Titre abrégé: Electrophoresis
Pays: Germany
ID NLM: 8204476
Informations de publication
Date de publication:
20 Jun 2024
20 Jun 2024
Historique:
revised:
03
05
2024
received:
01
03
2024
accepted:
28
05
2024
medline:
20
6
2024
pubmed:
20
6
2024
entrez:
20
6
2024
Statut:
aheadofprint
Résumé
Therapeutic messenger RNA (mRNA) has been demonstrated as a scalable and versatile vaccine platform for the rapid development and manufacture of new vaccine candidates. mRNA is synthesized enzymatically through in vitro transcription (IVT) using bacteriophage T7 RNA polymerase (T7 RNAP), a 99 kDa protein with high binding affinity for the promoter sequence and a low error rate. Post-IVT, mRNA is purified to remove impurities, but if T7 RNAP is insufficiently cleared, undesirable clinical side effects may result. Therefore, it is important to quantitate T7 RNAP concentrations in IVT and process intermediates to understand clearance during downstream purification. A high-throughput T7 RNAP assay was developed using Simple Western (SW), a capillary immunoassay technology, to quantitate concentrations as low as 5.3 ng/mL with good precision and accuracy. Compared to existing T7 RNAP immunoassays or total protein assays such as bicinchoninic acid assays or Bradford, the SW T7 RNAP assay is specific to T7 RNAP, requires <10 µL of sample volume, and consists of minimal sample handling and hands-on time. This work highlights the development and optimization of a highly sensitive and robust T7 RNAP quantitation assay using the SW platform.
Identifiants
pubmed: 38899564
doi: 10.1002/elps.202400044
doi:
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Informations de copyright
© 2024 Merck Sharp & Dohme LLC. Electrophoresis published by Wiley‐VCH GmbH.
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