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. 2024 Oct;45(19-20):1834-1839.
doi: 10.1002/elps.202400044. Epub 2024 Jun 20.

Detection of residual T7 RNA polymerase used in mRNA in vitro transcription by Simple Western

Yvonne Shieh  1 , Andrew R Swartz  2 , Richard R Rustandi  1
Affiliations

Detection of residual T7 RNA polymerase used in mRNA in vitro transcription by Simple Western

Yvonne Shieh et al. Electrophoresis. 2024 Oct.

Abstract

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.

Keywords: Simple Western; T7 RNA polymerase; capillary western; mRNA vaccine; purification.

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References

REFERENCES

    1. Pilkington EH, Suys EJA, Trevaskis NL, Wheatley AK, Zukancic D, Algarni A, et al. From influenza to COVID‐19: lipid nanoparticle mRNA vaccines at the frontiers of infectious diseases. Acta Biomater. 2021;131:16–40.
    1. Maruggi G, Zhang C, Li J, Ulmer JB, Yu D. mRNA as a transformative technology for vaccine development to control infectious diseases. Mol Ther. 2019;27:757–772.
    1. Henderson JM, Ujita A, Hill E, Yousif‐Rosales S, Smith C, Ko N, et al. Cap 1 messenger RNA synthesis with co‐transcriptional CleanCap® analog by in vitro transcription. Curr Protoc. 2021;1:e39.
    1. Mamat U, Wilke K, Bramhill D, Schromm AB, Lindner B, Kohl TA, et al. Detoxifying Escherichia coli for endotoxin‐free production of recombinant proteins. Microb Cell Fact. 2015;14:57.
    1. World Health Organization. Evaluation of the quality, safety and efficacy of messenger RNA vaccines for the prevention of infectious diseases: regulatory considerations. In: WHO expert committee on biological standardization: seventy‐fourth report. Geneva: World Health Organization; 2022: Annex 3 (WHO Technical Report Series, No. 1039. https://cdn.who.int/media/docs/default‐source/biologicals/vaccine‐standa...)

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