Letao Guo , Zhikai Liu , Shirong Song , Wang Yao , Mei Yang , Guangwen Chen
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Abstract
In vitro transcription (IVT) is the main manufacturing method to produce mRNA vaccines. In this study, a fed-batch strategy was systematically optimized for IVT process with dual goals of achieving a high reaction rate and maximizing the final mRNA yield simultaneously. Initially various experimental conditions were investigated including Mg2+, nucleotide triphosphates (NTP), dithiothreitol (DTT), spermidine, as well as the temperature and ionic strength. It was found that the concentrations of Mg2+ and NTP had a significant impact on IVT process. Subsequently, under the optimized conditions, dividing the IVT reaction into three distinct phases was proposed to enable more efficient transcription. By optimizing the concentrations of Mg2+ and NTP in two replenishment processes, our fed-batch strategy resulted in the production of 367.8 μg of mRNA with reduced dsRNA byproducts within 180 min. This was achieved under conditions of a final volume of 30 μL, 250 U T7 RNA polymerase (RNAP), and 2 μg DNA template. In conclusion, the IVT process using a fed-batch approach, rather than an excessive one-time NTP input, is an efficient method to improve productivity given a fixed amount of T7 RNAP and DNA template.
期刊介绍:
The Biochemical Engineering Journal aims to promote progress in the crucial chemical engineering aspects of the development of biological processes associated with everything from raw materials preparation to product recovery relevant to industries as diverse as medical/healthcare, industrial biotechnology, and environmental biotechnology.
The Journal welcomes full length original research papers, short communications, and review papers* in the following research fields:
Biocatalysis (enzyme or microbial) and biotransformations, including immobilized biocatalyst preparation and kinetics
Biosensors and Biodevices including biofabrication and novel fuel cell development
Bioseparations including scale-up and protein refolding/renaturation
Environmental Bioengineering including bioconversion, bioremediation, and microbial fuel cells
Bioreactor Systems including characterization, optimization and scale-up
Bioresources and Biorefinery Engineering including biomass conversion, biofuels, bioenergy, and optimization
Industrial Biotechnology including specialty chemicals, platform chemicals and neutraceuticals
Biomaterials and Tissue Engineering including bioartificial organs, cell encapsulation, and controlled release
Cell Culture Engineering (plant, animal or insect cells) including viral vectors, monoclonal antibodies, recombinant proteins, vaccines, and secondary metabolites
Cell Therapies and Stem Cells including pluripotent, mesenchymal and hematopoietic stem cells; immunotherapies; tissue-specific differentiation; and cryopreservation
Metabolic Engineering, Systems and Synthetic Biology including OMICS, bioinformatics, in silico biology, and metabolic flux analysis
Protein Engineering including enzyme engineering and directed evolution.
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Tris-HCl buffer (pH = 8.0, a stock concentration of 1M)