In recent years, the development and application of mRNA technology have continued to attract attention. mRNA drugs involve inoculating mRNA that encodes antigen proteins into the human body. By utilizing the genetic material inside human cells, they express and synthesize antigen proteins. This process induces and activates the body's immune system through antigen proteins, aiming to prevent and treat diseases. mRNA drugs have advantages such as safety, efficiency, and a short cycle. They can simultaneously induce humoral and cellular immunity and have been applied in various indications, including tumors, infectious diseases, rare diseases, cardiovascular diseases, and more.
The entire process of mRNA drug development can be roughly divided into several steps:
Sequence determination & design—mRNA in vitro transcription—encapsulation preparation
The conventional steps of mRNA in vitro transcription (IVT) include:
Plasmid DNA extraction & linearization—Plasmid DNA purification—In vitro transcription (co-transcriptional capping)—Purification—mRNA stock solution
The onepot mRNA in vitro transcription (IVT) process include:
Plasmid DNA extraction & linearization—In vitro transcription (co-transcriptional capping)—Purification—mRNA stock solution
Currently, most existing processes involve a single purification step for plasmid DNA after enzymatic cleavage before the IVT step. Yeasen Biotechlogy, based on a mature mRNA application development center, has developed the "Onepot mRNA Transcription" process. In this process, circular plasmid DNA is not purified after enzymatic cleavage; instead, it is directly used for in vitro transcription, resulting in a high-quality mRNA stock solution. This entire process, while ensuring product and process quality, shortens the duration of the existing process.
Process advantages:
- Process optimization, reducing IVT steps for simpler operation.
- Lower material costs, eliminating the need for post-cleavage purification and quality inspection.
- Maintaining mRNA quality and yield.
Data:
1. Yields and Integrity:
Using the 1μg of linearized 2K, 4K, and 9K plasmids, the onepot process can generate 150-200μg of mRNA.
| Length | Yields | Integrity |
| 2K | 200 μg | 94.00 % |
| 4K | 185 μg | 92.20 % |
| 9K | 150 μg | 87.50 % |
Integrity testing was performed using capillary electrophoresis (CE) to assess the integrity of fragments with lengths of 2K, 4K, and 9K. The integrity of 2K and 4K fragments was >92%, and the integrity of 9K fragments was >87%.
2. mRNA Capping Efficiency Detection
LC-MS was employed to assess the capping efficiency of the 2K sequence, revealing a capping efficiency of 99.5%.
Top image: HPLC UV chromatogram
Bottom image: Deconvoluted molecular weight spectrum
3. mRNA Polyadenylation Efficiency Detection
LC-MS was used to detect the polyadenylation efficiency of samples, showing results distributed normally.
Top image: Liquid chromatography TIC chromatogram
Bottom image: Deconvoluted molecular weight spectrum
4. mRNA Expression Assay
Transfection of 293T cells with mRNA products synthesized using both the conventional process (Left, linearized plasmids with purification) and the one-pot process (Right, linearized plasmids without purification) showed no difference in fluorescence protein expression after 24 hours of culture.
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