We developed a rapid chromatographic method which separates IVT reaction components, and can be used to monitor NTP consumption and mRNA production. With this method we can optimize IVT reaction, maximize mRNA yield while minimizing the formation of process impurities and the cost of the reaction. Our methodology was pioneering in the field of mRNA, showing how coupling analytical data of IVT reaction can lead to higher yields and lower costs.
The field is now exploring other inline process analytical tools, such as Raman spectroscopy, but chromatography still retains a pivotal place due to its resolving power and high speed. We showed how to capture high throughput analytical tools with design of experiment software to make a comprehensive optimization of IVT reaction. This led to highest every reported yield of IVT reaction of 25 grams per liter.
Our method allows to monitor both mRNA and NTP concentrations simultaneously with short readout times, therefore is suitable for near realtime IVT monitoring. The mRNA field is now moving towards continuing manufacturing of mRNA, and we are of course developing next generation tools to support this transition. To begin, thaw and preheat all the reagents, except enzymes, at 37 degrees Celsius for 15 minutes in a thermo block set at 300 revolutions per minute.
Mark each tube with a designated in vitro transcription or IVT number and time point. While the reagents are thawing, pipette two microliters of 100 millimolar EDTA into 0.5 milliliter sterile tubes. Now remove the enzymes from the freezer and store them temporarily in a cooler.
Prepare 50 to 100 microliters of the reaction mix with appropriate volumes of IVT reagents. Add T7 RNA polymerase as the last reagent when all IVT reagents are mixed. Immediately remove two microliters of the IVT mixture and pipette it into previously prepared 0.5 milliliter tubes containing two microliters EDTA.
Place the reaction tubes containing IVT mixtures in the thermo block and incubate them at 37 degrees Celsius. At each desired time point, remove two microliters of the IVT sample from the reaction mixture and pipette into tubes containing EDTA. After chromatographic analysis confirms the complete depletion of NTPs, quench bulk IVT reactions with EDTA to a final concentration of 50 millimolar.
For Fed-Batch IVT reaction, mix stocks of each NTP and magnesium chloride. After setting up the reaction, when the NTP concentration drops below 10%add an appropriate volume of the feed to the bulk IVT reaction. Once the desired mRNA production is achieved, inactivate the entire reaction with EDTA to a final concentration of 50 millimolar.
For column conditioning, equilibrate a 0.1 milliliter analytical column at room temperature for 12 hours prior to analysis. Attach the column to the chromatographic system, following the direction indicated on the column housing. Then flush the column with 50 column volumes of double distilled water followed by 50 column volumes of MPA at a flow rate of one milliliter per minute.
Then, run at least three blank samples injecting only MPA before analysis to establish the baseline. Now proceed to analyze the system suitability test sample and calibration curve samples. For the system suitability test, combine the capping reagent NTPs, plasmid DNA template, and the mRNA.
Now create the calibration standard using the diluted purified mRNA sample of known concentration with mobile phase A.If required, create standards with final NTP concentrations of 0.5, two, five, 10, 15, and 20 micromolar for generating the calibration curve for all NTPs. Determine the minimal dilution needed for IVT samples using the formula. Before diluting for chromatographic analysis, vortex and spin down the quenched IVT sample.
Pipette MPA and sodium chloride into a conical glass vial, then add the quenched IVT sample. Vortex the prepared sample and load it into the autosampler, set at four degrees Celsius. Inject 25 microliters of the diluted sample onto the analytical column and measure absorbance at 260 and 280 nanometers for each IVT sample.
After completing the analysis of all IVT samples, inject the system suitability test standard again to confirm system stability. Fast at-line analytics allows to monitor the production of mRNA over time. In this experiment, we compared the influence of IVT buffer composition on IVT kinetics.
IVT performed with buffer A resulted highest rate of mRNA production, followed by buffer B and buffer C.IVT reaction can also be performed as a Fed-Batch. Monitoring the consumption of NTPs allows to adjust the concentration by feeding NTPs and magnesium chloride to the reaction. Because feed additions dilute the IVT reaction, the mRNA concentration in IVT drops at each feed edition.
