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Recovery method of anti-reverse cap analog in mRNA in-vitro transcription process, anti-reverse cap analog and application

A recovery method and in vitro transcription technology, applied in the biological field, can solve problems such as the inability to encode proteins normally, and achieve the effect of reducing costs and improving utilization efficiency

Pending Publication Date: 2021-03-19
珠海丽凡达生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

And the cap can make the mRNA more stable and will not be degraded by exonuclease, and the mRNA without the cap cannot normally encode protein

Method used

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  • Recovery method of anti-reverse cap analog in mRNA in-vitro transcription process, anti-reverse cap analog and application
  • Recovery method of anti-reverse cap analog in mRNA in-vitro transcription process, anti-reverse cap analog and application
  • Recovery method of anti-reverse cap analog in mRNA in-vitro transcription process, anti-reverse cap analog and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1 Preliminary Recovery of Anti-Reverse Cap Analogs Using Reversed Phase High Performance Liquid Chromatography

[0067] This example illustrates the initial recovery of anti-reverse cap analogs by reverse phase high performance liquid chromatography and the confirmation of the final purified anti-reverse cap analogs.

[0068] Prepare 0.1M TEAA as the mobile phase, run at a column temperature of 45°C for 10 minutes at a flow rate of 1ml / min to recover anti-reverse cap analogs, and dilute the remaining mixed solution after the RNA transcription reaction by 2 times with 0.1M TEAA, The injection volume was 100 μl, and the peak elution time of each component was observed and the anti-reverse cap analog peak was collected. image 3 It is the liquid chromatogram of the remaining mixture after the RNA transcription reaction. It can be seen that the mixture contains residual NTP and anti-reverse cap analogs, and the two substances can be clearly separated. At this time, ...

Embodiment 2

[0069] Embodiment 2 Dialysis removes the method for NTP, TEAA

[0070] This implementation example illustrates the method of using dialysis to remove impurities in the chromatographic mobile phase from the chromatographically purified sample. The principle is to use TEAA (about 0.16KD) and the molecular weight of the chromatographic solvent to be small while the molecular weight of the anti-reverse cap analog (about 1KD) is relatively large, and to use a 0.5KD dialysis bag to selectively replace the remaining chromatographic mobile phase solvent For Nuclease-Free Water.

[0071] Boil the dialysis bag in purified water for 10 minutes, take it out for later use; add 1 volume of liquid-phase preliminary purified solution into the dialysis bag, and seal it; add 20 times the volume of Nuclease-Free Water into the beaker, and put it into a magnetic stirrer; Place the dialysis bag in the beaker, place the beaker on a magnetic stirrer, continue stirring, and dialyze for 3 hours; pour...

Embodiment 3

[0073] Example 3 Quantify the recovered and purified anti-reverse cap analogs

[0074] This example illustrates a method to quantify recovery of purified anti-reverse cap analogs.

[0075] Using a brand-new anti-reverse cap analog, serially diluted to 6.25mM, 3.13mM, 1.56mM, 0.78mM, 0.39mM, 0.20mM, using an ultra-micro spectrophotometer to measure its A260, and using Excel to plot a standard curve ( Such as Image 6 ); the sample to be tested is diluted in different multiples until its A260 falls within the range of the standard curve, and the concentration of the sample to be tested is calculated by the fitting equation of the standard curve.

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Abstract

The invention provides a recovery method of an anti-reverse cap analog in an mRNA in-vitro transcription process, the anti-reverse cap analog and application, and relates to the technical field of biology. The recovery method of the anti-reverse cap analog in the mRNA in-vitro transcription process provided by the invention is suitable for the in-vitro transcription process of any type of mRNA. According to the recovery method, the anti-reverse cap analog in reaction liquid after the mRNA transcription reaction is preliminarily separated through a high performance liquid chromatography; and then the anti-reverse cap analog can be obtained by sequentially purifying, concentrating and drying the crude product of the anti-reverse cap analog obtained through preliminary separation, so that therepeated utilization of the anti-reverse cap analog is realized. According to the recovery method, the mRNA production cost is effectively reduced; and the utilization efficiency of raw materials isimproved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for recovering anti-reverse cap analogs during mRNA in vitro transcription, anti-reverse cap analogs and applications. Background technique [0002] Messenger RNA (mRNA) drugs are a promising new treatment option with broad prospects in the fields of immunology, oncology and vaccines. mRNA contains instructions for cells to make proteins and send them to various parts of the body, so mRNA can be used as medicine to direct protein expression inside cells or protein secretion outside cells. Effective mRNA therapy requires efficient delivery of the mRNA to the patient and efficient production of the protein encoded by the mRNA in the patient. [0003] The basic structure of the mRNA stock solution product includes at least: the 5'cap structure and the specific mRNA sequence encoding the protein ( figure 1 ). Among them, the 5' cap structure of mRNA is particularly critical. ...

Claims

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Application Information

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IPC IPC(8): C07H21/02C07H1/06C12N15/10
CPCC07H21/02C07H1/06C12N15/10
Inventor 苏晓晔李爽雷奕欣朱雅婧彭育才
Owner 珠海丽凡达生物技术有限公司
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