Closed continuous mRNA preparation device

Abstract

The utility model provides a closed continuous mRNA (messenger Ribonucleic Acid) preparation device which comprises an IVT (Intermediate Valve Timing) reactor, wherein a first feed liquid discharge port of the IVT reactor is connected with an IVT product transfer container; a second feed liquid discharge port of the IVT product transfer container is connected with the chromatography module, and a liquid outlet of the chromatography module is connected with the ultrafiltration module transfer container; the ultrafiltration module transfer container is connected with a first ultrafiltration membrane bag; the first ultrafiltration membrane bag is connected with LNPs encapsulation equipment; the LNPs encapsulation equipment is connected with LNPs dilution and liquid change equipment, and the LNPs dilution and liquid change equipment is connected with the second ultrafiltration membrane bag; and the second ultrafiltration membrane bag is connected with a product storage container. According to the utility model, the whole process can be continuously carried out and the process is closed, so that the risk of mRNA degradation caused by the pollution of the exposed environment to mRNA is reduced.

Description

Technical Field

[0001] The utility model provides a closed continuous mRNA preparation device, belonging to the field of biotechnology. Background Art

[0002] mRNA technology has shown many advantages in the fields of medicine, vaccine development, and drug manufacturing. It has the advantages of rapid response, production flexibility, in vivo autonomous translation, high safety, wide application potential, and easy updating. It has become an important development direction in the field of modern biomedicine. Although mRNA has many advantages, there are many difficulties and challenges in the preparation and purification of mRNA. mRNA has poor stability and a short half-life. It is easy to degrade during the purification process. Measures must be taken to maintain the stability of mRNA during the extraction and purification process, such as using RNase inhibitors, low temperature operation, and avoiding mechanical shearing.

[0003] The linear DNA template is synthesized by in vitro transcription (IVT) to obtain the crude mRNA product. The main impurities include: T7 RNA polymerase, unused 5'-end cap structure analogs, residual DNA template, other residual proteins and free nucleotides, etc. Then, the relevant impurities are removed by chromatography techniques such as affinity chromatography, hydrophobic chromatography, ion exchange chromatography, molecular exclusion chromatography, etc. Next, tangential flow filtration is used to achieve the purpose of concentration and liquid exchange, and finally lipid nanoparticle encapsulation (LNPs) is used to encapsulate, protect and effectively deliver mRNA.

[0004] Since mRNA is unstable and easily degraded by various enzymes or non-enzymatic chemical degradation processes, environmental RNA enzymes may destroy mRNA, so it is best to operate in a closed environment without nucleases to minimize the time of exposure to the environment. Existing technologies, from IVT to chromatography to tangential flow filtration to nanolipid particle encapsulation, all require the transfer of containers and contact with the environment, increasing the risk of contact with contaminants such as nucleases. Utility Model Content

[0005] The utility model at least reduces one of the problems existing in the prior art to a certain extent. The utility model aims to provide a continuous purification process from in vitro transcription to final nanolipid particle encapsulation in a closed container to avoid contact with external pollutants that may cause mRNA degradation, thereby improving efficiency and safety.

[0006] The utility model proposes a continuous purification system, which can realize closed and continuous in vitro transcription, chromatography purification, tangential flow filtration, and nanolipid particle encapsulation. It improves the purification efficiency and reduces the risk of instability and easy degradation during the purification process. The specific technical solution is:

[0007] A closed continuous mRNA preparation device comprises an IVT reactor, wherein a first liquid discharge port of the IVT reactor is connected to an IVT product transfer container;

[0008] The second liquid discharge port of the IVT product transfer container is connected to the chromatography module, and the liquid outlet of the chromatography module is connected to the ultrafiltration module transfer container;

[0009] The ultrafiltration module transfer container is connected to the first ultrafiltration membrane package;

[0010] The first ultrafiltration membrane package is connected to the LNPs encapsulation device;

[0011] The LNPs encapsulation device is connected to the LNPs dilution and fluid replacement device, and the LNPs dilution and fluid replacement device is connected to the second ultrafiltration membrane package;

[0012] The second ultrafiltration membrane package is connected to the product storage container.

[0013] Furthermore, the IVT reactor is provided with a first liquid inlet, a second liquid inlet, a first exhaust port, a first liquid discharge port, a first agitator, a first temperature control system and a pH detection sensor; the first liquid discharge port is connected to the IVT product transfer container through a first four-way valve.

[0014] The IVT product transfer container includes a third liquid inlet, a fourth liquid inlet, a second exhaust port, a second liquid discharge port, a second agitator, a second temperature control system, and a first precision weighing system; the third liquid inlet is connected to the first four-way valve through the second four-way valve and the first pump;

[0015] The second liquid discharge port of the IVT product transfer container is connected to the chromatography module through the third four-way valve, and the liquid outlet of the chromatography module is connected to the ultrafiltration module transfer container.

[0016] The ultrafiltration module transfer container includes a fifth liquid inlet, a third exhaust port, a third liquid discharge port, a third agitator, and a second precision weighing system;

[0017] The third liquid discharge port is connected to the fifth four-way valve;

[0018] The fifth liquid inlet is connected to the chromatography module and the tee through the fourth four-way valve; the tee is also respectively connected to the ultrafiltration membrane package and the ultrafiltration infusion pump on the infusion pipeline; a transmembrane pressure control valve and a second pressure sensor are also provided on the reflux pipeline between the tee and the first ultrafiltration membrane package; the first ultrafiltration membrane package is also provided with an ultrafiltration permeable end; the first ultrafiltration membrane package is also connected to the fifth four-way valve through the first pressure sensor and the second pump.

[0019] The fifth four-way valve is also connected to the LNPs liquid inlet of the LNPs encapsulation device through the third pump and the filter, and the mRNA stock solution is obtained after the LNPs are completed; the LNPs encapsulation device is connected to the LNPs dilution and liquid replacement device.

[0020] The LNPs dilution and liquid replacement equipment includes an LNPs liquid storage container, the liquid inlet of the LNPs liquid storage container is connected to the second ultrafiltration membrane package through a fourth pump and a fourth pressure sensor; the reflux pipeline of the second ultrafiltration membrane package is connected to the LNPs liquid storage container through a third pressure sensor and a fifth pump; the second ultrafiltration membrane package is also connected to a product storage container.

[0021] The utility model can carry out the entire process continuously and in a closed process, thereby reducing the risk of mRNA degradation caused by exposure to the environment and contamination of mRNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a structural schematic diagram of the utility model. DETAILED DESCRIPTION

[0023] The specific technical solution of the utility model is explained in conjunction with the accompanying drawings.

[0024] like Figure 1 As shown, a closed continuous mRNA preparation device comprises an IVT reactor 1, wherein the IVT reactor 1 is provided with a first liquid inlet 1-1, a second liquid inlet 1-2, a first exhaust port 1-3, a first liquid discharge port, a first agitator 1-4, a first temperature control system and a pH detection sensor; the first liquid discharge port is connected to an IVT product transfer container 2 via a first four-way valve 1-5;

[0025] The IVT product transfer container 2 includes a third liquid inlet 2-1, a fourth liquid inlet 2-2, a second exhaust port 2-3, a second liquid discharge port, a second agitator 2-4, a second temperature control system, and a first precision weighing system. The third liquid inlet 2-1 is connected to the first four-way valve 1-5 through the second four-way valve 2-6 and the first pump 2-7;

[0026] The second liquid discharge port of the IVT product transfer container 2 is connected to the chromatography module 3 through the third four-way valve 2-5, and the liquid outlet of the chromatography module 3 is connected to the ultrafiltration module transfer container 4;

[0027] The ultrafiltration module transfer container 4 includes a fifth liquid inlet 4-1, a third exhaust port 4-4, a third liquid discharge port, a third agitator 4-3, and a second precision weighing system.

[0028] The third liquid discharge port is connected to the fifth four-way valve 4-5;

[0029] The fifth liquid inlet 4-1 is connected to the chromatography module 3 and the tee 4-12 through the fourth four-way valve 4-2; the tee 4-12 is also connected to the ultrafiltration membrane package 4-8 and the ultrafiltration infusion pump 4-13 on the infusion pipeline respectively; a transmembrane pressure control valve 4-11 and a second pressure sensor 4-10 are also provided on the reflux pipeline between the tee 4-12 and the first ultrafiltration membrane package 4-8; the first ultrafiltration membrane package 4-8 is also provided with an ultrafiltration permeation end 4-9; the first ultrafiltration membrane package 4-8 is also connected to the fifth four-way valve 4-5 through the first pressure sensor 4-7 and the second pump 4-6;

[0030] The fifth four-way valve 4-5 is also connected to the LNPs inlet 5-3 of the nanolipid particle LNPs encapsulation device 5 through the third pump 5-1 and the filter 5-2, and the mRNA stock solution is obtained after the LNPs are completed. The LNPs encapsulation device 5 is connected to the LNPs dilution and liquid replacement device 6, and the LNPs dilution and liquid replacement device 6 includes an LNPs storage container 6-1, and the liquid inlet of the LNPs storage container 6-1 is connected to the second ultrafiltration membrane package 6-6 through the fourth pump 6-3 and the fourth pressure sensor 6-5; the reflux line of the second ultrafiltration membrane package 6-6 is connected to the LNPs storage container 6-1 through the third pressure sensor 6-4 and the fifth pump 6-2; the second ultrafiltration membrane package 6-6 is also connected to the product storage container 6-7.

[0031] Implementation: IVT raw materials are injected into the IVT reactor 1 through the first liquid inlet 1-1 and the second liquid inlet 1-2. After the IVT reaction is completed, the feed liquid enters the IVT product transfer container 2 through the first four-way valve 1-5 and the second four-way valve 2-6 connected to the first feed liquid discharge port. According to the loading conditions required for chromatography, regulating liquid is added through the first pump 2-7 via the second four-way valve 2-6 to regulate the IVT product. The regulated feed liquid enters the chromatography module 3 through the second feed liquid discharge port and the third four-way valve 2-5. After the chromatography is completed, the chromatography feed liquid enters the ultrafiltration module transfer container 4 through the fourth four-way valve 4-2, and the ultrafiltration module transfer container 4 enters the first ultrafiltration membrane package 4-8 through the third feed liquid discharge port and the second pump 4-6. The reflux liquid of the first ultrafiltration membrane package 4-8 enters the ultrafiltration module transfer container 4 through the fifth liquid inlet 4-1 through the three-way 4-12 and the fourth four-way valve 4-2 to circulate, and the ultrafiltration replenishment liquid enters the ultrafiltration module transfer container 4 through the three-way 4-12 and the fourth four-way valve 4-2 through the ultrafiltration replenishment pump 4-13 to complete the ultrafiltration process. The ultrafiltration process controls the transmembrane pressure through the first pressure sensor 4-7, the second pressure sensor 4-10, and the transmembrane pressure control valve 4-11. The ultrafiltration feed liquid passes through the third pump 5-1 and the 0.2um filter 5-2 to enter the LNPs encapsulation device 5, and the stock solution is obtained after the LNPs encapsulation is completed. After the LNPs encapsulation, the stock solution enters the LNPs dilution and liquid replacement device 6, and the stock solution dilution operation is performed. The diluted stock solution is passed through an ultrafiltration device consisting of a fourth pump 6-3, a fifth pump 6-2 and a second ultrafiltration membrane package 6-6 connected to the outlet and inlet of the LNPs dilution and liquid exchange equipment 6 to concentrate and exchange the LNPs, dilute to the target concentration, and discharge to the storage container 6-7 through a three-way valve to obtain the LNPs final product.

Claims

1. A closed continuous mRNA preparation device, characterized in that: It comprises an IVT reactor (1), wherein a first liquid discharge port of the IVT reactor (1) is connected to an IVT product transfer container (2); The second liquid discharge port of the IVT product transfer container (2) is connected to the chromatography module (3), and the liquid outlet of the chromatography module (3) is connected to the ultrafiltration module transfer container (4); The ultrafiltration module transfer container (4) is connected to the first ultrafiltration membrane package (4-8); The first ultrafiltration membrane package (4-8) is connected to the LNPs encapsulation device (5); The LNPs encapsulation device (5) is connected to the LNPs dilution and fluid replacement device (6), and the LNPs dilution and fluid replacement device (6) is connected to the second ultrafiltration membrane package (6-6); The second ultrafiltration membrane package (6-6) is connected to the product storage container (6-7).

2. A closed continuous mRNA preparation device according to claim 1, characterized in that: The IVT reactor (1) is provided with a first liquid inlet (1-1), a second liquid inlet (1-2), a first exhaust port (1-3), a first liquid discharge port, a first agitator (1-4), a first temperature control system and a pH detection sensor; the first liquid discharge port is connected to the IVT product transfer container (2) via a first four-way valve (1-5).

3. A closed continuous mRNA preparation device according to claim 2, characterized in that: The IVT product transfer container (2) comprises a third liquid inlet (2-1), a fourth liquid inlet (2-2), a second exhaust port (2-3), a second liquid discharge port, a second agitator (2-4), a second temperature control system, and a first precision weighing system; the third liquid inlet (2-1) is connected to the first four-way valve (1-5) via a second four-way valve (2-6) and a first pump (2-7); The second liquid discharge port of the IVT product transfer container (2) is connected to the chromatography module (3) via the third four-way valve (2-5), and the liquid outlet of the chromatography module (3) is connected to the ultrafiltration module transfer container (4).

4. A closed continuous mRNA preparation device according to claim 3, characterized in that: The ultrafiltration module transfer container (4) comprises a fifth liquid inlet (4-1), a third exhaust port (4-4), a third liquid discharge port, a third stirrer (4-3), and a second precision weighing system; The third liquid discharge port is connected to the fifth four-way valve (4-5); The fifth liquid inlet (4-1) is connected to the chromatography module (3) and the tee (4-12) via the fourth four-way valve (4-2); the tee (4-12) is also respectively connected to the ultrafiltration membrane package (4-8) and the ultrafiltration rehydration pump (4-13) on the rehydration pipeline; a transmembrane pressure control valve (4-11) and a second pressure sensor (4-10) are also provided on the reflux pipeline between the tee (4-12) and the first ultrafiltration membrane package (4-8); the first ultrafiltration membrane package (4-8) is also provided with an ultrafiltration permeation end (4-9); the first ultrafiltration membrane package (4-8) is also connected to the fifth four-way valve (4-5) via the first pressure sensor (4-7) and the second pump (4-6).

5. A closed continuous mRNA preparation device according to claim 4, characterized in that: The fifth four-way valve (4-5) is also connected to the LNPs liquid inlet (5-3) of the LNPs encapsulation device (5) through the third pump (5-1) and the filter (5-2), and the mRNA stock solution is obtained after the LNPs are completed; the LNPs encapsulation device (5) is connected to the LNPs dilution and liquid replacement device (6).

6. A closed continuous mRNA preparation device according to claim 5, characterized in that: The LNPs dilution and liquid replacement device (6) comprises an LNPs liquid storage container (6-1); the liquid inlet of the LNPs liquid storage container (6-1) is connected to the second ultrafiltration membrane package (6-6) via a fourth pump (6-3) and a fourth pressure sensor (6-5); the return line of the second ultrafiltration membrane package (6-6) is connected to the LNPs liquid storage container (6-1) via a third pressure sensor (6-4) and a fifth pump (6-2); and the second ultrafiltration membrane package (6-6) is also connected to a product storage container (6-7).

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