CN116554995A - mRNA continuous in vitro transcription reaction system and continuous transcription method - Google Patents
mRNA continuous in vitro transcription reaction system and continuous transcription method Download PDFInfo
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Abstract
The invention relates to the technical field of biological products, in particular to a continuous in-vitro transcription reaction system and a continuous transcription method of mRNA. The continuous reaction and harvesting system for preparing mRNA comprises: an mRNA in vitro transcription reaction (IVT) system, a cooling module, a continuous chromatography device, a heating module and an online detection system. In the mRNA continuous IVT reaction and harvesting method, the DNA template used is immobilized on the microsphere. After IVT starts reacting for a period of time, the reaction liquid flows into a continuous chromatographic device loaded with oligo dT chromatographic filler through the cooling module, mRNA is captured and purified through the continuous chromatographic device, and the rest components of the reaction liquid flow through and then flow back to the IVT reaction container through the heating module to complete circulation. The system facilitates maintaining the IVT reaction at an optimal state, while increasing mRNA production while reducing consumption of DNA templates, enzymes and cap analogues.
Description
Technical Field
The invention relates to the technical field of biological products, in particular to a continuous in-vitro transcription reaction system and a continuous transcription method of mRNA.
Background
The preparation of mRNA comprises the steps of fermentation and purification of plasmid, linearization and purification of DNA template, in vitro transcription (in vitro transcription, IVT) and purification of mRNA, wherein IVT of mRNA is a core process step and is a main cost factor at present. The IVT reaction is essentially an enzymatic synthesis reaction in which the cap analogue and NTP are enzymatically synthesized into mRNA, and thus also follows the general rules of enzymatic reaction kinetics, such as decreasing substrate concentration and increasing product concentration as the reaction proceeds, these factors inhibit the forward progress of the reaction, and factors that reduce enzymatic activity also lead to a decrease in reaction rate. Therefore, the method for improving the mRNA synthesis efficiency is to supplement NTP in the reaction system, add magnesium ions and adjust pH to keep the enzyme in the optimal catalytic condition. There are studies (document Pregeljc, janja Skok, tina Vodopivec, et al, incrusting yield of in vitro transcription reaction with at-line high pressure liquid chromatography monitoring [ J ]. Biotechnology and Bioengineering, 2022) showing that by supplementing NTPs and cap analogues in the IVT reaction, a higher yield and higher ratio of capping rate can be achieved than in the batch reaction mode by maintaining the GTP to cap analogue ratio at 1:4. However, the fed-batch can only supplement the raw materials for reaction, can not remove the reaction product from the reaction system in time, and can not remove the inhibition of high product concentration on the reaction.
After the IVT reaction is finished, oligo dT affinity chromatography purification is needed, and because oligo dT affinity chromatography packing is expensive, a large amount of capital is needed to purchase the packing in the existing batch production mode, and the early investment is high, so that flexible production of different varieties is not facilitated. Because the IVT reaction liquid can be directly subjected to oligo dT affinity chromatography purification after being cooled, the method has the potential of integrating with a continuous chromatography system, thereby reducing the scale of chromatography, saving the cost, capturing mRNA generated in the IVT reaction in time, removing the mRNA from the IVT reaction system, promoting forward progress of the IVT reaction and improving the yield of the mRNA.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide a novel continuous in-vitro transcription reaction system for mRNA, which can be matched with a microsphere immobilized DNA template, and can quickly separate the DNA template from mRNA products after transcription is completed, so that the DNA template can be recovered in time, recycling can be realized, large-scale fermentation is realized, and in-vitro transcription efficiency is remarkably improved.
In order to solve the technical problems and achieve the purposes, the following technical scheme is as follows:
in a first aspect, the present invention provides a continuous reaction and harvesting system for preparing mRNA by in vitro transcription, comprising, in order from the direction of flow of the reaction system: the system comprises an mRNA in-vitro transcription reaction device, a cooling module and a continuous chromatography device, and further comprises a heating module and a process analysis module;
the liquid outlet of the continuous chromatographic device is connected with the mRNA in-vitro transcription reaction device through a temperature-raising module to form a loop;
the mRNA in-vitro transcription reaction device is provided with a microsphere interception device;
the process analysis module is used for realizing the real-time analysis of the reaction liquid by the high performance liquid chromatography HPLC or ion migration mass spectrometry through the online sampling module.
In an alternative embodiment, the mRNA in vitro transcription reaction apparatus uses microsphere immobilized DNA templates for mRNA transcription;
the microsphere interception device is selected from a magnetic force control module arranged in the mRNA in-vitro transcription reaction device or a microfiltration device arranged at the liquid outlet end of the mRNA in-vitro transcription reaction device.
Preferably, the microfiltration device is a tangential microfiltration device.
Preferably, the microfiltration device is provided with a hollow fiber column with a pore diameter of 0.2-0.5 μm.
In an alternative embodiment, the continuous chromatographic apparatus is provided with 3 to 8 chromatographic columns for continuous liquid inlet and outlet.
Preferably, the chromatography column is an oligo dT affinity chromatography column.
In an alternative embodiment, the cooling mode of the cooling module is selected from air cooling, water cooling or semiconductor refrigeration; the heating mode of the heating module is selected from water bath or metal bath.
In a second aspect, the present invention provides a continuous in vitro transcription reaction and harvesting method for mRNA, suitable for use in the continuous in vitro transcription reaction and harvesting system for preparing mRNA according to any of the previous embodiments, comprising the steps of:
adding the microsphere immobilized DNA template and a transcription reaction system into an mRNA in-vitro transcription reaction device, after the transcription reaction is completed, intercepting the microsphere immobilized DNA template by a microsphere interception device, cooling by a cooling module, then entering a continuous chromatography device for capturing and purifying mRNA, and returning the flowing-through liquid to the mRNA in-vitro transcription reaction device by a heating module, wherein a chromatographic column in the continuous chromatography device is regenerated and balanced, and repeating the mRNA transcription reaction of the next round.
In an alternative embodiment, the microsphere immobilized DNA template consists of a single stranded DNA template specifically bound to a microsphere immobilized oligonucleotide fragment immobilized to the microsphere via a linker fragment.
Preferably, the oligonucleotide is 40-80 nucleotides in length and has a Tm value greater than 45℃for binding to a single stranded DNA template.
In an alternative embodiment, the components of the transcription reaction system include: 0-500 mM buffer salt, 100-500 mM NaCl, 1-100 mM DTT, 1-10 RNase Inhibitor and 10U, mg 2+ 1～20mM、Spermidine 0.1～0.5mM；T7 RNA Polymerase 10～200U；NTP 5～20mM。
Preferably, the components of the transcription reaction system include: 20-200 mM buffer salt,NaCl100～300mM、DTT 10～50mM、RNase Inhibitor 2～5U、Mg 2+ 5～20mM、Spermidine 0.1～0.2mM；T7 RNA Polymerase 50～100U；NTP 5～10mM。
Preferably, the single-stranded DNA template is added at 0.1-10. Mu.g/ml, and the feed NTPs and magnesium ions are added in parallel.
Preferably, the single-stranded DNA template is added in an amount of 0.5 to 2. Mu.g/ml.
Preferably, the feeding is performed by supplementing the NTPs such that the molar concentration ratio of cap analogue to NTPs is 4:1, the magnesium ion concentration and pH were maintained at initial values.
In an alternative embodiment, the temperature reduction module reduces the temperature of the liquid in the pipeline to 2-25 ℃. The temperature rising module rises the temperature of the liquid in the pipeline to 25-45 ℃.
In an alternative embodiment, the mRNA capture method used by the continuous chromatographic apparatus is Oligo dT affinity chromatography.
Preferably, the method of each column of Oligo dT affinity chromatography is: column balance: 3-10 CV balance buffer solution; loading: 1-5 CV transcription reaction liquid; pre-washing: 1-2 CV of balance buffer solution; eluting: 1-2 CV of eluent A; column cleaning: 10CV eluent B; and (3) post-treatment and regeneration of the filler: 1-2 CV water, 3CV 0.5M sodium hydroxide and 1-2 CV water.
In an alternative embodiment, the process analysis module samples and detects the content and the pH value of each component in the mRNA in-vitro transcription reaction device in real time, and dynamically adjusts the reaction system and the chromatographic system through process analysis and control.
Compared with the prior art, the invention has the beneficial effects that:
(1) The continuous reaction and harvesting system for preparing mRNA by in vitro transcription provided by the invention reduces the scale of chromatographic packing by utilizing a continuous chromatographic device, reduces the purchase cost of single-batch production of mRNA affinity chromatographic packing, and is beneficial to flexible production of different varieties.
(2) The continuous reaction and harvesting method for preparing mRNA by in vitro transcription provided by the invention is suitable for the continuous reaction and harvesting system for preparing mRNA by in vitro transcription, and can timely separate mRNA generated by the reaction by using the microsphere immobilized DNA template, reduce the inhibition of the product on enzymatic reaction, and facilitate maintaining the IVT reaction under the optimal reaction condition by combining fed-batch feeding, so that the IVT reaction is more efficient and the cost is reduced.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of a continuous reaction and harvesting system for preparing mRNA by in vitro transcription according to an embodiment of the present invention;
FIG. 2 is a schematic diagram of the principle of the microsphere immobilized DNA template provided by the embodiment of the invention;
FIG. 3 is a schematic diagram of a magnetic force module according to an embodiment of the present invention trapping magnetic beads so as to retain DNA templates in a reaction vessel;
FIG. 4 is a schematic diagram of a heating module and a cooling module according to an embodiment of the present invention;
fig. 5 is a schematic diagram of a continuous chromatography method of 3 columns according to an embodiment of the present invention.
Icon: 1-IVT reaction system; a 2-IVT reaction vessel; 3-a hollow fiber microfiltration device; 4-feeding a liquid supplementing pipeline; 5-heating module; 6-overflow pipeline; 7-a buffer container; 8-a cooling module; 9-an online sampling module; 10-a process analysis module; 11-a continuous chromatographic device; 12-Oligo dT column.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention. The components of the embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations.
Thus, the following detailed description of the embodiments of the invention, as presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
It should be noted that: like reference numerals and letters denote like items in the following figures, and thus once an item is defined in one figure, no further definition or explanation thereof is necessary in the following figures.
In the description of the present invention, it should also be noted that, unless explicitly specified and limited otherwise, the terms "disposed," "mounted," "connected," and "connected" are to be construed broadly, and may be, for example, fixedly connected, detachably connected, or integrally connected; can be mechanically or electrically connected; can be directly connected or indirectly connected through an intermediate medium, and can be communication between two elements. The specific meaning of the above terms in the present invention will be understood in specific cases by those of ordinary skill in the art.
In a specific embodiment, the invention provides in a first aspect a continuous reaction and harvesting system for preparing mRNA by in vitro transcription, comprising in order, in the direction of flow of the reaction system: the system comprises an mRNA in-vitro transcription reaction device, a cooling module and a continuous chromatography device, and further comprises a heating module and a process analysis module;
the liquid outlet of the continuous chromatographic device is connected with the mRNA in-vitro transcription reaction device through a temperature-raising module to form a loop;
the mRNA in-vitro transcription reaction device is provided with a microsphere interception device;
the process analysis module is used for realizing the real-time analysis of the reaction liquid by the high performance liquid chromatography HPLC or ion migration mass spectrometry through the online sampling module.
In an alternative embodiment, the mRNA in vitro transcription reaction apparatus uses microsphere immobilized DNA templates for mRNA transcription;
the microsphere interception device is selected from a magnetic force control module arranged in the mRNA in-vitro transcription reaction device or a microfiltration device arranged at the liquid outlet end of the mRNA in-vitro transcription reaction device.
Preferably, the microfiltration device is a tangential microfiltration device.
Preferably, the microfiltration device is provided with a hollow fiber column with a pore diameter of 0.2-0.5 μm.
In an alternative embodiment, the continuous chromatographic apparatus is provided with 3 to 8 chromatographic columns for continuous liquid inlet and outlet.
Preferably, the chromatography column is an oligo dT affinity chromatography column.
In an alternative embodiment, the cooling mode of the cooling module is selected from air cooling, water cooling or semiconductor refrigeration; the heating mode of the heating module is selected from water bath or metal bath.
In a second aspect, the present invention provides a continuous in vitro transcription reaction and harvesting method for mRNA, suitable for use in the continuous in vitro transcription reaction and harvesting system for preparing mRNA according to any of the previous embodiments, comprising the steps of:
adding the microsphere immobilized DNA template and a transcription reaction system into an mRNA in-vitro transcription reaction device, after the transcription reaction is completed, intercepting the microsphere immobilized DNA template by a microsphere interception device, cooling by a cooling module, then entering a continuous chromatography device for capturing and purifying mRNA, and returning the flowing-through liquid to the mRNA in-vitro transcription reaction device by a heating module, wherein a chromatographic column in the continuous chromatography device is regenerated and balanced, and repeating the mRNA transcription reaction of the next round.
In an alternative embodiment, the microsphere immobilized DNA template consists of a single stranded DNA template specifically bound to a microsphere immobilized oligonucleotide fragment immobilized to the microsphere via a linker fragment.
Preferably, the oligonucleotide is 40-80 nucleotides in length and the Tm value for the binding to a single stranded DNA template is higher than 45 ℃.
In an alternative embodiment, the components of the transcription reaction system include: 0-500 mM buffer salt, 100-500 mM NaCl, 1-100 mM DTT, 1-10 RNase Inhibitor and 10U, mg 2+ 1～20mM、Spermidine 0.1～0.5mM；T7 RNA Polymerase 10～200U；NTP 5～20mM。
Preferably, the components of the transcription reaction system include: 20-200 mM buffer salt, 100-300 mM NaCl, 10-50 mM DTT and 2-5U, mg RNase Inhibitor 2+ 5～20mM、Spermidine 0.1～0.2mM；T7 RNA Polymerase 50～100U；NTP 5～10mM。
Preferably, the single-stranded DNA template is added at 0.1-10. Mu.g/ml, and the feed NTPs and magnesium ions are added in parallel.
Preferably, the single-stranded DNA template is added in an amount of 0.5 to 2. Mu.g/ml.
Preferably, the feeding method is to supplement the NTPs such that the molar concentration ratio of cap analogue to NTPs is 4:1, the magnesium ion concentration and pH were maintained at initial values.
In an alternative embodiment, the temperature reduction module reduces the temperature of the liquid in the pipeline to 2-25 ℃. The temperature rising module rises the temperature of the liquid in the pipeline to 25-45 ℃.
In an alternative embodiment, the mRNA capture method used by the continuous chromatographic apparatus is Oligo dT affinity chromatography.
Preferably, the method of each column of Oligo dT affinity chromatography is: column balance: 3-10 CV balance buffer solution; loading: 1-5 CV transcription reaction liquid; pre-washing: 1-2 CV of balance buffer solution; eluting: 1-2 CV of eluent A; column cleaning: 10CV eluent B; and (3) post-treatment and regeneration of the filler: 1-2 CV water, 3CV 0.5M sodium hydroxide and 1-2 CV water.
In an alternative embodiment, the process analysis module samples and detects the content and the pH value of each component in the mRNA in-vitro transcription reaction device in real time, and dynamically adjusts the reaction system and the chromatographic system through process analysis and control.
Some embodiments of the present invention are described in detail below with reference to the accompanying drawings. The following embodiments and features of the embodiments may be combined with each other without conflict.
In the following examples and comparative examples, the DNA template (SEQ ID NO. 1) used was single-stranded DNA obtained by PCR amplification, in which the 3' -end was a 40nt linker (SEQ ID NO. 2), the target gene sequence (SEQ ID NO. 3) was after the linker, and the DNA template was immobilized on the microspheres by base-pairing with the chemically coupled oligonucleotide (SEQ ID NO. 4) on the custom microspheres.
In the following examples, the components of the IVT reaction system used were: HEPES (hydroxyethylpiperazine ethylsulfuric acid buffer) 50mM, naCl300mM, magnesium acetate 7mM, sodium acetate 50mM, DTT (dithiothreitol) 40mM, spermidine 0.2mM, RNase Inhibitor 4U, T RNA polymerase 100U, pyrophosphatase 2U, NTP each 5mM, cap1 (Cap analogue) 25mM, DNA template (SEQ ID NO. 1) 1. Mu.g/ml, pH 7.4.
In the following examples, oligo dT chromatography solutions were used comprising: HEPES 50mM, naCl300mM, pH 7.4; and (3) eluting the pure water.
Wherein, SEQ ID NO.1:
TGGGGGGGGTTTTTTTTTTTTTTTTTTTATATGCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTATATGCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCCGCCCACTCAGACTTTATTCAAAGACCACGGGGGTACGGGTGCAGGAAGGGGAGGAGGGGCTGGGGGGAGGCCCAAGGGGCAAGAAGCATGGCCACCGAGGCTCCAGCTTACTATGCCTTAAGAACATTGGTGAGCAGCATAAGGCCTGCATATCCAAGGAGGATGACAATAATGATGAACAGCCACTTCTTTGCAGGGTAGCACTGGGGATTTTTTAGAAACTCACGACAGAATGTGCAGTCCACTGCATCACAACCATCTGGGAACATGCAACTACCCTCAAATGTGAATTCAGTTCCATCTACCAATGCCCCTTTGACGTGGATTGCTGTTTTTCCAACCAATGATCCTGAGTGGAATTGAATCTCCGTG
CTTTTCTTACCACTTGTGGCAGAACTGCAAAAGTGAGAAGCGCAGAC
AGTAGCTGACTTGAGCTCACTGGTTAGAGTGATCAGCCTCACACCCCC
ATTTATGCATTCTAGATGGCAGCCAGTGCATTGACCAATCCTTTGCTCT
GGCTGGCTCACCTCCAGTGTTGCCATCATTCTGGAGAAACCATAGCAC
TTTGGTCTGACACGCATCCCTCCATATGACACAACGACTTCCCCTGGC
TTGTGCACAAGTGAGCATCTACACTTGGCTTCAGATGCCTCTTCCCCA
TGTTCACAACCTGCCACCTTGCAAAACTGCATGTCCCCCCTGCATGAT
CCACTGGCCTTGCAAGCCTTTTCTTCTGATTCAGAACACGGCCCAGTC
CCTTCCTTATAGCACACAAAATCCTTGGATGAGCTCTCTTGTACCCATT
TGGTCTTGTGCTCCCTCATGATGATTTTATGAGAGTGGCCAACATCCAT
CCATGCAACATCAAAGGGCTGGGGTAGACTCTCACCCTCACACTGGT
TGAACACAATGCCATCAATTTTGCATATGTCTGGGAATTCACTTTGGCT
AAAGCTCTTCAGCTCCAGAAATGTCTCCTCAGGGATGGGTGTCATTGG
CTGACAAGCAACATCACCTATCCAGAGAACCCTATCCGAGGGCAGAA
GACCAGAACTAGTCCCGGAGTCTGAACTGCAGAGCTCACCTGACTCA
GTCCTAGACTTCCCACTACCACAGCCCCAGCCTACGTATCCATCAAAA
GTTGGCCGTTCAAAAAAGACTTTCCCCTTGTATGAAGGCCCAGGAAC
TATCATATCAGACCCCTTGGCCTTCTTCACGACAAGCATCCCAAGCCA
ATCAGCATCACAGGGGCTTAGAAGCCCACTCCACCTTGAGTAAGAATT
TTCAGCTGGGTAGTAGCTCACCTGAGAGCGTCCACCTCGCTGCCCTAC
GTAGCCCTGAAATTGTGAGTGGTTTCTAAGCCAAGACGAAACATGTAT
CAGCCGATCTATCTGACAAATCTTCTCAGAGTAGCCAAGCAGGTGGG
GTATGTCAGCACTCTTGTTTGAGTGGATGGGTCCTGCGCAAATGATTG
GGCCCGTGTCCCCACCGCATTGAATGACTAAGCAGATCAGAGAGGAG
AACCAGATGACTTTCATCATGGTGGCGGGTTCTCTCTGAGTCTGTGGG
GACCAGAAGAATACTAGTTTATTCTCGGAAGGAAGGAAGGGGTTGGAGCATTTGACTTGGAACAAT。
SEQ ID NO.2：
GGAAGGAAGGAAGGGGTTGGAGCATTTGACTTGGAACAAT。
SEQ ID NO.3：
TGGGGGGGGTTTTTTTTTTTTTTTTTTTATATGCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTATATGCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCCGCCCACTCAGACTTTATTCAAAGACCACGGGGGTACGGGTGCAGGAAGGGGAGGAGGGGCTGGGGGGAGGCCCAAGGGGCAAGAAGCATGGCCACCGAGGCTCCAGCTTACTATGCCTTAAGAACATTGGTGAGCAGCATAAGGCCTGCATATCCAAGGAGGATGACAATAATGATGAACAGCCACTTCTTTGCAGGGTAGCACTGGGGATTTTTTAGAAACTCACGACAGAATGTGCAGTCCACTGCATCACAACCATCTGGGAACATGCAACTACCCTCAAATGTGAATTCAGTTCCATCTACCAATGCCCCTTTGACGTGGATTGCTGTTTTTCCAACCAATGATCCTGAGTGGAATTGAATCTCCGTGCTTTTCTTACCACTTGTGGCAGAACTGCAAAAGTGAGAAGCGCAGACAGTAGCTGACTTGAGCTCACTGGTTAGAGTGATCAGCCTCACACCCCCATTTATGCATTCTAGATGGCAGCCAGTGCATTGACCAATCCTTTGCTCTGGCTGGCTCACCTCCAGTGTTGCCATCATTCTGGAGAAACCATAGCAC
TTTGGTCTGACACGCATCCCTCCATATGACACAACGACTTCCCCTGGC
TTGTGCACAAGTGAGCATCTACACTTGGCTTCAGATGCCTCTTCCCCA
TGTTCACAACCTGCCACCTTGCAAAACTGCATGTCCCCCCTGCATGAT
CCACTGGCCTTGCAAGCCTTTTCTTCTGATTCAGAACACGGCCCAGTC
CCTTCCTTATAGCACACAAAATCCTTGGATGAGCTCTCTTGTACCCATT
TGGTCTTGTGCTCCCTCATGATGATTTTATGAGAGTGGCCAACATCCAT
CCATGCAACATCAAAGGGCTGGGGTAGACTCTCACCCTCACACTGGT
TGAACACAATGCCATCAATTTTGCATATGTCTGGGAATTCACTTTGGCT
AAAGCTCTTCAGCTCCAGAAATGTCTCCTCAGGGATGGGTGTCATTGG
CTGACAAGCAACATCACCTATCCAGAGAACCCTATCCGAGGGCAGAA
GACCAGAACTAGTCCCGGAGTCTGAACTGCAGAGCTCACCTGACTCA
GTCCTAGACTTCCCACTACCACAGCCCCAGCCTACGTATCCATCAAAA
GTTGGCCGTTCAAAAAAGACTTTCCCCTTGTATGAAGGCCCAGGAAC
TATCATATCAGACCCCTTGGCCTTCTTCACGACAAGCATCCCAAGCCA
ATCAGCATCACAGGGGCTTAGAAGCCCACTCCACCTTGAGTAAGAATT
TTCAGCTGGGTAGTAGCTCACCTGAGAGCGTCCACCTCGCTGCCCTAC
GTAGCCCTGAAATTGTGAGTGGTTTCTAAGCCAAGACGAAACATGTAT
CAGCCGATCTATCTGACAAATCTTCTCAGAGTAGCCAAGCAGGTGGG
GTATGTCAGCACTCTTGTTTGAGTGGATGGGTCCTGCGCAAATGATTG
GGCCCGTGTCCCCACCGCATTGAATGACTAAGCAGATCAGAGAGGAG
AACCAGATGACTTTCATCATGGTGGCGGGTTCTCTCTGAGTCTGTGGG
GACCAGAAGAATACTAGTTTATTCTC。
SEQ ID NO.4：
ATTGTTCCAAGTCAAATGCTCCAACCCCTTCCTTCCTTCC。
example 1
The continuous reaction and harvesting system for preparing mRNA by in vitro transcription provided in this example, as shown in FIGS. 1, 2, 4 and 5, comprises: an IVT reaction system (1), a cooling module (8), a continuous chromatography device (11), a heating module (5) and a process analysis module (10);
the liquid outlet of the IVT reaction container (2) in the IVT reaction system (1) is connected with the liquid inlet of the continuous chromatographic device (11) through a water-cooling jacket, and the liquid outlet of the continuous chromatographic device (11) is connected with the IVT reaction system (1) through a water-bath jacket to form a loop.
The IVT reaction vessel (2) adopts a blue cap bottle with the volume of 0.1L, the cap is provided with a liquid outlet pipeline and a liquid inlet pipeline, two ends of the IVT reaction vessel (2) are connected with a hollow fiber microfiltration device (3), the interception aperture of a hollow fiber column is 0.2 mu m, and the membrane area is 16cm 2 。
The continuous chromatographic device (11) can adopt a commercial continuous chromatographic system or a customized continuous chromatographic system existing in the market, is provided with 4 oligo dT chromatographic columns (12) with the specification of 1ml to capture and purify mRNA generated by IVT, and flows through the liquid and returns to an IVT reaction container (2) to recover raw materials such as enzyme, NTPs and the like. The purified mRNA was subjected to downstream processing and continuous chromatography as shown in FIG. 5.
A buffer container (7) is arranged between the continuous chromatography device (11) and the hollow fiber microfiltration device (3) and is used for buffering the difference of liquid flow rates between the continuous chromatography device and the hollow fiber microfiltration device. The speed of the produced filtered liquid is higher than the sample loading speed of the continuous chromatographic device (11) by adjusting the liquid inlet flow speed and the transmembrane pressure of the hollow fiber microfiltration device (3), so that no flow break is ensured. An overflow channel (6) is arranged at the middle height position of the buffer container (7) and is connected with the IVT reaction device, and when excessive solution is accumulated and the liquid level is higher than the overflow channel (6), overflow returns to the IVT reactor.
The process analysis module (10) adopts ion migration mass spectrometry to analyze the content of four nucleotides and the content of cap analogues, detects the content of magnesium ions through atomic absorption broad spectrum, and detects the pH value through a pH meter. The nucleotide and cap analogue contents in the IVT reaction system are supplemented through a feeding and fluid replacement pipeline (4), so that the ratio of cap analogue to GTP is maintained to be 4:1, the four nucleotide content was maintained at 5mM, magnesium ion was supplemented, the concentration was maintained at 7.5mM, and the pH was maintained at 7.4.
The cooling module (8) adopts a water bath jacket temperature control mode to cool IVT reaction liquid to room temperature; the temperature rising module (5) adopts a water bath jacket temperature control mode to rise chromatographic flow penetrating liquid to 37 ℃.
The raw materials required by IVT are sequentially added into a blue cap bottle, stirred and heated to 37 ℃, finally enzyme and template are added, the total volume is 100ml, and IVT reaction is started. And simultaneously, starting up and preprocessing the hollow fiber microfiltration device (3), the layer continuous chromatography device (11) and the like. After the IVT reaction starts for 1h, pumping the reaction liquid into the hollow fiber micro-filtration device (3), returning the reflux liquid into the IVT reaction system (1), and enabling the filtrate of the hollow fiber micro-filtration device (3) to enter the continuous chromatographic device (11) through the cooling module (8). And (3) feeding the sample into the chromatographic system by 1-3 CV, feeding the sample flow through liquid and the 1CV pre-washing liquid, heating the sample flow through liquid and the 1CV pre-washing liquid, and returning the sample flow through liquid and the 1CV pre-washing liquid to the IVT reaction container (2), so that the volume of the solution in the whole system is not increased. The chromatographic eluate is collected for storage or downstream processing.
The operation time of the whole system is 6h. After the completion of the reaction, the whole IVT reaction solution was filtered to a continuous chromatography apparatus (11) to collect purified mRNA.
Example 2
The continuous reaction and harvesting system for preparing mRNA by in vitro transcription provided in this example, as shown in FIGS. 1, 2, 3, 4 and 5, comprises: an IVT reaction system (1), a cooling module (8), a continuous chromatography device (11), a heating module (5) and a process analysis module (10);
the liquid outlet of the IVT reaction system (1) is connected with the liquid inlet of the continuous chromatographic device (11) through a water-cooling jacket, and the liquid outlet of the continuous chromatographic device (11) is connected with the IVT reaction system (1) through a water-bath jacket to form a loop.
The IVT reaction container (2) adopts a solution bag with the volume of 0.1L, and two ends of the bag are provided with a liquid outlet pipeline and a liquid inlet pipeline. The difference between this example and example 1 is that the IVT reaction system (1) eliminates the hollow fiber microfiltration device (3) shown in fig. 1, but adds a magnetic module at the bottom of the IVT reaction vessel (2), as shown in fig. 3, the magnetic beads with DNA templates adsorbed are uniformly spread in the IVT reaction vessel (2).
The continuous chromatographic device (11) can adopt a commercial continuous chromatographic system or a customized continuous chromatographic system existing in the market, is provided with 4 oligo dT chromatographic columns (12) with the specification of 1ml to capture and purify mRNA generated by IVT, and flows through the liquid and returns to an IVT reaction container (2) to recover raw materials such as enzyme, NTPs and the like. The purified mRNA was subjected to downstream processing and continuous chromatography as shown in FIG. 5.
The process analysis module (10) adopts ion migration mass spectrometry to analyze the content of four nucleotides and the content of cap analogues, detects the content of magnesium ions through atomic absorption broad spectrum, and detects the pH value through a pH meter. The nucleotide and cap analogue contents in the IVT reaction system are supplemented through a feeding and fluid replacement pipeline (4), so that the ratio of cap analogue to GTP is maintained to be 4:1, the four nucleotide content was maintained at 5mM, magnesium ion was supplemented, the concentration was maintained at 7.5mM, and the pH was maintained at 7.4.
The cooling module (8) adopts a water bath jacket temperature control mode to cool IVT reaction liquid to room temperature; the temperature rising module (5) adopts a water bath jacket temperature control mode to rise chromatographic flow penetrating liquid to 37 ℃.
The raw materials required by IVT are sequentially added into a blue cap bottle, stirred and heated to 37 ℃, finally enzyme and template are added, the total volume is 100ml, and IVT reaction is started. And simultaneously, the continuous chromatographic device (11) and the like are started and pretreated. After 1h from the start of the IVT reaction, the reaction solution is pumped into the continuous chromatographic apparatus (11) and the reflux solution is returned to the IVT reaction system (1). And (3) feeding the sample into the chromatographic system by 1-3 CV, feeding the sample flow through liquid and the 1CV pre-washing liquid, heating the sample flow through liquid and the 1CV pre-washing liquid, and returning the sample flow through liquid and the 1CV pre-washing liquid to the IVT reaction container (2), so that the volume of the solution in the whole system is not increased. The chromatographic eluate is collected for storage or downstream processing.
The operation time of the whole system is 6h. After the completion of the reaction, the whole IVT reaction solution was filtered to a continuous chromatography apparatus (11) to collect purified mRNA.
Comparative example 1
The in vitro transcription method for preparing mRNA provided in this comparative example comprises:
1. the device comprises: a 100ml blue cap bottle and a stirring and heating system;
2. the operation method comprises the following steps: the IVT reaction system used comprises the following components: HEPES 50mM, naCl300mM, magnesium acetate 7mM, sodium acetate 50mM, DTT 40mM, spermidine 0.2mM, RNase Inhibitor 4U, T RNA polymerase 100U, pyrophosphatase 2U, NTP each 10mM, cap125mM, DNA template (SEQ ID NO. 1) 1. Mu.g/ml, pH 7.4. The raw materials required by IVT are sequentially added into a blue cap bottle, stirred and heated to 37 ℃, finally enzyme and template are added, the total volume is 100ml, and IVT reaction is started. After 3h of reaction, the reaction was terminated.
Comparative example 2
The in vitro transcription method for preparing mRNA provided in this comparative example comprises:
1. the device comprises: a 100ml blue cap bottle and a stirring and heating system;
2. the operation method comprises the following steps: the IVT reaction system used comprises the following components: HEPES 50mM, naCl300mM, magnesium acetate 7mM, sodium acetate 50mM, DTT 40mM, spermidine 0.2mM, RNase Inhibitor 4U, T RNA polymerase 100U, pyrophosphatase 2U, NTP each 10mM, cap125mM, DNA template (SEQ ID NO. 1) 1. Mu.g/ml, pH 7.4. The raw materials required by IVT are sequentially added into a blue cap bottle, stirred and heated to 37 ℃, finally enzyme and template are added, the total volume is 100ml, and IVT reaction is started. Sampling and detecting the NTPs content and the Cap1 content after 1h and 2h of reaction, then supplementing the NTPs and the Cap1 to the initial concentration, and stopping the reaction after 3 h.
TABLE 1 mRNA yield after completion of IVT reaction (100 ml reaction system)
| Experimental group | Example 1 | Example 2 | Comparative example 1 | Comparative example 2 |
| mRNA production | 1.02g | 0.89g | 0.32g | 0.68g |
| Capping rate | 90％ | 89％ | 91％ | 93％ |
Table 1 shows that the yield of mRNA obtained by the continuous reaction and harvesting system of example 1 and example 2 provided by the present invention is higher than that of comparative example 1 and comparative example 2. The mRNA preparation method provided by the invention has higher yield. The capping rates of the various groups are close.
In addition, the yield of example 2 was close to that of example 1, probably because the DNA template adsorption microspheres were immobilized on the bottom of the reaction bag, and the contact with the enzyme and NTP was not sufficient as in example 1.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (10)
1. A continuous reaction and harvesting system for preparing mRNA by in vitro transcription, characterized in that it comprises, in sequence, in terms of the flow direction of the reaction system: the system comprises an mRNA in-vitro transcription reaction device, a cooling module and a continuous chromatography device, and further comprises a heating module and a process analysis module;
the liquid outlet of the continuous chromatographic device is connected with the mRNA in-vitro transcription reaction device through a temperature-raising module to form a loop;
the mRNA in-vitro transcription reaction device is provided with a microsphere interception device;
the process analysis module is used for realizing the real-time analysis of the reaction liquid by the high performance liquid chromatography HPLC or ion migration mass spectrometry through the online sampling module.
2. The continuous reaction and harvesting system for preparing mRNA by in vitro transcription according to claim 1, wherein the mRNA in vitro transcription reaction apparatus uses microsphere immobilized DNA templates for mRNA transcription;
the microsphere interception device is selected from a magnetic force control module arranged in the mRNA in-vitro transcription reaction device, or a microfiltration device arranged at the liquid outlet end of the mRNA in-vitro transcription reaction device;
preferably, the microfiltration device is a tangential microfiltration device;
preferably, the microfiltration device is provided with a hollow fiber column with a pore diameter of 0.2-0.5 μm.
3. The continuous reaction and harvesting system for preparing mRNA by in vitro transcription according to claim 1, wherein the continuous chromatographic device is provided with 3-8 chromatographic columns for continuous liquid inlet and liquid outlet;
preferably, the chromatography column is an oligo dT affinity chromatography column.
4. The continuous reaction and harvesting system for preparing mRNA by in vitro transcription according to claim 1, wherein the cooling means of the cooling module is selected from air cooling, water cooling or semiconductor cooling; the heating mode of the heating module is selected from water bath or metal bath.
5. A continuous in vitro transcription reaction and harvesting method of mRNA, suitable for use in the continuous reaction and harvesting system for preparing mRNA by in vitro transcription according to any one of claims 1 to 4, characterized in that it comprises the following steps:
adding the microsphere immobilized DNA template and a transcription reaction system into an mRNA in-vitro transcription reaction device, after the transcription reaction is completed, intercepting the microsphere immobilized DNA template by a microsphere interception device, cooling by a cooling module, then entering a continuous chromatography device for capturing and purifying mRNA, and returning the flowing-through liquid to the mRNA in-vitro transcription reaction device by a heating module, wherein a chromatographic column in the continuous chromatography device is regenerated and balanced, and repeating the mRNA transcription reaction of the next round.
6. The method of claim 5, wherein the microsphere immobilized DNA template consists of a single-stranded DNA template and microsphere immobilized oligonucleotide fragments specifically bound to the microsphere via a linker fragment;
preferably, the oligonucleotide is 40-80 nucleotides in length and has a Tm value greater than 45℃for binding to a single stranded DNA template.
7. The continuous in vitro transcription reaction and harvest method of mRNA according to claim 5, wherein the components of the transcription reaction system comprise: 0-500 mM buffer salt, 100-500 mM NaCl, 1-100 mM DTT, 1-10 RNase Inhibitor and 10U, mg 2+ 1～20mM、Spermidine 0.1～0.5mM；T7 RNA Polymerase 10～200U；NTP 5～20mM；
Preferably, the transcription is reversedThe reaction system comprises the following components: 20-200 mM buffer salt, 100-300 mM NaCl, 10-50 mM DTT and 2-5U, mg RNase Inhibitor 2+ 5～20mM、Spermidine 0.1～0.2mM；T7 RNA Polymerase 50～100U；NTP 5～10mM；
Preferably, the added single-stranded DNA template is 0.1-10 mug/ml, and the feeding NTPs and magnesium ions are added in parallel;
preferably, the added single-stranded DNA template is 0.5-2. Mu.g/ml;
preferably, the feeding is performed by supplementing the NTPs such that the molar concentration ratio of cap analogue to NTPs is 4:1, the magnesium ion concentration and pH were maintained at initial values.
8. The continuous in vitro transcription reaction and harvest method of mRNA according to claim 5, wherein the cooling module reduces the temperature of the liquid in the pipeline to 2-25 ℃; the temperature rising module rises the temperature of the liquid in the pipeline to 25-45 ℃.
9. The continuous in vitro transcription reaction and harvest method of mRNA according to claim 5, wherein the mRNA capture method used by the continuous chromatography device is Oligo dT affinity chromatography;
preferably, the method of each column of Oligo dT affinity chromatography is: column balance: 3-10 CV balance buffer solution; loading: 1-5 CV transcription reaction liquid; pre-washing: 1-2 CV of balance buffer solution; eluting: 1-2 CV of eluent A; column cleaning: 10CV eluent B; and (3) post-treatment and regeneration of the filler: 1-2 CV water, 3CV 0.5M sodium hydroxide and 1-2 CV water.
10. The continuous in vitro transcription reaction and harvesting method of mRNA according to claim 5, wherein the process analysis module samples and detects the content and pH value of each component in the device for the in vitro transcription reaction of mRNA in real time, and dynamically adjusts the reaction system and the chromatographic system through process analysis and control.
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