CN112521437A - Recovery method of anti-reverse cap analogue in mRNA in-vitro transcription process, anti-reverse cap analogue and application - Google Patents
Recovery method of anti-reverse cap analogue in mRNA in-vitro transcription process, anti-reverse cap analogue and application Download PDFInfo
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Abstract
The invention provides a recovery method of an anti-reverse cap analogue in an mRNA in-vitro transcription process, the anti-reverse cap analogue and application, and relates to the technical field of biology. The recovery method effectively reduces the production cost of mRNA and improves the utilization efficiency of raw materials.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a recovery method of an anti-reverse cap analogue in an mRNA in-vitro transcription process, the anti-reverse cap analogue and application.
Background
Messenger RNA (mRNA) drugs are a novel treatment scheme with great potential and have wide prospects in the fields of immunology, oncology, vaccine and the like. mRNA contains instructions for cells to make and deliver proteins to various parts of the body, and thus the use of mRNA as a drug can direct intracellular protein expression or extracellular protein secretion. Effective mRNA therapy requires efficient delivery of mRNA to a patient and efficient production of the protein encoded by the mRNA in the patient.
The basic structure of the mRNA stock product at least comprises: the 5' cap structure and the specific mRNA sequence encoding the protein (FIG. 1). Among these, the 5' cap structure of mRNA is particularly critical. Ribosomes in cells translate the sequence of mRNA into protein via the 5' cap structure of the mRNA. And the cap can make mRNA more stable and not be degraded by exonuclease, and mRNA without the cap can not normally code protein. Therefore, in the production of mRNA products, it is important to ensure that mRNA has a 5' cap structure.
The production of the mRNA stock is carried out by an in vitro enzyme-catalyzed reaction. Specifically, in the reaction, T7 RNA transcriptase is completed by transcription reaction using linearized DNA plasmid as template, Anti-Reverse Cap Analog (ARCA) as example, and nucleoside triphosphate mixture NTPs (including adenosine triphosphate ATP, uridine triphosphate UTP, cytidine triphosphate CTP and guanosine triphosphate GTP) as raw materials (FIG. 2). Wherein the cap structure is a blue mark in the figure. The capping of mRNA is accomplished by simultaneous reaction of ARCA anti-reverse cap analogs (caps) and NTPs during the reaction of DNA transcription of mRNA, a process referred to as co-transcription.
As a result of the reaction process, it was found that the co-transcription reaction produced a proportion of uncapped products due to the similarity in chemical structure between the anti-reverse cap analogue and Guanosine Triphosphate (GTP) and the same reactivity of T7 RNA transcriptase to both. This product is an ineffective impurity that should be avoided as much as possible during the production of mRNA drugs.
In order to increase the capping ratio of the reaction product, the capping efficiency was secured to about 80% by increasing the amount of ARCA (ARCA: GTP ═ 4: 1) in the actual production process. For a typical mRNA product containing hundreds to thousands of bases, the transcription reaction may utilize only one thousand times the availability of the anti-reverse cap analog in the reaction starting material compared to other NTPs materials, since the synthesized mRNA contains only one cap structure (produced by the anti-reverse cap analog) and hundreds to thousands of bases (produced by the NTPs materials). In addition, the raw material cost of the anti-reverse cap analogue is far higher than that of other reaction raw materials, so that the development of the recycling technology of the anti-reverse cap analogue compound has great industrial significance.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
It is a first object of the present invention to provide a method for recovering an anti-reverse cap analog during in vitro transcription of mRNA, which alleviates at least one of the technical problems of the prior art.
It is a second object of the present invention to provide an anti-reverse cap analog recovered by the above recovery method.
The third purpose of the invention is to provide the application of the recovered anti-reverse cap analogue in-vitro transcription of mRNA.
The invention provides a method for recovering anti-reverse cap analogues in the in-vitro transcription process of mRNA, which comprises the steps of separating the anti-reverse cap analogues in a reaction liquid after the transcription reaction of the mRNA through a high performance liquid chromatography to obtain crude anti-reverse cap analogues, and purifying, concentrating and drying the crude anti-reverse cap analogues in sequence to obtain the anti-reverse cap analogues.
Further, the anti-reverse cap analogs include one or more of ARCA, m7G (5') ppp (5') (2'ome a) pG, m7G (5') ppp (5') (2' ome g) pG, m7(3'ome g) (5') ppp (5') (2' ome g) pG, m7(3'ome g) (5') ppp (5') (2' ome a) pG, mCAP, dmCAP, tmCAP, or dmCAP.
Further, the chromatography comprises adsorption chromatography, partition chromatography, ion exchange chromatography, gel chromatography or reversed-phase high performance liquid chromatography, preferably reversed-phase high performance liquid chromatography.
Further, the crude anti-reverse cap analog was purified by dialysis.
Further, the purified anti-reverse cap analog was concentrated and dried by vacuum centrifugation.
Further, after the reaction solution after the mRNA transcription reaction is pretreated, the anti-reverse cap analog is separated by high performance liquid chromatography.
Further, the pretreatment comprises diluting the reaction solution after the mRNA transcription reaction with a mobile phase;
preferably, the mobile phase comprises TEAA, preferably 0.05-0.2M TEAA;
preferably, the dilution factor is 1-3, preferably 2.
Further, the high performance liquid chromatography comprises the following steps:
(a) pretreatment of reaction liquid: diluting the reaction solution after mRNA transcription reaction by 1-3 times with a mobile phase to obtain a reaction solution to be separated;
(b) separating the reaction liquid to be separated obtained in the step (a) by adopting reverse-phase high performance liquid chromatography, wherein the chromatographic condition at least meets one of the following conditions:
the mobile phase is 0.05-0.2M TEAA; the flow rate is 0.8-1.2 ml/min; the column temperature is 42-48 ℃; the sample volume of the reaction solution to be separated is 80-120 mul.
The present invention also provides an anti-inversion cap analog recovered using the above recovery method.
In addition, the invention also provides the application of the anti-reverse cap analogue in-vitro transcription of mRNA.
The recovery method of the anti-reverse cap analogue in the mRNA in-vitro transcription process is suitable for the in-vitro transcription process of any type of mRNA, the recovery method initially separates the anti-reverse cap analogue in a reaction liquid after the mRNA transcription reaction through a high performance liquid chromatography, and then obtains the anti-reverse cap analogue after sequentially purifying, concentrating and drying an anti-reverse cap analogue crude product obtained by the initial separation, thereby realizing the repeated utilization of the anti-reverse cap analogue. The recovery method effectively reduces the production cost of mRNA, and improves the utilization efficiency of raw materials through recovery and reutilization.
Moreover, the anti-reverse cap analogue recovered by the recovery method provided by the invention can normally play the role in the subsequent new transcription reaction, obtains the same effect as a brand-new cap, and can be used for synthesizing high-quality mRNA products again.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a schematic diagram showing the basic structure of a mRNA stock product;
FIG. 2 is a schematic diagram showing a transcription reaction process using an anti-reverse cap analog (ARCA) as an example and a nucleoside triphosphate mixture NTPs as raw materials;
FIG. 3 is a liquid chromatogram of the initial reaction raw material remaining after the RNA co-transcription reaction, whose IDs are respectively confirmed by comparing with liquid chromatograms of an NTP mixture and an anti-reverse cap analog;
FIG. 4 is a liquid chromatogram of an anti-reverse cap analog recovered by chromatography, with retention times consistent with standards;
FIG. 5 is a liquid chromatogram superimposed before and after recovery and purification of an anti-reverse cap analog to determine anti-reverse cap analog recovery;
FIG. 6 is a standard curve for the determination of anti-inversion cap analog concentration by UV spectrophotometry.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Unless defined otherwise herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by one of ordinary skill in the art. The meaning and scope of a term should be clear, however, in the event of any potential ambiguity, the definition provided herein takes precedence over any dictionary or extrinsic definition. In this application, unless otherwise indicated, the use of the term "including" and other forms is not limiting.
Generally, the nomenclature used, and the techniques thereof, in connection with the cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly employed in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature used in connection with the analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein, and the laboratory procedures and techniques thereof, are those well known and commonly employed in the art.
Based on the fact that the utilization efficiency of raw materials used for mRNA in vitro transcription is only 0.05% of the feeding amount of the raw materials, the raw material cost of the anti-reverse cap analogue is always high, the invention provides a method for recovering the anti-reverse cap analogue in the mRNA in vitro transcription process, the anti-reverse cap analogue in a reaction liquid after mRNA transcription reaction is separated through high performance liquid chromatography to obtain a crude product of the anti-reverse cap analogue, and the crude product of the anti-reverse cap analogue is purified, concentrated and dried in sequence to obtain the anti-reverse cap analogue.
Anti-reverse cap analogs, also known as cap structure analogs, are important starting materials for increasing the 5' cap structure of mRNA during in vitro transcription of mRNA. In the present invention, the anti-reverse cap analogs that can be recovered can be, for example, but are not limited to, one or more of ARCA, m7G (5') ppp (5') (2'OMeA) pG, m7G (5') ppp (5') (2' OMeG) pG, m7(3'OMeG) (5') ppp (5') (2' OMeG) pG, m7(3'OMeG) (5') ppp (5') (2' OMeA) pG, mCAP, dmCAP, tmCAP, or dmCAP.
The high-phase liquid chromatography has the characteristics of high separation efficiency and high sensitivity, and the anti-reverse cap analogue in the reaction liquid after the mRNA transcription reaction is separated by using the high performance liquid chromatography, so that the separation effect can be greatly improved; and the high performance liquid chromatography has wide application range, and is suitable for the in vitro transcription process of any type of mRNA when the anti-reverse cap analogue is separated. The present invention is not limited to a specific type of high performance liquid chromatography, and any type of chromatography that can perform a separation function may be used, and examples thereof include, but are not limited to, adsorption chromatography, partition chromatography, ion exchange chromatography, gel chromatography, and reverse-phase high performance liquid chromatography.
After the initial separation by high performance liquid chromatography, the crude anti-reverse cap analogue is obtained, and then the obtained crude anti-reverse cap analogue is purified, concentrated and dried in sequence to obtain the anti-reverse cap analogue. The purification method is not limited, and any purification method capable of effectively removing other small molecular impurities and residual solvents and retaining the anti-reverse cap analogue in the prior art is suitable for the invention. The present invention also does not limit the way of concentration and drying, and all techniques that can achieve the concentration and drying effects without affecting the yield of the anti-reverse cap analog can be used in the present invention.
The recovery method of the anti-reverse cap analogue in the mRNA in-vitro transcription process is suitable for the in-vitro transcription process of any type of mRNA, the recovery method initially separates the anti-reverse cap analogue in a reaction liquid after the mRNA transcription reaction through a high performance liquid chromatography, and then obtains the anti-reverse cap analogue after sequentially purifying, concentrating and drying an anti-reverse cap analogue crude product obtained by the initial separation, thereby realizing the repeated utilization of the anti-reverse cap analogue. The recovery method effectively reduces the production cost of mRNA and improves the utilization efficiency of raw materials.
In some preferred embodiments, the crude anti-reverse cap analog is purified by dialysis.
By utilizing the size difference of molecular weight, the method of dialysis can remove chromatographic mobile phase impurities contained in a sample after chromatographic purification, effectively remove other small molecular impurities and residual solvent, and achieve the aim of purifying and recycling the obtained anti-reverse cap analogue.
In some preferred embodiments, the purified anti-reverse cap analog is concentrated and dried using vacuum centrifugation.
The vacuum centrifugal concentration mode is suitable for more sensitive biological samples, such as protein, nucleic acid and other substances. Therefore, in the present embodiment, concentration and drying are performed by vacuum centrifugation, and it is possible to obtain a high-concentration sample without causing problems such as cross contamination, sample loss, sample denaturation, reduction in sample activity, or oxidation.
In some preferred embodiments, the anti-reverse cap analogs are isolated by high performance liquid chromatography after pretreatment of the reaction solution after the mRNA transcription reaction.
The pretreatment preferably comprises diluting the reaction solution after the mRNA transcription reaction with a mobile phase.
Preferably, the mobile phase comprises TEAA, preferably 0.05-0.2M TEAA, for example it may be, but not limited to, 0.05M TEAA, 0.1M TEAA, 0.15M TEAA or 0.2M TEAA.
Preferably, the dilution factor is 1-3, such as but not limited to 1, 2 or 3, preferably 2.
By using a specific mobile phase to dilute the reaction solution by a specific multiple, the anti-reverse cap analogue in the reaction solution can be separated more accurately and efficiently in the subsequent high performance liquid chromatography reaction.
In some preferred embodiments, the high performance liquid chromatography comprises the steps of:
(a) pretreatment of reaction liquid: diluting the reaction solution after mRNA transcription reaction by 1-3 times with a mobile phase to obtain a reaction solution to be separated;
(b) separating the reaction liquid to be separated obtained in the step (a) by adopting reverse-phase high performance liquid chromatography, wherein the chromatographic condition at least meets one of the following conditions:
the mobile phase is 0.05-0.2M TEAA; the flow rate is 0.8-1.2 ml/min; the column temperature is 42-48 ℃; the sample volume of the reaction solution to be separated is 80-120 mul.
Wherein, the concentration of the mobile phase can be, but is not limited to, 0.05M, 0.1M, 0.15M or 0.2M; the flow rate may be, for example, but is not limited to, 0.8ml/min, 0.9ml/min, 1ml/min, 1.1ml/min, or 1.2 ml/min; the column temperature may be, for example, but not limited to, 42 ℃, 43 ℃, 44 ℃, 45 ℃, 46 ℃, 47 ℃, or 48 ℃; the amount of the reaction solution to be separated may be, for example, but not limited to, 80. mu.l, 90. mu.l, 100. mu.l, 110. mu.l or 120. mu.l.
By further optimizing and limiting the conditions of the high-phase liquid chromatography, the chromatographic separation effect can be better, and the recovery efficiency of the anti-reverse cap analogue can be improved.
Preferably, the mobile phase is 0.1M TEAA, the flow rate is 1ml/min, the column temperature is 45 ℃, and the sample volume of the reaction solution to be separated is 100 μ l.
When the above chromatographic conditions are simultaneously satisfied, the separation efficiency of the anti-reverse cap analog is higher.
According to a second aspect of the present invention, there is also provided an anti-inversion cap analogue recovered using the recovery method described above.
The anti-reverse cap analogue recovered by the recovery method provided by the invention can normally play the role in the subsequent new transcription reaction, obtains the same effect as the brand-new anti-reverse cap analogue, and can be used for synthesizing high-quality mRNA products again.
In addition, the invention also provides the application of the anti-reverse cap analogue in-vitro transcription of mRNA.
When the method is applied, the recovered anti-reverse cap analogue is redissolved for quantification, then the dosage of the recovered anti-reverse cap analogue is adjusted according to production requirements, and the recovered anti-reverse cap analogue is put into the next mRNA in vitro transcription reaction for recycling.
Specifically, Tris-HCl solution can be used for redissolution, and ultraviolet spectrophotometry can be used for quantification.
The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
Example 1 Primary recovery of anti-reverse Cap analogs Using reverse phase high Performance liquid chromatography
This example illustrates the method of primary recovery of the anti-reverse cap analog by reverse phase high performance liquid chromatography and validation of the final purified anti-reverse cap analog.
Preparing 0.1M TEAA as a mobile phase, operating at the column temperature of 45 ℃ for 10min at the flow rate of 1ml/min for recovering the anti-reverse cap analogue, diluting the remaining mixed solution after RNA transcription reaction by 2 times by using 0.1M TEAA, wherein the sample amount is 100 mu l, observing the peak-out time of each component and collecting the peak of the anti-reverse cap analogue. FIG. 3 is a liquid chromatogram of the remaining mixture after RNA transcription reaction, and it can be seen that the mixture contains residual NTP and anti-reverse cap analog, and the two substances can be clearly separated, and collection is performed only according to the peak-off time of the anti-reverse cap analog. FIG. 4 is a liquid chromatogram of the finally recovered and purified anti-reverse cap analogue, in which no other impurity peaks can be observed, and only the anti-reverse cap analogue peak is observed, indicating that the recovered anti-reverse cap analogue has a high purity. FIG. 5 is a liquid chromatogram superimposed before and after recovery and purification of the anti-reverse cap analog, where it can be seen that the peak positions of the anti-reverse cap analog before and after recovery are consistent, again indicating that the anti-reverse cap analog is recovered and not other residual materials.
Example 2 method for removing NTP and TEAA by dialysis
This example illustrates the use of dialysis to remove chromatographic mobile phase impurities from a chromatographically purified sample. The principle is to use a dialysis bag of 0.5KD to selectively replace the residual mobile phase chromatographic solvent with nucleic-Free Water, using TEAA (about 0.16KD) and the chromatographic solvent with smaller molecular weight and the anti-reverse cap analog (about 1KD) with larger molecular weight.
Boiling the dialysis bag in purified water for 10min, and taking out for use; adding 1 volume of the liquid phase primary purified solution into a dialysis bag, and sealing; adding 20 times volume of nucleic-Free Water into a beaker, and adding a magnetic stirrer; placing the dialysis bag in the beaker, placing the beaker on a magnetic stirrer, continuously stirring, and dialyzing for 3 h; the Water in the beaker was poured out and replaced with a new 20-fold volume of Nuclear-Free Water and dialyzed overnight.
The solution obtained by dialysis was dispensed into 1.5mL of EP tubes, 1mL per tube, and placed in a vacuum centrifugal concentrator (Christ, RVC 2-18CD plus) with the open, set at 25 ℃, the vacuum pump was turned on, concentrated overnight, and dried. This was reconstituted with the appropriate amount of 100mM Tris-HCl solution.
Example 3 quantification of recovered purified anti-reverse cap analogs
This example illustrates a method for quantifying the recovery of purified anti-reverse cap analogs.
Gradient dilution to 6.25mM, 3.13mM, 1.56mM, 0.78mM, 0.39mM, 0.20mM using the novel anti-reverse cap analogue, A260 thereof was determined using an ultramicro spectrophotometer, and standard curves were plotted using Excel (see FIG. 6); diluting the sample to be detected by different times until the A260 of the sample to be detected falls in the range of the standard curve, and calculating the concentration of the sample to be detected by a fitting equation of the standard curve.
Example 4 Using recovered and purified anti-reverse cap analog and a completely new anti-reverse cap analog to establish a reaction separately, the yield of the reaction was compared
This example used the recovery of purified anti-reverse cap analogue and a completely new anti-reverse cap analogue to establish reactions separately, comparing the yields of the reactions.
Reactions were established using recovered and purified anti-reverse cap analogs and brand new anti-reverse cap analogs, respectively, and after completion of the reactions, mRNA was purified and quantified, and the yields were compared (see table 1), showing that recovery of purified anti-reverse cap analogs did not affect RNA transcription reactions and could achieve yields consistent with brand new anti-reverse cap analogs.
TABLE 1 comparison of reaction yields using recovered purified anti-reverse cap analogs versus using all-new anti-reverse cap analogs
Example 5 comparison of protein expression at cellular level of mRNA prepared from recovered purified anti-reverse cap analog and novel anti-reverse cap analog
This example illustrates protein expression at the cellular level of mRNA prepared by cell transfection, ELISA method to compare recovered purified anti-reverse cap analog with a novel anti-reverse cap analog.
HEK293 cells plated at a density of 4X 105One/well, 5.0% CO at 37 ℃2The incubator was overnight. Then, the medium was changed, the cell plate was taken out and the medium was aspirated, and fresh medium was added thereto, and the mixture was placed at 37 ℃ and 5.0% CO2An incubator. Solution A: mu.g of mRNA was mixed in 100. mu.l of Opti-MEM medium, solution B: mixing 3 μ l Lipo MessengerMax in 100 μ l Opti-MEM medium, mixing solution A and solution B, standing at room temperature for 20min, adding into cell plate, mixing, standing at 37 deg.C with 5.0% CO2Culturing in an incubator for 24 h. Then the culture supernatant is gently aspirated, 1ml PBS/hole is added for washing, 50 mu l of lysate 1 is added, the cell is scraped down by a cell scraper and transferred into a 1.5ml centrifuge tube, the mixture is evenly mixed by oscillation, and is centrifuged for 1 to 2 hours at the centrifugal force of 17000g and the supernatant is sucked for protein detection at the temperature of 4 ℃.
The expression of the corresponding protein was detected by enzyme-linked immunosorbent assay (ELISA). The protein monoclonal antibody is used as a coating antibody, another antibody combined with horseradish peroxidase (HRP) is used as a detection antibody, and TMB is used as a display antibodyThe color substrate solution is reacted for 10min in the dark, and then 2M H is added2SO4The reaction was stopped, with maximum absorption at 450nm and read using a Molecular Devices plate reader.
This example illustrates the substantial agreement of protein expression at the cellular level of mRNA prepared from recovered purified anti-reverse cap analog and novel anti-reverse cap analog by comparing the average absorbance of the samples to the protein expression level as shown in Table 2.
TABLE 2 comparison of protein expression levels in cells of mRNA prepared using recovered purified anti-reverse cap analogs and mRNA prepared by standard reactions for ELISA assays
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. A method for recovering anti-reverse cap analogues in the in vitro transcription process of mRNA is characterized in that the anti-reverse cap analogues in reaction liquid after the transcription reaction of the mRNA are separated through high performance liquid chromatography to obtain crude anti-reverse cap analogues, and the crude anti-reverse cap analogues are purified, concentrated and dried in sequence to obtain the anti-reverse cap analogues.
2. The recovery method of claim 1, wherein the anti-reverse cap analog comprises one or more of ARCA, m7G (5') ppp (5') (2'ome a) pG, m7G (5') ppp (5') (2' ome g) pG, m7(3'ome g) (5') ppp (5') (2' ome g) pG, m7(3'ome g) (5') ppp (5') (2' ome a) pG, mCAP, dmCAP, tmCAP, or dmCAP.
3. The recovery process according to claim 1, wherein the chromatography comprises adsorption chromatography, partition chromatography, ion exchange chromatography, gel chromatography or reversed phase high performance liquid chromatography, preferably reversed phase high performance liquid chromatography.
4. The recovery method of claim 1, wherein the crude anti-reverse cap analog is purified by dialysis.
5. The recovery method of claim 1, wherein the purified anti-reverse cap analog is concentrated and dried by vacuum centrifugation.
6. The recovery method according to claim 1, wherein the reaction solution after the mRNA transcription reaction is pretreated, and then the anti-reverse cap analogue is separated by high performance liquid chromatography.
7. The recovery method according to claim 6, wherein the pretreatment comprises diluting the reaction solution after the mRNA transcription reaction with a mobile phase;
preferably, the mobile phase comprises TEAA, preferably 0.05-0.2M TEAA;
preferably, the dilution factor is 1-3, preferably 2.
8. The recovery method according to any one of claims 1 to 7, wherein the high performance liquid chromatography comprises the steps of:
(a) pretreatment of reaction liquid: diluting the reaction solution after mRNA transcription reaction by 1-3 times with a mobile phase to obtain a reaction solution to be separated;
(b) separating the reaction liquid to be separated obtained in the step (a) by adopting reverse-phase high performance liquid chromatography, wherein the chromatographic condition at least meets one of the following conditions:
the mobile phase is 0.05-0.2M TEAA, preferably 0.1M TEAA; the flow rate is 0.8-1.2ml/min, preferably 1 ml/min; the column temperature is 42-48 ℃, preferably 45 ℃; the amount of the reaction solution to be separated is 80 to 120. mu.l, preferably 100. mu.l.
9. The anti-reverse cap analog recovered by the recovery process of any one of claims 1-8.
10. Use of an anti-reverse cap analogue as claimed in claim 9 for in vitro transcription of mRNA.
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