阅读说明：本技术 基于智能控制的rna酶解液层析分离方法及系统 (RNA enzymolysis liquid chromatographic separation method and system based on intelligent control ) 是由 陈修足 王军 邱蔚然 徐浩 邱志云 于 2021-09-24 设计创作，主要内容包括：本发明公开了一种基于智能控制的RNA酶解液层析分离方法及系统,通过实时在线检测和智能化控制对层析分离工艺进一步改进,在RNA酶解液层析分离方法过程中,通过RNA酶解液层析分离系统的控制系统控制RNA酶解液依次进行上样、水洗、洗脱得到四种核苷酸溶液,再将四种核苷酸溶液进行浓缩、结晶、干燥处理,实现层析分离全流程无人化操作,过程控制更加便捷,省时省力,降低劳动成本,减少人为误差,避免生产受到损失。本发明在上样过程中通过多层或多根层析柱排列组合方式,通过实时在线监测和智能化控制,可使上样量增加10～15％,并使洗脱结晶产品纯度提高至99％以上。(The invention discloses an RNA enzymolysis liquid chromatography separation method and system based on intelligent control, which further improve the chromatography separation process through real-time online detection and intelligent control. In the invention, the sample loading amount can be increased by 10-15% by a multi-layer or multi-layer chromatography column arrangement and combination mode in the sample loading process and by real-time online monitoring and intelligent control, and the purity of an eluted and crystallized product is improved to more than 99%.)

1. The RNA enzymolysis liquid chromatographic separation method based on intelligent control is characterized in that an RNA enzymolysis liquid chromatographic separation system is adopted to carry out chromatographic separation on RNA enzymolysis liquid, and the RNA enzymolysis liquid chromatographic separation method comprises the following steps:

(1) the control system of the RNA enzymatic hydrolysate chromatographic separation system pumps the filtered RNA enzymatic hydrolysate into a column chromatography system, and four nucleotides, namely 5 '-CMP, 5' -AMP, 5 '-UMP and 5' -GMP in the RNA enzymatic hydrolysate are adsorbed into the column chromatography system;

(2) washing, namely washing the column chromatography system with deionized water after the sample loading is finished, and detecting the column chromatography system with a nucleic acid protein detector;

(3) eluting, eluting strongly basic anion exchange resin in a column chromatography system by using an eluent after water washing is finished, detecting the effluent liquid of the column chromatography system at regular time by using an HPLC (high performance liquid chromatography) detector, judging the type of the nucleotide, respectively collecting four nucleotide solutions of 5 '-CMP (chemical mechanical polishing), 5' -AMP (AMP), 5 '-UMP (UMP) and 5' -GMP (GMP), and respectively concentrating, crystallizing and drying the four nucleotide solutions to obtain a crystallized finished product.

2. The RNA enzymatic hydrolysate chromatographic separation method based on intelligent control as claimed in claim 1,

in the step (1):

in the sample loading process, the concentration of the RNA enzymolysis liquid is 1-3%, the volume of the RNA enzymolysis liquid is 8-12 times of the volume of a resin column in the column chromatography system, and the flow rate of the RNA enzymolysis liquid is 80-120L/h; and/or

In the sample loading process, a nucleic acid protein detector is adopted to detect the effluent liquid of the column chromatography system, when A260 in the effluent liquid of the column chromatography system is less than 1, the waste is discharged, otherwise, the sample loading is finished; and/or

In the step (2):

in the water washing process, the volume of the deionized water is 4-10 times of the volume of the resin column in the column chromatography system; and/or

In the washing process, a nucleic acid protein detector is adopted to detect the effluent liquid of the column chromatography system, and when A260 in the effluent liquid of the column chromatography system is less than 1, waste is discharged; otherwise, starting the collector; and/or

In the step (3):

the elution is a step elution or a gradient elution.

3. The RNA enzymatic hydrolysate chromatographic separation method based on intelligent control as claimed in claim 2, wherein when gradient elution is adopted in the step (2):

the column chromatography system comprises 1 strong acid cation exchange resin and a strong base anion exchange resin unit consisting of a plurality of strong base anion exchange resins which are sequentially connected in series, and a one-way valve is arranged between the strong acid cation exchange resin and the strong base anion exchange resin unit; and/or

The eluent is a sodium chloride solution with the mass percentage concentration of 0-6%, the volume of the eluent is 10-20 times of that of a strong acid cation exchange resin column of the column chromatography system, and the flow rate of the eluent is 12-24L/h; and/or

The four nucleotides were collected as follows: in the gradient elution process, an HPLC detector is used for detecting the effluent of the strongly basic anion exchange resin unit of the column chromatography system at regular time, when 5 '-CMP is detected in the effluent and the concentration is more than or equal to 0.4g/L, the collector starts to collect 5' -CMP, when 5 '-AMP is detected in the effluent, the collector starts to collect 5' -AMP, when 5 '-UMP is detected in the effluent, the collector starts to collect 5' -UMP, when 5 '-GMP is detected in the effluent, the collector starts to collect 5' -GMP, and when the concentration of 5 '-GMP is less than or equal to 10g/L, the collector stops collecting 5' -GMP.

4. The RNA enzymatic hydrolysate chromatographic separation method based on intelligent control as claimed in claim 2, wherein when stepwise elution is adopted in the step (2):

the column chromatography system comprises a plurality of layers of stacked strong base anion exchange resins or a plurality of strong base anion exchange resins connected in series; one-way valves are arranged between each layer/each layer of strong-base anion exchange resin; and/or

The eluent is formic acid solution and/or sodium formate solution and/or sodium chloride solution and/or hydrochloric acid solution, and the flow rate of the eluent is 12-24L/h; and/or

The four nucleotides were collected as follows: in the step of step elution, an HPLC (high performance liquid chromatography) detector is adopted to detect the effluent liquid of each layer or each piece of strong-base anion exchange resin of the column chromatography system at regular time, 0.01mol/L formic acid solution is adopted as the eluent to carry out step elution on 5 '-CMP, and 5' -CMP with the concentration of 10-50 g/L is collected; using 0.1mol/L formic acid solution as eluent to elute the 5 '-AMP in a stepwise manner, and collecting the 5' -AMP with the concentration of 10-50 g/L; using 0.1mol/L formic acid solution and 0.1mol/L sodium formate solution as eluent to elute the 5' -UMP in stages; and (3) adopting a sodium chloride solution with pH of 3.0 and mass percentage concentration as an eluent to elute GMP in a segmented manner, and collecting GMP with the concentration of 10-50 g/L.

5. A RNA enzymolysis liquid chromatographic separation system is characterized by comprising a column chromatography system, a sample loading mechanism, a water washing mechanism, an eluent mechanism, a collector, a detection mechanism and a control system;

the column chromatography system comprises 1 strong-acid cation exchange resin and a strong-base anion exchange resin unit, wherein the strong-base cation exchange resin unit is formed by a plurality of strong-base anion exchange resins which are connected in series; a one-way valve is arranged between the strong acid cation exchange resin unit and the strong base anion exchange resin unit;

the detection mechanism detects the effluent liquid of the column chromatography system and transmits the detection information to the control system;

the control system receives detection information of the detection mechanism and generates a water washing instruction, an elution instruction and a collection instruction according to the detection information; and the control system is respectively connected with the sample loading mechanism, the water washing mechanism, the eluent mechanism, the column chromatography system, the collector and the detection mechanism.

6. The RNA enzymatic hydrolysis chromatographic separation system according to claim 5, wherein at least 2 strongly basic anion exchange resins are provided in the strongly basic anion exchange resin unit; and/or

The strong-acid cation exchange resin is Na type, and the strong-base anion exchange resin is Cl type; and/or

The control system comprises a sample loading controller for controlling the sample loading mechanism, a water washing controller for controlling the water washing mechanism and an elution controller for controlling the eluent mechanism; and/or

The detection mechanism comprises a nucleic acid protein detector and an HPLC detector which are respectively connected with the liquid outlet of the column chromatography system; timing switches are arranged on pipelines between the nucleic acid protein detector and the liquid outlet of the column chromatography system; and/or

The RNA enzymolysis liquid chromatographic separation system also comprises a monitoring system, and the monitoring system comprises a camera device for monitoring the chromatographic separation process of the RNA enzymolysis liquid and a display connected with the camera device.

7. The RNA enzymatic hydrolysate chromatographic separation system is characterized by comprising a column chromatography system, a sample loading mechanism, a water washing mechanism, an eluent mechanism, a collector, a detection mechanism and a control system;

the column chromatography system comprises a plurality of layers of stacked strong base anion exchange resins or a plurality of strong base anion exchange resins connected in series; one-way valves are arranged among the layers/the plurality of layers of strong-base anion exchange resins;

the detection mechanism detects the effluent liquid of each layer/each strong-base anion exchange resin and transmits the detection information to the control system;

the control system receives detection information of the detection mechanism and generates a water washing instruction, an elution instruction and a collection instruction according to the detection information; and the control system is respectively connected with the sample loading mechanism, the water washing mechanism, the eluent mechanism, the column chromatography system, the collector and the detection mechanism through electric signals.

8. The RNA enzymatic hydrolysate chromatographic separation system according to claim 7, wherein the strongly basic anion exchange resin is provided with at least 3 layers or 3 pieces; and/or

The strong-base anion exchange resin is Cl type; and/or

The control system comprises a sample loading controller for controlling the sample loading mechanism, a water washing controller for controlling the water washing mechanism and an elution controller for controlling the eluent mechanism; and/or

The detection mechanism comprises a nucleic acid protein detector and a first HPLC detector which are respectively communicated with each layer/each strong basic anion exchange resin liquid outlet, and a second HPLC detector which is communicated with the last layer or the last strong basic anion exchange resin liquid outlet; a one-way valve is arranged on a pipeline between the second HPLC detector and the last layer or the last strong-base anion exchange resin liquid outlet; and/or

The RNA enzymolysis liquid chromatographic separation system also comprises a monitoring system, and the monitoring system comprises a camera device for monitoring the chromatographic separation process of the RNA enzymolysis liquid and a display connected with the camera device.

Technical Field

The invention belongs to the field of mononucleotide preparation, and particularly relates to an RNA enzymolysis liquid chromatographic separation method and system based on intelligent control.

Background

In recent years, with research and development of small nucleic acid drugs and successful popularization and application of mRNA vaccines, 4 kinds of 5 ' -nucleotides, i.e., 5 ' -AMP (5 ' -adenylic acid), 5 ' -GMP (5 ' -guanylic acid), 5 ' -CMP (5 ' -cytidylic acid) and 5 ' -UMP (5 ' -uridylic acid), which are the most basic raw materials, are increasingly in demand; wherein, the biological source of RNA is mainly to culture yeast by molasses fermentation, and then to extract RNA, feed protein, coenzyme I (or coenzyme A) and the like from the yeast, and the RNA obtained by the way is favored by 5' -nucleotide obtained after the enzymolysis of RNA due to the biological safety; the process uses abundant and cheap cane sugar resources, and becomes protein, RNA and high value-added products with scarce resources, so that the process is more in line with the direction of circular economy and sustainable development.

RNA can be hydrolyzed into 45 ' -nucleotides such as 5 ' -AMP, 5 ' -GMP, 5 ' -CMP, 5 ' -UMP and the like through phosphodiesterase or nuclease P1, the mixture of the 45 ' -nucleotides needs to be separated through cation exchange resin or anion exchange resin chromatography, single nucleotide can be obtained after loading, water washing, stepwise elution or gradient elution in sequence, then the single 5 ' -nucleotide can be obtained through concentration, crystallization and drying, and the method can be applied to infant milk powder nutrition enhancers, medical intermediates, small nucleic acid drugs, vaccines and the like.

At present, the process of producing 4 kinds of 5' -nucleotides by enzymolysis of RNA is well established, and for example, the relevant literature for separation by cation chromatography is as follows: document 1 (capital brewery, group 824 of Chinese academy of sciences: production of 5 ' -mononucleotide and adenosine triphosphate, scientific Press, 1971), document 2 (national loyalty, fermentation industry, 1997.35, 836-844), document 3 (Lidellite, production of 5 ' -nucleotide using 5 ' -phosphodiesterase enzyme method, Master thesis of the university of eastern Richardson, 2002), document 4 (Chinese patent publication No. CN1286259A), and document 5 (Chinese patent CN 100347310C); the relevant literature for separation by anion chromatography is as follows: document 6(CN 1177859C), document 7 (chinese patent CN 108752405B), and the like; since 4 kinds of 5' -nucleotides contain amino groups except uridylic acid, the pH is slightly acidic, such as pH of about 2-4, and the amino group has a positive charge; all nucleotides have phosphate groups and enol groups, so that the nucleotides have strong negative charges when the pH is 6-7, and the first phosphate group, the second phosphate group and the enol group are dissociated when the pH is more than 10, so that the negative charges are stronger. Thus, more resin is used for separating nucleotides by cation resin chromatography, while less resin is used for anion chromatography. According to the calculation of theory and practical application, the dosage of the medicine is different by about 5 times.

In actual production, except for using cation exchange resin in early small-batch production, anion chromatography separation is used in the prior large-batch production process such as Japan and the like; however, in the anion chromatographic separation process, the chromatographic separation period is long, the process control is relatively complex, and the chromatographic separation period is generally 6 days or even longer; because anion chromatographic separation belongs to the continuous operation process, four shifts and three shifts are needed during production, time and labor are wasted, night shifts are needed, artificial errors are easily caused, the yield is often reduced, and the production is lost.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide the RNA enzymolysis liquid chromatography separation method and the RNA enzymolysis liquid chromatography separation system based on intelligent control, the chromatography separation process is further improved through real-time online detection and intelligent control, the unmanned operation of the whole chromatography separation process is realized, the process control is more precise and convenient, the time and the labor are saved, the labor cost is reduced, the human error is reduced, and the production loss is avoided; in the invention, the real-time online detection of a nucleic acid protein detector (short for detector) is used for monitoring whether the nucleotide flows out in the processes of sample loading and water washing so as to know whether the process is normally operated; the elution process was controlled and four nucleotide solutions of 5 '-CMP, 5' -AMP, 5 '-UMP and 5' -GMP were collected by timed on-line sampling and HPLC detector analysis.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides an RNA enzymolysis liquid chromatographic separation method based on intelligent control, which adopts an RNA enzymolysis liquid chromatographic separation system to carry out chromatographic separation on RNA enzymolysis liquid, and comprises the following steps:

(1) the control system of the RNA enzymatic hydrolysate chromatographic separation system pumps the filtered RNA enzymatic hydrolysate into a column chromatography system, and four nucleotides, namely 5 '-CMP, 5' -AMP, 5 '-UMP and 5' -GMP in the RNA enzymatic hydrolysate are adsorbed into the column chromatography system;

(2) washing, namely washing the column chromatography system with deionized water after the sample loading is finished, and detecting the column chromatography system with a nucleic acid protein detector;

(3) eluting, eluting strongly basic anion exchange resin in a column chromatography system by using an eluent after water washing is finished, detecting the effluent liquid of the column chromatography system at regular time by using an HPLC (high performance liquid chromatography) detector, judging the type of the nucleotide, respectively collecting four nucleotide solutions of 5 '-CMP (chemical mechanical polishing), 5' -AMP (AMP), 5 '-UMP (UMP) and 5' -GMP (GMP), and respectively concentrating, crystallizing and drying the four nucleotide solutions to obtain a crystallized finished product.

Preferably, in the step (1):

in the sample loading process, the concentration of the RNA enzymolysis liquid is 1-3%, the volume of the RNA enzymolysis liquid is 8-12 times of the volume of a resin column in the column chromatography system, and the flow rate of the RNA enzymolysis liquid is 80-120L/h; and/or

In the sample loading process, a nucleic acid protein detector is adopted to detect the effluent liquid of the column chromatography system, when A260 in the effluent liquid of the column chromatography system is less than 1, the waste is discharged, otherwise, the sample loading is finished; and/or

In the step (2):

in the water washing process, the volume of the deionized water is 4-10 times of the volume of the resin column in the column chromatography system; and/or

In the washing process, a nucleic acid protein detector and an HPLC (high performance liquid chromatography) detector are adopted to detect the effluent liquid of the column chromatography system, and when A260 in the effluent liquid of the column chromatography system is less than 1, waste is discharged; otherwise, starting the collector; and/or

In the step (3):

the elution is a step elution or a gradient elution.

Preferably, when gradient elution is used in step (2):

the column chromatography system comprises 1 strong acid cation exchange resin and a strong base anion exchange resin unit consisting of a plurality of strong base anion exchange resins which are sequentially connected in series, and a one-way valve is arranged between the strong acid cation exchange resin and the strong base anion exchange resin unit; and/or

The eluent is a sodium chloride solution with the mass percentage concentration of 0-6%, the volume of the eluent is 10-20 times of the volume of a strong acid cation exchange resin column of the column chromatography system, and the flow rate of the eluent is 12-24L/h; and/or

The four nucleotides were collected as follows: in the gradient elution process, an HPLC detector is used for detecting the effluent of the strongly basic anion exchange resin unit of the column chromatography system at regular time, when 5 '-CMP is detected in the effluent and the concentration is more than or equal to 0.4g/L, the collector starts to collect 5' -CMP, when 5 '-AMP is detected in the effluent, the collector starts to collect 5' -AMP, when 5 '-UMP is detected in the effluent, the collector starts to collect 5' -UMP, when 5 '-GMP is detected in the effluent, the collector starts to collect 5' -GMP, and when the concentration of 5 '-GMP is less than or equal to 10g/L, the collector stops collecting 5' -GMP.

Preferably, when step (2) employs stepwise elution:

the column chromatography system comprises a plurality of layers of stacked strong base anion exchange resins or a plurality of strong base anion exchange resins connected in series; one-way valves are arranged between each layer/each layer of strong-base anion exchange resin; and/or

The eluent is formic acid solution and/or sodium formate solution and/or sodium chloride solution and/or hydrochloric acid solution, and the flow rate of the eluent is 12-24L/h; and/or

The four nucleotides were collected as follows: in the step of step elution, an HPLC (high performance liquid chromatography) detector is adopted to detect the effluent liquid of each layer or each piece of strong-base anion exchange resin of the column chromatography system at regular time, 0.01mol/L formic acid solution is adopted to carry out step elution on 5 '-CMP, and the 5' -CMP with the concentration of 10-50 g/L is collected; eluting 5 '-AMP by adopting 0.1mol/L formic acid solution, and collecting the 5' -AMP with the concentration of 10-50 g/L; eluting 5' -UMP by adopting 0.1mol/L formic acid solution and 0.1mol/L sodium formate solution; and (3) eluting GMP by using a sodium chloride solution with the pH of 3.0 and the mass percentage concentration of 3%, and collecting the GMP with the concentration of 10-50 g/L.

The invention provides a RNA enzymolysis liquid chromatographic separation system, which comprises a column chromatography system, a sample loading mechanism, a water washing mechanism, an eluent mechanism, a collector, a detection mechanism and a control system, wherein the sample loading mechanism is used for loading RNA enzymolysis liquid;

the column chromatography system comprises 1 strong-acid cation exchange resin and a strong-base anion exchange resin unit, wherein the strong-base cation exchange resin unit is formed by a plurality of strong-base anion exchange resins which are connected in series; a one-way valve is arranged between the strong acid cation exchange resin unit and the strong base anion exchange resin unit;

the detection mechanism detects the effluent liquid of the column chromatography system and transmits the detection information to the control system;

the control system receives detection information of the detection mechanism and generates a water washing instruction, an elution instruction and a collection instruction according to the detection information; and the control system is respectively connected with the sample loading mechanism, the water washing mechanism, the eluent mechanism, the column chromatography system, the collector and the detection mechanism.

Preferably, at least 2 strong base anion exchange resins are arranged in the strong base anion exchange resin unit; and/or

The strong-acid cation exchange resin is Na type, and the strong-base anion exchange resin is Cl type; and/or

The control system comprises a sample loading controller for controlling the sample loading mechanism, a water washing controller for controlling the water washing mechanism and an elution controller for controlling the eluent mechanism; and/or

The detection mechanism comprises a nucleic acid protein detector and an HPLC detector which are respectively connected with the liquid outlet of the column chromatography system; timing switches are arranged on pipelines between the nucleic acid protein detector and the liquid outlet of the column chromatography system; and/or

The RNA enzymolysis liquid chromatographic separation system also comprises a monitoring system, and the monitoring system comprises a camera device for monitoring the chromatographic separation process of the RNA enzymolysis liquid and a display connected with the camera device.

The third aspect of the invention provides a RNA enzymolysis liquid chromatographic separation system, which comprises a column chromatography system, a sample loading mechanism, a water washing mechanism, an eluent mechanism, a collector, a detection mechanism and a control system;

the column chromatography system comprises a plurality of layers of stacked strong base anion exchange resins or a plurality of strong base anion exchange resins connected in series; one-way valves are arranged among the layers/the plurality of layers of strong-base anion exchange resins;

the detection mechanism detects the effluent liquid of each layer/each strong-base anion exchange resin and transmits the detection information to the control system;

the control system receives detection information of the detection mechanism and generates a water washing instruction, an elution instruction and a collection instruction according to the detection information; and the control system is respectively connected with the sample loading mechanism, the water washing mechanism, the eluent mechanism, the column chromatography system, the collector and the detection mechanism through electric signals.

Preferably, the strongly basic anion exchange resin is provided with at least 3 layers or 3 groups; and/or

The strong-base anion exchange resin is Cl type; and/or

The control system comprises a sample loading controller for controlling the sample loading mechanism, a water washing controller for controlling the water washing mechanism and an elution controller for controlling the eluent mechanism; and/or

The detection mechanism comprises a nucleic acid protein detector and a first HPLC detector which are respectively communicated with each layer/each strong basic anion exchange resin liquid outlet, and a second HPLC detector which is communicated with the last layer or the last strong basic anion exchange resin liquid outlet; a one-way valve is arranged on a pipeline between the second HPLC detector and the last layer or the last strong-base anion exchange resin liquid outlet; and/or

The RNA enzymolysis liquid chromatographic separation system also comprises a monitoring system, and the monitoring system comprises a camera device for monitoring the chromatographic separation process of the RNA enzymolysis liquid and a display connected with the camera device.

The invention has the following beneficial effects:

1. the RNA enzymolysis liquid chromatography separation method and system based on intelligent control further improve the chromatography separation process through real-time online detection and intelligent control, the control system is utilized to carry out sampling, washing and elution on the RNA enzymolysis liquid to obtain 4 nucleotide solutions, and then the nucleotide solutions are concentrated, crystallized and dried, so that the whole chromatography separation process is unmanned, the process control is more convenient and faster, the time and the labor are saved, the labor cost is reduced, the human errors are reduced, and the production loss is avoided;

2. the RNA enzymolysis liquid chromatographic separation method and system based on intelligent control not only get rid of complicated manual operation, but also are an innovation of the whole chromatographic separation process, can further finely control the process, improve the sample loading amount by 10-15% (namely capacity) and the product quality (more than 99%), and are more energy-saving and environment-friendly.

Drawings

Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:

FIG. 1 is a schematic structural diagram of a chromatographic separation method for RNA enzymolysis liquid based on intelligent control provided by a second aspect of the invention;

FIG. 2 is a schematic structural diagram of a RNA enzymatic hydrolysate chromatographic separation system provided by a third aspect of the invention;

FIG. 3 is a schematic diagram of another RNA enzymatic hydrolysate chromatographic separation system provided by the third aspect of the invention;

FIG. 4 is a logic control diagram of an intelligent chromatography separation system based on an intelligent control RNA enzymolysis liquid chromatography separation method provided by the first aspect of the invention;

FIG. 5 is a flow chart of the RNA enzymolysis liquid chromatography separation method based on intelligent control provided by the first aspect of the invention.

Detailed Description

In order to better understand the technical scheme of the invention, the technical scheme of the invention is further explained by combining the embodiment.

Referring to fig. 1, the RNA enzymatic hydrolysate chromatographic separation system provided by the present invention includes a column chromatography system, a sample loading mechanism, a water washing mechanism, an eluent mechanism, a collector 110, a detection mechanism, and a control system 115; wherein the column chromatography system comprises 1 strong acid cation exchange resin 105 and a strong base anion exchange resin unit composed of multiple strong base anion exchange resins (such as 106, 107) connected in series; a one-way valve K4 is arranged between the strong acid cation exchange resin 105 and the strong base anion exchange resin units (106, 107); the detection mechanism detects the effluent of the column chromatography system and transmits the detection information to the control system 115; the control system 115 receives the detection information of the detection mechanism and generates a water washing instruction, an elution instruction and a collection instruction according to the detection information; the control system 115 is respectively connected with the sample loading mechanism, the water washing mechanism, the eluent mechanism, the column chromatography system, the collector 110 and the detection mechanism through electric signals.

As shown in fig. 1, the sample loading mechanism includes a sample loading tank 101, a pump a connected to the sample loading tank 101, and a check valve K1 connected to the pump a; the water washing mechanism comprises a water washing tank 102, a pump B connected with the water washing tank 102 and a one-way valve K2 connected with the pump B; the eluent mechanism comprises a first elution tank 103, a second elution tank 104, a pump C connected with the first elution tank 103 and the second elution tank 104, and a one-way valve K3 connected with the pump C; wherein, the one-way valve K1, the one-way valve K2 and the one-way valve K3 are all three-way one-way valves.

As shown in connection with fig. 1, the strongly basic anion exchange resin in the strongly basic anion exchange resin unit is provided with at least 2, such as 2, strongly basic anion exchange resins 106, 107 in series; wherein, the strong acid cation exchange resin 105 and the strong base anion exchange resins 106 and 107 in the column chromatography system are regenerated by NaOH or HCl and washed by deionized water to be nearly neutral; wherein the strong acid cation exchange resin 105 is Na type, and the strong base anion exchange resins 106 and 107 are Cl type;

as shown in fig. 1, 4, and 5, the control system 115 includes a loading controller 111, a water washing controller 112, and an elution controller 113; the loading controller 111 controls the opening and closing of the pump A according to a loading instruction of the control system 115, and is used for pumping the RNA enzymatic hydrolysate into the column chromatography system, wherein the loading instruction comprises loading time, loading amount, flow rate of the pump A, opening and closing of a one-way valve K1 and the like; the water washing controller 112 controls the opening and closing of the pump B according to a water washing instruction of the control system 115, and is used for pumping deionized water into the column chromatography system for water washing, wherein the water washing instruction comprises the opening and closing of the one-way valve K2, the usage amount of the deionized water, the flow rate of the pump B and the like; the elution controller 113 controls the on-off of the pump C according to an elution instruction of the control system 115, and is used for pumping the eluent into the column chromatography system for elution, wherein the elution instruction comprises the type, concentration and usage amount of the eluent, the flow rate of the pump C, the on-off of the one-way valve K3, the one-way valve K4 and the like; the collector 110 collects the eluted nucleotides into corresponding containers according to the collection instructions of the control system 115.

Referring to fig. 1, the detection mechanism includes a nucleic acid protein detector 108 and an HPLC detector 109 respectively connected to the liquid outlet of the column chromatography system; timing switches are arranged on pipelines between the nucleic acid protein detector 108 and the HPLC detector 109 and the liquid outlet of the column chromatography system;

referring to fig. 1, the RNA enzymatic solution chromatographic separation system further comprises a monitoring system 114, and the monitoring system 114 comprises a camera device for monitoring the RNA enzymatic solution chromatographic separation process and a display connected with the camera device.

Referring to fig. 2 and 3, another RNA enzymatic hydrolysate chromatographic separation system provided by the present invention is partially the same as the control principle in fig. 1, and mainly differs in the design of a column chromatography system and a detection mechanism, and the RNA enzymatic hydrolysate chromatographic separation system shown in fig. 2 and 3 includes a column chromatography system, a sample loading mechanism, a water washing mechanism, an eluent mechanism, a collector 110, a detection mechanism, and a control system 115; wherein the column chromatography system comprises stacked multiple layers of strongly basic anion exchange resins (see FIG. 2) or multiple strongly basic anion exchange resins in series (see FIG. 3); one-way valves 3, 5 and 7 are arranged among the layers/a plurality of layers of strong-base anion exchange resins; the detection mechanism detects the effluent of each layer/layer of strong base anion exchange resin and transmits the detection information to the control system 115; the control system 115 receives the detection information of the detection mechanism and generates a water washing instruction, an elution instruction and a collection instruction according to the detection information; the control system 115 is respectively connected with the sample loading mechanism, the water washing mechanism, the eluent mechanism, the column chromatography system, the collector and the detection mechanism.

As shown in fig. 2 and 3, the strongly basic anion exchange resin is provided with at least 3 layers or 3, such as 4 layers in fig. 2: 1#, 2#, 3#, 4 of strong basic anion exchange resin in fig. 3 are arranged as follows: 1#, 2#, 3#, 4 #. Wherein, the strongly basic anion exchange resins 1#, 2#, 3#, 4# are regenerated by NaOH or HCl, and washed by deionized water to be nearly neutral, and the strongly basic anion exchange resins 1#, 2#, 3#, 4# are Cl type; the inlet ends of each layer or each layer of strong basic anion exchange resin 1#, 2#, 3#, 4# of the column chromatography system are communicated with the sample loading mechanism, the water washing mechanism and the eluent mechanism, and the pipelines on the inlet ends are provided with one-way valves, namely, the one-way valves 1, 10, 11, 12 (see fig. 2 or fig. 3).

As shown in fig. 2 and 3, the control system 115 includes a sample application controller 111 for controlling the sample application mechanism, a water washing controller 112 for controlling the water washing mechanism, and an elution controller 113 for controlling the eluent mechanism; the loading controller 111 pumps the RNA enzymatic hydrolysate into the column chromatography system according to a loading instruction of the control system 115, wherein the loading instruction comprises loading time, loading amount, flow rate of a pump, opening and closing of a one-way valve and the like; the washing controller 112 pumps deionized water into the column chromatography system for washing according to a washing instruction of the control system 115, wherein the washing instruction comprises opening and closing of a one-way valve, usage amount of the deionized water, flow rate of a pump and the like; the elution controller 113 pumps the eluent into the column chromatography system for elution according to an elution instruction of the control system 115, wherein the elution instruction comprises the type, concentration and usage amount of the eluent, the flow rate of a pump, the opening and closing of a one-way valve and the like; the collector 110 collects the eluted nucleotides into corresponding containers according to the collection instructions of the control system 115.

Referring to FIGS. 2 and 3, the detection mechanism includes a nucleic acid protein detector 14 (abbreviated as "detector") and a first HPLC detector 15 respectively connected to each layer/each outlet of the strongly basic anion exchange resin, and a second HPLC detector 16 connected to the last layer or the last outlet of the strongly basic anion exchange resin; the pipeline between the second HPLC detector and the last layer or the last strong-base anion exchange resin 4# liquid outlet is provided with a one-way valve 9.

Referring to fig. 2 and 3, the collector 110 is disposed at the outlet of the strong base anion exchange resin 4# for collecting nucleotide solution with qualified concentration, wherein the collector 110 is disposed at the outlet of the strong base anion exchange resin 4#, the pipeline between the collector 110 and the strong base anion exchange resin 4# is provided with the check valve 9 and the check valve 13, and the pipeline between the check valve 9 and the check valve 13 is provided with a sampling branch for the second HPLC detector.

Referring to fig. 2 and 3, the RNA enzymatic hydrolysate chromatographic separation system further comprises a monitoring system 114, and the monitoring system 114 comprises a camera device for monitoring the RNA enzymatic hydrolysate chromatographic separation process and a display connected with the camera device.

With reference to fig. 1, fig. 2, and fig. 3, the RNA enzymolysis liquid chromatography separation method and system based on intelligent control have the general concept: removing suspended particles from the RNA enzymolysis solution through a plate frame or centrifugation, and adjusting the pH to 10.0 by using 2mol/L NaOH; the whole chromatographic separation process can be divided into 3 processes of sample loading, water washing and gradient elution (or step elution). Referring to FIG. 1, the sample is a plurality of ion exchange resins connected in series after the filtered RNA enzymolysis solution passes through regeneration, wherein 115 is a strong acid cation exchange resin (the cation exchange resin is regenerated by HCl and washed to be neutral by deionized water and is Cl type) for removing impurities such as calcium and magnesium ions, partial pigments and soluble macromolecular proteins; 116. 117 is strongly basic anion exchange resin (anion exchange resin is regenerated by NaOH, washed by deionized water to be neutral and in Na type), the function of the anion exchange resin is to exchange and adsorb products (5 '-CMP, 5' -AMP, 5 '-UMP, 5' -GMP and the like) containing phosphate bonds on a column through the action of the ion exchange resin, effluent liquid of 115, 116 and 117 can be connected with a nucleic acid protein detector to measure the A260 value (namely the absorbance of ultraviolet light with the wavelength of 260 nm) of the effluent liquid on line in the loading process, the effluent liquid belongs to a normal range when the A260 is less than 1 (the concentration can also be converted into a concentration according to the A260, and the concentration C is less than or equal to 0.04g/L and belongs to the normal range), waste can be discharged, otherwise, the effluent liquid needs to be connected into another channel container to be recycled; after the sample loading is finished, deionized water is used for washing, the process also passes through the columns 115, 116 and 117 which are connected in series, impurities which are not adsorbed on the columns are washed, the effluent liquid also needs to pass through a nucleic acid protein detector for online detection of ultraviolet absorbance at 260nm, and similarly, the sample loading process is also adjusted and controlled through the A260 value in the effluent liquid; after water washing, 4 kinds of nucleotides can be eluted respectively by gradient elution in fig. 1 or stepwise elution in fig. 2 or fig. 3, the kinds of nucleotides are judged according to the peak-out time of the chromatogram detected by the HPLC detector in table 1, and the nucleotides are collected respectively according to the concentration C of the corresponding nucleotides.

TABLE 1 HPLC Peak times and concentrations C of certain 5' -nucleotides

Wherein the nucleic acid protein detector can adopt the nucleic acid protein detector existing in China, such as Shanghai Huxi HD-21-1, with the range of 0.1-2 (unit); in the processes of sample loading and water washing, the effluent liquid has lower nucleotide concentration, so that the online detection can be directly realized; in the elution process, the concentration of the nucleotide is higher and is usually within the range of 10-70g/L, the nucleotide can be measured by diluting 500-fold, and at the moment, an intelligent mechanical arm is needed to perform online sampling and dilution at fixed time (set for 0.5-1h), and then an HPLC (high performance liquid chromatography) with an automatic sample introduction device is added for measurement. And then calculated by the control system 115 and regulated according to the process parameters.

With reference to fig. 1 to 5, the RNA enzymatic hydrolysate chromatographic separation method based on intelligent control of the present invention performs chromatographic separation on RNA enzymatic hydrolysate by using an RNA enzymatic hydrolysate chromatographic separation system to realize intelligent control of the whole process of RNA enzymatic hydrolysate, and the RNA enzymatic hydrolysate chromatographic separation method includes the following steps:

(1) the control system 115 of the RNA enzymatic hydrolysate chromatographic separation system pumps the filtered RNA enzymatic hydrolysate into a column chromatography system, and the four nucleotides of 5 '-CMP, 5' -AMP, 5 '-UMP and 5' -GMP in the RNA enzymatic hydrolysate are adsorbed into the column chromatography system;

the specific process is as follows: combining the control system 115 of the RNA enzymatic hydrolysate chromatographic separation system shown in FIG. 1, the loading controller 111 controls the pump A and the one-way valve K1 (the one-way valve is a three-way one-way valve) to load, the loading process is strong acid cation exchange resin 115 → strong base anion exchange resin 106 → strong base anion exchange resin 107 → nucleic acid protein detector 108 → waste discharge, the volume of the RNA enzymatic hydrolysate is about 8-12 times of the volume of the resin in the column chromatographic system during loading, the flow rate of the pump A is set to be 80-120L/h (namely the flow rate of the RNA enzymatic hydrolysate), the loading start time is set by the loading controller 111, and after loading, the pump A automatically stops working and starts the pump B for water washing; in the sample loading process, after the RNA enzymolysis liquid is adsorbed by 115, 116 and 117 columns of a column chromatography system, a nucleic acid protein detector is adopted to detect the A260 value of the effluent liquid every hour, when the A260 value is less than 1, the waste is discharged, otherwise, the RNA enzymolysis liquid is collected by a collector 110 and recycled;

or the RNA enzymolysis liquid chromatography separation system shown in FIG. 2 or FIG. 3 can be used for sample loading, when the sample loading starts, the sample loading controller 111 controls the one-way valve 1 and the one-way valve 2 to be opened, when the A260 detected by the nucleic acid protein detector 14 is more than or equal to 1, the sample loading controller 111 automatically closes the one-way valve 2, opens the one-way valve 3 and the one-way valve 4, continues the sample loading of the second layer or the second strong-base anion exchange resin 2#, and so on until the sample loading of the fourth layer.

(2) Washing, namely washing the column chromatography system with deionized water after the sample loading is finished, and detecting the effluent of the column chromatography system at regular time by using a nucleic acid protein detector and an HPLC (high performance liquid chromatography) detector;

the specific process is as follows: referring to fig. 1, after the sample loading is finished, the water washing controller 112 controls the pump B and the check valve K2 (the check valve is a three-way check valve) to perform water washing, the water washing process is strong acid cation exchange resin 115 → strong base anion exchange resin 106 → strong base anion exchange resin 107 → nucleic acid protein detector 108 → waste discharge, when the water washing is performed, the flow rate of the pump B is set to be 80-120L/h, the deionized water passes through the columns 115, 116 and 117 of the column chromatography system, the column chromatography system effluent is sampled and detected at regular time by using the nucleic acid protein detector 108 and the HPLC detector 109, wherein the a260 value of the effluent is detected by the nucleic acid protein detector 108 every hour, when the a260 is less than 1, the waste discharge is performed, and the pump B is automatically stopped after the water washing is performed by using deionized water with 4-10 times the volume of the resin columns (that is, the volume of the deionized water is 4-10 times of the volume of each resin column in the column chromatography system), the elution controller 113 controls the pump C to elute. Detecting the effluent of the column chromatography system by using a nucleic acid protein detector, and discharging waste when A260 of the effluent of the column chromatography system is less than 1; otherwise, the collector starts collecting, and simultaneously feeds back information to the control system 115, and the sample loading amount needs to be adjusted;

the RNA enzymolysis solution chromatography separation system shown in FIG. 2 or FIG. 3 can also be used for water washing, and the principle is the same as the above.

(3) Eluting, namely eluting strongly basic anion exchange resin in a column chromatography system by using an eluent after water washing is finished, and respectively collecting four nucleotide solutions of 5 '-CMP, 5' -AMP, 5 '-UMP and 5' -GMP;

the specific process is as follows:

gradient elution: in conjunction with the control system 115 of the RNA enzymatic hydrolysate chromatographic separation system shown in fig. 1, the column chromatography system comprises 1 strong-acid cation exchange resin 105 and a strong-base anion exchange resin unit composed of a plurality of strong-base anion exchange resins (such as 2 strong-base anion exchange resins 106, 107) which are connected in series in sequence, and a one-way valve K4 is arranged between the strong-acid cation exchange resin and the strong-base anion exchange resin unit; after the water washing is finished, the elution controller 113 controls the one-way valve K3 to be opened and the one-way valve K4 to be closed, separates the strong-acid cation exchange resin 105 from the strong-base anion exchange resin 106, and controls the pump C to perform elution, wherein the elution at this time adopts gradient elution, the elution solution adopts 0-5 wt% of sodium chloride solution, and the gradient elution process is as follows: strong base anion exchange resin 106 → strong base anion exchange resin 107 → nucleic acid protein detector 108 → collector 110, when gradient elution is carried out, the pump flow rate is set to be 12-24L/h, the elution controller 113 controls the pump C to be automatically started, after the eluent passes through 106 and 107, the mechanical arm can be adopted to sample and dilute at regular time and the detection is carried out by HPLC detector 109, wherein:

when the concentration C of the nucleotide in the effluent liquid is less than 0.4g/L, discharging waste;

when the concentration C detected by the HPLC detector 109 in the effluent is more than or equal to 0.4g/L, the collector 110 starts to collect CMP;

when the effluent is HPLC-detected as AMP, the collector 110 starts to collect AMP;

when the effluent is HPLC-detected to be UMP, the trap 110 begins to collect UMP;

when the effluent is detected to be GMP by HPLC, the collector 110 starts to collect GMP;

when the concentration of GMP C is less than or equal to 10g/L, the collector 110 stops collecting GMP.

Step (3) of step elution: eluting with RNA enzymolysis solution chromatographic separation system shown in FIG. 2 or FIG. 3 (step elution), and as shown in FIG. 2 or FIG. 3, column chromatography system comprises stacked multiple layers of strong base anion exchange resin or multiple strong base anion exchange resins connected in series (such as 4 stacked layers or 4 columns connected in series); one-way valves are arranged among the layers/multiple layers of strong basic anion exchange resins, namely, in figure 2 or figure 3, the one-way valves 3, 4, 5 and 7 are respectively arranged among the strong basic anion exchange resins 1#, 2#, 3#, and 4 #; after the water washing is finished, the eluent is formic acid solution and/or sodium formate solution and/or sodium chloride solution, and the flow rate of the eluent is 12-24L/h; during the stepwise elution, the effluent of each layer or each strongly basic anion exchange resin is periodically tested by a nucleic acid protein detector and an HPLC tester:

the method comprises the steps of eluting 5 '-CMP by stages by using 0.01mol/L formic acid solution, collecting the 5' -CMP with the concentration of 10-50 g/L, namely opening the one-way valve 1, the one-way valve 3, the one-way valve 5, the one-way valve 7 and the one-way valve 9, eluting the 5 '-CMP by using 0.01mol/L formic acid solution, closing the one-way valve 1 and the one-way valve 3 and opening the one-way valve 10 when the first HPLC detector 15 detects that the concentration of the strong basic anion exchange resin 1# outlet 5' -CMP is less than or equal to 0.05g/L, closing the one-way valve 5 and opening the one-way valve 11 when the first HPLC detector 15 detects that the concentration of the strong basic anion exchange resin 2# outlet 5 '-CMP is less than or equal to 0.05g/L, and opening the one-way valve 11 when the first HPLC detector 15 detects that the concentration of the strong basic anion exchange resin 3# outlet 5' -CMP is less than or equal to 0.05g/L, closing the one-way valve 7 and opening the one-way valve 12; while eluting, sampling and detecting the concentration of 5' -CMP at the outlet of the one-way valve 9 by a second HPLC detector 16 at fixed time, and opening the one-way valve 13 when the concentration is in the range of 10-50 g/L, and collecting all the samples for later use;

collecting 5 ' -AMP, 5 ' -UMP and GMP by the same method after the collection of the 5 ' -CMP is finished, wherein the 5 ' -AMP is eluted by 0.1mol/L formic acid solution, and the 5 ' -AMP with the concentration of 10-50 g/L is collected; eluting 5' -UMP by adopting 0.1mol/L formic acid solution and 0.1mol/L sodium formate solution; eluting the 5 '-GMP by using a sodium chloride solution with the pH of 3.0 and the concentration of 3 wt%, and collecting the 5' -GMP with the concentration of 10-50 g/L.

And (3) concentrating, crystallizing and drying the four separated nucleotide solutions respectively to obtain corresponding crystals: respectively concentrating the collected 5 '-CMP, 5' -AMP and 5 '-UMP to 10-20%, decolorizing, filtering, then respectively adding 2-3 times of 95% ethanol to obtain a crystalline product, filtering and drying to respectively obtain single crystalline products with the content of more than 99%, namely 5' -CMP, 5 '-AMP and 5' -UMP, directly decolorizing and filtering due to the fact that the concentration of a collected solution of 5 '-GMP is high, adding 2-3 times of 95% ethanol to obtain a crystalline product, filtering and drying to obtain more than 99% of 5' -GMP.

Whether the step elution or the gradient elution is adopted, the RNA enzymolysis liquid chromatography separation system shown in the figure 2 or the figure 3 can be adopted for loading, namely when the first layer or the first column is saturated, the second layer or the second column can be automatically switched, and by analogy, 4 layers or 5 layers or more can be superposed, or 4 groups or 5 groups or more can be connected in series. Thus, with the 4-layer or 4-column combination shown in FIG. 2 or FIG. 3, the first layer or column is loaded with 100% of the sample through the column, the second layer or column is loaded with 75% of the sample through the column, the third layer or column is loaded with 50% of the sample through the column, and the fourth layer or column is loaded with 25% of the sample through the column. The enzymolysis liquid contains a plurality of impurities and has great pollution to the chromatographic column, so that the pollution of the chromatographic column can be reduced to the minimum degree by multi-layer and multi-root fractional sample loading, and the method is very favorable for increasing the sample loading amount, namely increasing the yield, improving the separation efficiency and improving the product quality. The general factory is fed once a day because the separation period is long, if the separation period is 6 days, 6 sets of columns are in operation, and if online detection and intelligent control are not available, manual monitoring or control is difficult to achieve.

During elution, the step of elution in chinese patent CN1177859C can be performed according to the RNA enzymatic hydrolysate chromatographic separation system shown in fig. 2 or fig. 3, that is, when the first layer or the first 5' -CMP is completely eluted during the first elution with lower concentration, the first layer or the first CMP can be disconnected, the second layer or the second CMP can be washed, and so on. When the second, higher concentration elutes 5' -AMP, the same can be followed separately as above. 5 '-UMP and 5' -GMP and so on; thus, impurities adsorbed on the column can be eluted as little as possible, and the yield and the product quality are improved. When the gradient elution mode of Chinese patent CN108752405A is adopted, because the chromatographic belt is continuously pushed and eluted, the chromatographic belt cannot be layered or sectionally eluted, and can only be connected together no matter the chromatographic belt is overlapped or serially connected, but the pollution is reduced in the same way as the sectionally eluted chromatographic belt when the chromatographic belt is loaded.

The RNA enzymolysis liquid chromatographic separation method and system based on intelligent control not only get rid of complicated manual operation, but also are an innovation of the whole chromatographic separation process, can further finely control the process, improve the sample loading amount by 10-15% (namely capacity) and the product quality (more than 99%), and are more energy-saving and environment-friendly.

The RNA enzymolysis liquid chromatography separation method and system based on intelligent control of the invention are further described by combining specific examples.

Example 1

In this embodiment, the RNA enzymolysis solution chromatography separation system shown in fig. 1 is adopted to perform chromatography separation on RNA enzymolysis solution, and the specific process is as follows:

1) enzymolysis

Weighing 14kg of RNA, fixing the volume, dissolving in 200L of water, heating to 72 ℃, adding 0.6kg of phosphodiesterase, carrying out heat preservation reaction for 4h, hydrolyzing the RNA into 4 nucleotides such as 5 '-AMP, 5' -GMP, 5 '-CMP, 5' -UMP and the like, carrying out enzyme inactivation with the conversion rate of 75%, rapidly cooling to below 45 ℃, adjusting the pH value to 10.0 by using 6mol/L NaOH, adding diatomite, filtering, diluting the clarified filtrate to 1600L by using deionized water, and reserving for later use.

2) Chromatographic separation of sample

Preparation work: referring to FIG. 1, column chromatography system comprises 732 column packed cation exchange resin 60L in column 105, column 106 and column 107 packed anion exchange resin 60L of 201X7 respectively, and is regenerated with 1.2mol/LHCl or NaOH, and washed with deionized water to near neutral, to obtain Na-type strongly acidic cation exchange resin 105 and Cl-type strongly basic anion exchange resins 106 and 107.

And starting a control system 115, starting a pump A by a loading controller 111, connecting RNA enzymolysis liquid in series by 105, 106 and 107 columns for loading, and finishing loading at the flow rate of 80-120L/h for about 20-30 h. The sample loading controller 111 turns off the pump a, the water washing controller 112 turns on the pump B to perform water washing at the same flow rate, after the water washing is finished, the pump B is turned off, and the elution controller 113 turns on the pump C to start gradient elution. Gradient elution conditions: 300L of water is filled in the elution tank 103, 300L of NaCl solution with the mass percentage concentration of 5% is adopted in the elution tank 104, the total amount is 600L, the NaCl solution with the mass percentage concentration of 0-5 is formed, the flow rate is controlled to be 12-24L/h, and the elution is finished within about 50-100 h; during elution, the strongly acidic cation exchange resin 105 is cut off, and the strongly basic anion exchange resins 106 and 107 are directly eluted in series. The effluent was sampled periodically, diluted automatically, and the sample was automatically injected through HPLC detector 109 and collected by controlled elution start collector fractions. When a 5 '-CMP peak appears and the concentration C is more than or equal to 0.4g/L, starting to collect 5' -CMP; starting to collect 5 '-AMP when a peak of 5' -AMP appears; collecting 5 '-UMP when a peak of 5' -UMP appears; starting to collect 5 '-GMP when a 5' -GMP peak appears; the collection was stopped when the concentration of 5' -GMP C was < 10 g/L.

3) Concentrating, crystallizing, and drying

Respectively concentrating the collected 5 '-CMP, 5' -AMP and 5 '-UMP liquid to 10-20%, decolorizing, filtering, adding 2-3 times of 95% ethanol to obtain crystallized product, filtering, and drying to obtain 2kg of 5' -CMP with content of 99.2%; 2.1kg of 5' -AMP, content 99.5%; 2.1kg of 5' -UMP, content 99.1%. The concentration of 5 '-GMP is higher, the concentration of the general eluent is 50-80 g/L, concentration is not needed, direct decolorization and filtration can be carried out, 2-3 times of 95% ethanol is added for crystallization and drying, and 2.2kg of 5' -GMP with the content of 99.1% can be obtained.

Example 2

The treated nucleotides were prepared by increasing the feed rate by 10% as in example 1, i.e., 15.4kg of RNA and 0.66kg of enzyme, and finally diluted to 1760L for use.

Each layer was loaded with about 30L of a regenerated anion exchange resin in Cl form for a total of 120L according to the RNA enzymatic solution chromatography separation system shown in FIG. 2. The first layer can be loaded by 370-420L, the one-way valve 1 and the one-way valve 2 are started to be loaded, when the nucleic acid protein detector detects that A260 is more than or equal to 1, the controller automatically closes the valve 2, the one-way valve 3 and the one-way valve 4 are started, and the second layer is loaded continuously; when the effluent A260 is more than or equal to 1, closing the one-way valve 4, opening the one-way valve 5 and the one-way valve 6, and continuing to sample the third layer; when the effluent A260 is more than or equal to 1, closing the check valve 6, opening the check valve 7 and the check valve 8, and continuing the sample loading of the fourth layer. Wherein, the second layer can be loaded with 800-840L, the third layer can be loaded with 1240-1300L, the fourth layer can be loaded with 1760L or even more, and the total loading amount can be increased by 10-15%.

After the completion of the loading, the sample was washed with 600L purified water.

The gradient elution procedure of example 1 was then followed.

4 kinds of nucleotides are respectively concentrated, crystallized and dried to finally obtain 2.2kg of 5' -CMP with the content of 99.4 percent; 2.3kg of 5' -AMP, content 99.6%; 2.4kg of 5 '-UMP, a content of 99.3%, 2.5kg of 5' -GMP, a content of 99.3%.

Example 3

In this embodiment, the RNA enzymolysis solution is chromatographically separated by using the RNA enzymolysis solution chromatographic separation system shown in FIG. 2;

enzymolysis, loading and washing were carried out as in example 2.

A sectional elution method is adopted during elution.

The one-way valve 1, the one-way valve 3, the one-way valve 5, the one-way valve 7 and the one-way valve 9 are opened firstly, 0.01mol/L formic acid is used for eluting 5 '-CMP, when the first HPLC detector 15 detects that the concentration of the 5' -CMP at the first layer outlet is less than or equal to 0.05g/L, the one-way valve 1 and the one-way valve 3 are closed, the one-way valve 10 is opened, when the concentration of the 5 '-CMP at the second layer outlet is less than or equal to 0.05g/L, the valve 5 is closed, the valve 11 is opened, and when the concentration of the 5' -CMP at the third layer outlet is less than or equal to 0.05g/L, the one-way valve 7 is closed, and the one-way valve 12 is opened. Meanwhile, the second HPLC detector 16 at the outlet of the one-way valve 9 samples and detects the 5' -CMP concentration at fixed time, and when the concentration is in the range of 10-50 g/L, the one-way valve 13 is opened, and all the samples are collected for standby.

After the 5 ' -CMP is collected, opening the one-way valve 1, the one-way valve 3, the one-way valve 5, the one-way valve 7 and the one-way valve 9, eluting 5 ' -AMP with 0.1mol/L formic acid, closing the one-way valve 13 and opening the one-way valve 10 when the first HPLC detector 15 detects that the concentration of the 5 ' -AMP at the first layer outlet is less than or equal to 0.05 g/L. The same applies below. When the concentration of 5 '-AMP at the outlet of the one-way valve 9 is within the range of 10-50 g/L, all the 5' -AMP is collected for later use.

After the collection of 5 '-AMP, the 5' -UMP was eluted with 0.1mol/L formic acid and 0.1mol/L sodium formate, and the procedure was as described above. When the concentration of 5' -UMP at the outlet of the one-way valve 9 is within the range of 10-50 g/L, all the UMP is collected for later use.

After completion of the collection of 5 '-UMP, 5' -GMP was eluted with 3% NaCl pH3.0, and the procedure was as described above. And finally, when the concentration of 5' -GMP at the outlet of the one-way valve 9 is within the range of 10-50 g/L, completely collecting the components for later use.

4 kinds of nucleotides are respectively concentrated, crystallized and dried to finally obtain 2.4kg of 5' -CMP with the content of 99.3 percent; 2.4kg of 5' -AMP, content 99.6%; 2.5kg of 5' -UMP, content 99.5%; 2.5kg of 5' -GMP, content 99.5%.

Example 4

In this example, the RNA enzymolysis solution is chromatographically separated according to the RNA enzymolysis solution chromatographic separation system shown in FIG. 3;

treated RNA was prepared as in example 3 and the final RNA hydrolysate was diluted to 1760L for use. Then, the sample was loaded in the tandem method shown in FIG. 3. Each was packed with 30L of anion exchange resin and regenerated in Cl form for a total of 120L. The first column can be loaded by 370-420L, valves 1 and 2 are started to be loaded, when the effluent concentration A260 is larger than or equal to 1, the valve 2 is closed, and valves 3 and 4 are started; when the effluent A260 of the valve 4 is more than or equal to 1, closing the valve 4 and opening the valves 5 and 6; when the effluent concentration A260 of the valve 6 is more than or equal to 1, the valve 6 is closed, and the valves 7 and 8 are opened. The first column can sample 370-420L, the second column can sample 800-840L, the third column can sample 1240-1300L, the fourth column can sample 1760L, and the total sample loading amount can be increased by 10-15%.

After the loading, the sample is washed with 600L of purified water (or deionized water).

Then, the elution was carried out in a gradient manner in accordance with the method described in example 2.

4 kinds of nucleotides are respectively concentrated, crystallized and dried to finally obtain 2.3kg of 5' -CMP with the content of 99.6 percent; 2.3kg of 5' -AMP, content 99.8%; 2.5kg of 5' -UMP, content 99.6%; 2.5kg of 5' -GMP, content 99.5%.

Example 5

In this example, the RNA enzymolysis solution chromatography separation system shown in FIG. 3 performs chromatography separation on RNA enzymolysis solution;

the enzymatic hydrolysis, loading and washing were carried out as in example 4.

A sectional elution method is adopted during elution.

Firstly opening the one-way valve 1, the one-way valve 3, the one-way valve 5, the one-way valve 7 and the one-way valve 9, eluting 5 '-CMP with 0.01mol/L formic acid, closing the one-way valve 1 and the one-way valve 3 and opening the one-way valve 10 when the first HPLC detector detects that the concentration of the 5' -CMP of the first outlet is less than or equal to 0.05g/L, closing the one-way valve 5 and opening the one-way valve 11 when the concentration of the 5 '-CMP of the second outlet is less than or equal to 0.05g/L, and closing the one-way valve 7 and opening the one-way valve 12 when the concentration of the 5' -CMP of the third outlet is less than or equal to 0.05 g/L. Meanwhile, the second HPLC detector at the outlet of the check valve 9 samples and detects the 5' -CMP concentration at fixed time, and when the concentration is in the range of 1-5%, the check valve 13 is opened, and all the samples are collected for standby.

After the 5 ' -CMP is collected, opening the one-way valve 1, the one-way valve 3, the one-way valve 5, the one-way valve 7 and the one-way valve 9, eluting 5 ' -AMP with 0.1mol/L formic acid, and closing the one-way valve 13 and opening the one-way valve 10 when the first HPLC detector detects that the concentration of the 5 ' -CMP at the first outlet is less than or equal to 0.05 g/L. The same applies below. When the concentration of 5 '-AMP at the outlet of the one-way valve 9 is in the range of 1% -5%, all the 5' -AMP is collected for standby.

After the collection of 5 '-AMP, the 5' -UMP was eluted with 0.1mol/L formic acid and 0.1mol/L sodium formate, and the procedure was as described above. When the concentration of 5' -UMP at the outlet of the one-way valve 9 is within the range of 10-50 g/L, all the UMP is collected for later use.

After completion of the collection of 5 '-UMP, 5' -GMP was eluted with 3% NaCl pH3.0, and the procedure was as described above. And finally, when the concentration of 5' -GMP at the outlet of the one-way valve 9 is within the range of 10-50 g/L, completely collecting the components for later use.

4 kinds of nucleotide are respectively concentrated, crystallized and dried to finally obtain 2.3kg of 5' -CMP with the content of 99.5 percent; 2.4kg of 5' -AMP, the content is 99.5%; 2.5kg of 5' -UMP with the content of 99.5 percent; 2.6kg of 5' -GMP with the content of 99.5 percent.

It should be understood by those skilled in the art that the above embodiments are only for illustrating the present invention and are not to be used as a limitation of the present invention, and that changes and modifications to the above described embodiments are within the scope of the claims of the present invention as long as they are within the spirit and scope of the present invention.