A fully automatic chromatography purification system

Abstract

The present invention proposes a fully automatic chromatography purification system, including: a sample tank, a loading system, an eluent tank, an infusion pump, a chromatographic column, an online detection device with a full-wavelength UV-visible detector and an online ultra-high performance liquid chromatograph, a target pure product tank, an impure product recovery tank and a solvent mixing tank; the data detected by the online detection device is transmitted to the control cabinet computer to control the opening and closing of multiple valves. The closed-loop fully automatic chromatography purification system proposed in the present invention can accurately determine the purity of the fraction through online UV-visible detection and online UHPLC detection, and realize automatic determination and automatic collection of target pure products, impure products (recoverable for secondary purification) and waste liquid, which not only improves efficiency and reduces the space occupied by the collection tank, but also improves the yield; the system can also realize automatic solvent recovery and automatic liquid preparation, thereby realizing the fully automatic operation of the entire chromatography purification process.

Description

Technical Field

[0001] The present invention relates to the technical field of a device and method for purifying polypeptides, and in particular to a high performance liquid chromatography device and method for purifying polypeptides. Background Art

[0002] High performance liquid chromatography is an effective separation and analysis method, and is also an important high-efficiency preparative separation technology. High performance liquid chromatographs are generally composed of a reservoir, a pump, an injector, a chromatographic column, a detector, and a recorder. The mobile phase in the reservoir is pumped into the system by a high-pressure pump, and the sample solution enters the mobile phase through the injector and is carried into the chromatographic column (stationary phase) by the mobile phase. Since the components in the sample solution have different distribution coefficients in the two phases, when they move relative to each other in the two phases, after repeated adsorption-desorption distribution processes, the components have large differences in movement speed, and are separated into individual components and flow out of the column in turn. When passing through the detector, the sample concentration is converted into an electrical signal and transmitted to the recorder, and the data is printed out in the form of a graph.

[0003] First, if Figure 1 As shown, the existing high performance liquid chromatograph detects fractions by ultraviolet single wavelength or multi-wavelength detection. Due to the large injection volume of preparative chromatographic separation, a whole peak that has not been completely separated is presented on the ultraviolet-visible detector, and it is even more impossible to determine the content or concentration of the target and impurities in each time period. Therefore, the only way is to collect all the fractions in separate tanks, and then offline detect the content and concentration of the target in the fractions collected in each tank to determine whether the fractions collected in each tank are qualified, and merge the fractions in the tanks whose test results are within the qualified range. There are two ways to collect fractions using a fraction collector. One is that the collector is driven by a motor to make a linear motion so that each fraction collecting tank moves to the fraction outlet conduit and collects a certain amount of fraction (US8858899B2 and US4422151A); the other is that the fraction outlet conduit is arranged on a rotating arm and can move forward and backward along the rotating arm, and the rotating arm can be driven by a motor to make a rotating motion. Through the rotating motion of the rotating arm and the forward and backward movement of the fraction outlet conduit along the rotating arm, the fraction outlet conduit passes through the inlet of each fraction collecting tank in turn and injects the fraction into the collecting tank (US11686712B2, a liquid fraction collector for a liquid chromatography system).

[0004] The problems with the current online detection and fraction collection devices and technologies are as follows: ① In order to avoid mixing qualified fractions with unqualified fractions, the fraction collection period needs to be shortened as much as possible, so many collection tanks are needed, which not only takes up a lot of space, but also leads to a lot of post-processing (offline detection, merging and cleaning of collection tanks, etc.) and product losses caused by post-processing (product residues in the tank during the merging process, product deterioration, mixing errors and cross-contamination risks, etc.); and if the collection frequency is too low in order to reduce the number of collection tanks, the yield of qualified products will decrease; ② When the fraction collector collects the fraction, it is necessary to use a linear motor or a rotating arm drive motor to match the fraction outlet conduit and the fraction collection tube one by one. This mechanical transmission has a high failure rate and uncertainty when working continuously for a long time, resulting in the loss of collected products, especially for high-value products, which will cause greater losses. Therefore, this method of segmented collection, offline detection, and re-merging of qualified products is not only labor-intensive and inefficient, but also prone to errors and losses.

[0005] Secondly, the fractions obtained after separation and purification by the existing high-performance liquid chromatography, those without the target substance or with low target substance content are directly treated as waste liquid. The general way to deal with waste liquid is to combine the fractions that do not need to be purified and then concentrate them. The solid is handed over to a qualified hazardous chemical treatment agency for harmless treatment as a hazardous chemical. The liquid is measured for its proportion and then transferred to the mobile phase for recycling after being configured to be qualified. The waste liquid generated when using high-performance liquid chromatography technology to separate and purify the target substance will increase the floor space due to storage problems on the one hand, and increase the manpower and equipment investment due to waste liquid treatment on the other hand. Handing it over to a hazardous chemical treatment agency for treatment will also increase the cost of waste liquid treatment. Therefore, it is necessary to further improve the waste liquid recovery system. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a closed-loop fully automatic chromatography purification system integrating the following parts, including: a chromatography system main device, an online detection device, an automatic sample collection device and a control system. The control system is connected to the above devices, and the connection includes a strong current connection and a weak current connection.

[0007] (1) The main device of the chromatography system includes a sample tank, a sample loading pump, an eluent tank, an infusion pump, and a chromatography column. The sample tank and the eluent tank are connected to the inlet of the chromatography column through pipelines, and the outlet of the chromatography column is connected to the online detection device and the automatic sample collection device. The fully automatic chromatography purification system is controlled by a control system.

[0008] (2) An online detection device including a full-wavelength UV-visible detector and an online ultra-high performance liquid chromatograph. The online detection also includes an automatic sampling device and a data processing and data transmission device.

[0009] (3) Automatic sample collection device controlled by online test results,

[0010] (4) Automatic solvent recovery device, including solvent recovery and online liquid preparation,

[0011] (5) A control system consisting of a control cabinet that collects data, analyzes and integrates the control of the entire system, and a central controller for remote control. The remote control center communicates with the control cabinet, and transmits data and commands to the control cabinet; the control cabinet is connected to each device to collect data and perform electrical control.

[0012] The purification system provided by the present invention is as follows Figure 2 As shown:

[0013] (1) The inlet of the chromatographic column is connected to the sample loading device and the high-pressure elution device through a switching valve. The sample loading device includes a sample tank and a sample loading pump behind the tank. The high-pressure elution device includes an eluent tank and a high-pressure constant-current pump behind the tank (with a pressure resistance of not less than 2 MPa). The sample loading pump, the high-pressure constant-current pump and the switching valve are all controlled by a central controller and a control cabinet. The eluent can also be prepared online to achieve gradient elution. The specific implementation method is to load the elution solvents A and B into the eluent tanks A and B respectively, and each eluent tank is followed by a high-pressure constant-current pump A and B.

[0014] (2) The outlet of the chromatographic column is connected to an online detection device. The online detection device includes: a full-wavelength ultraviolet-visible detector, an automatic sampling device, an online ultra-high performance liquid chromatograph, and a data processing and data transmission device. The full-wavelength ultraviolet-visible detector scans the fractions prepared by the high-performance chromatography to detect whether the fractions contain the chromatographic peaks of the target. The fractions without the chromatographic peaks of the target are collected and entered into the solvent recovery tank, and the fractions with the chromatographic peaks of the target are alternately collected and entered into the first collection buffer tank or the second collection buffer tank; the online sampling device includes the first collection buffer tank, the second collection buffer tank, an analytical sample sampling and online diluter, and multiple control valves; the online sampler realizes alternate sampling in the first collection buffer tank and the second collection buffer tank through the control valve, and the analytical sample enters the ultra-high performance liquid chromatograph after online dilution for analysis and detection, and determines the purity of the target and the content of each impurity in the corresponding fraction solution; the data obtained by the detection is transmitted to the control center after data processing, and then the switching valve of the automatic fraction collection device is controlled by the control system to transfer the corresponding fractions in the buffer tank to the target pure product tank, the impure product recovery tank and the waste liquid tank according to the predetermined purity standard.

[0015] The first collection buffer tank and the second collection buffer tank can also be directly connected to the outlet of the chromatographic column through a valve. In this configuration, the online ultra-high performance liquid chromatograph can still achieve the purpose of detecting the purity of the fraction and controlling the collection of the fraction, but there are the following two disadvantages: First, all sections of liquid flowing out of the chromatographic column must be detected by the online ultra-high performance liquid chromatograph, which increases the workload of the high performance liquid chromatography; second, all the previous and subsequent fractions, including the fractions with and without chromatographic peaks, must pass through the buffer tank and the fraction collection device, causing the entire device to be easily cross-contaminated; therefore, the present invention preferably uses a full-wavelength ultraviolet-visible detector and an online ultra-high performance liquid chromatograph in series at the same time. The time bottleneck of the online ultra-high performance liquid chromatograph detection determines the width of the fraction segmentation, thereby affecting the purity and yield of the fraction, so the liquid chromatograph used needs to ensure that the time of each analysis does not exceed 10 minutes, and the preferred time of each analysis is within 5 minutes. A liquid chromatograph that meets the above requirements is defined as an ultra-high performance liquid chromatograph. To achieve the above object, the present invention proposes to use a core-shell type analytical chromatographic column for rapid analysis and detection, which not only ensures rapid analysis results but also avoids the use of an ultra-high pressure pump.

[0016] (3) Connected to the online detection device is an automatic sample collection device, including: a target pure product tank, an impure product recovery tank and a waste liquid tank, and a switching valve controlled by a control system based on the analysis data of the online ultra-high performance liquid phase.

[0017] (4) The automatic solvent recovery and online liquid preparation device of the present invention can be embedded in the entire system as an option to achieve fully automatic recycling of the solvent; it can also be used offline alone, and then the recovered solvent is prepared in the required proportion and then added to the eluent tank. The automatic solvent recovery device includes: a solvent mixing tank, a solvent buffer tank and a solvent tank group; the input end of the solvent buffer tank is connected to the target pure product tank, the impure product recovery tank and the waste liquid tank through a pipeline, and a pump and a valve are arranged on the pipeline; the output end of the solvent buffer tank is connected to the first input end of the solvent mixing tank through a pipeline, and a high-precision flow meter is arranged on the pipeline; the solvent tank group is connected to the second input end of the solvent mixing tank through a pipeline, and a pump and a valve are arranged on the pipeline; the solvent mixing tank is also connected to the eluent tank through a pipeline; the target pure product tank, the impure product recovery tank and the waste liquid tank are respectively provided with a vacuum distillation device on the connecting pipeline with the solvent buffer tank; the solvent is recovered to the solvent buffer tank through online vacuum vacuum distillation, and the mass flow meter is used to detect and calculate the ratio of different solvents in the solution during the transfer to the solvent mixing tank, and the ratio is immediately adjusted to the set ratio in the solvent mixing tank by adding an appropriate amount of specific solvent from the solvent tank group. There is also a solvent recovery tank behind the full-wavelength ultraviolet-visible detector in parallel with the first collection buffer tank and the second collection buffer tank, which is used to collect solvents or fractions that do not contain target substances. The outlet end of the solvent recovery tank is connected to the solvent buffer tank and the eluent tank respectively through a switching valve; a reduced pressure distillation device is arranged on the pipeline connected to the solvent buffer tank; when the solvent composition and proportion in the solvent recovery tank are exactly the same as those in the eluent tank and does not contain impurities, the solvent can be directly transported to the eluent tank; when the solvent in the solvent recovery tank is impure or the proportion of the solvent composition is inconsistent with that in the eluent tank, the solvent is input into the solvent buffer tank through reduced pressure distillation.

[0018] The present invention adopts the following technical means to achieve more accurate and highly automated chromatographic separation and purification by using the above-mentioned device.

[0019] On the one hand, a fully automatic chromatographic purification system is provided, comprising: an automatic control device, a sample loading device, a high-pressure elution device, a chromatographic column, an online detection device, and an automatic fraction collection device; the sample tank and the eluent tank are respectively connected to the inlet end of the chromatographic column through a pipeline and then through a switching valve, and an injection pump and a high-pressure infusion pump are respectively arranged on the pipeline; the chromatographic column is preferably a dynamic axial pressurized chromatographic column to improve the separation effect; the chromatographic column is filled with a chromatographic stationary phase, preferably a spherical silica gel matrix chromatographic stationary phase; the filling length of the chromatographic column is 20cm-100cm; further, in addition to the tailing agent, two or more silanes are selected on the surface of the silica gel matrix for bonding modification (excluding alkylsilyl groups with a total carbon content of not more than 5 for tailing), and the proportion of any one of them is not less than 10%; one of the two silanes is a polar silane, and the other is a non-polar silane, or the two are different non-polar silanes.

[0020] The sample solution to be purified enters the chromatographic column from the sample tank through the injection pump; then the switching valve will be switched to connect the high-pressure elution device with the chromatographic column under the control of the automatic control device, and the eluent in the eluent tank will be continuously pumped into the chromatographic column; while the eluent continues to flow in the chromatographic column, the sample continues to dynamically exchange between the eluent and the stationary phase, thereby achieving gradual separation.

[0021] Furthermore, a guard column can be added in front of the chromatographic column; further, the guard column can be filled with fillers with larger particle sizes to prevent colloids in the sample from precipitating and contaminating the chromatographic column. At the same time, when the colloid precipitates, the back pressure of the guard column and the back pressure of the entire chromatographic system can be prevented from being too high; further, the sample solution and pure water can be introduced alternately to reduce sample diffusion.

[0022] The chromatographic column fraction outlet is provided with a main pipeline and three branches formed by the end of the main pipeline. The three branches are respectively connected to the solvent recovery tank, the first collection buffer tank and the second collection buffer tank. Valves are arranged on the three branches.

[0023] The fraction solution flowing out from the lower end of the chromatographic column enters the full-wavelength UV-Vis detector. Before the chromatographic peak containing the target object is found, all solutions flowing through the full-wavelength UV-Vis detector enter the solvent recovery tank; the solution fraction containing the target object is detected to enter the first collection buffer tank and the second collection buffer tank alternately.

[0024] Furthermore, the online detection device also includes an online ultra-high performance liquid chromatograph, which is connected to the first collection buffer tank and the second collection buffer tank and is used to detect the purity of the target object in the fractions of the first collection buffer tank and the second collection buffer tank.

[0025] Further, the online ultra-high performance liquid chromatograph is electrically connected to the control cabinet, and the control cabinet is configured to control the opening and closing of the valves of the first branch, the second branch, the third branch, the fourth branch, the fifth branch and the sixth branch according to the result of the online ultra-high performance liquid chromatograph;

[0026] The first branch is a connecting pipeline between the first collecting buffer tank and the target pure product tank; the second branch is a connecting pipeline between the first collecting buffer tank and the impure product recovery tank; the third branch is a connecting pipeline between the second collecting buffer tank and the target pure product tank; the fourth branch is a connecting pipeline between the second collecting buffer tank and the impure product recovery tank, the fifth branch is a connecting pipeline between the first collecting buffer tank and the waste liquid tank, and the sixth branch is a connecting pipeline between the second collecting buffer tank and the waste liquid tank.

[0027] The online sampling device alternately takes samples from the first collection buffer tank and the second collection buffer tank, and after online quantitative dilution, enters the ultra-high performance liquid chromatograph to determine the purity of the fraction; when the purity of the fraction meets the preset standard, the valve connected to the first or third branch is opened, and the fraction that meets the purity requirement is automatically collected and enters the target pure product tank; when the purity of the fraction does not meet the purity standard but the concentration and purity meet the recovery and re-purification standard, the fraction enters the non-pure product recovery tank via the second or fourth branch; when the concentration or purity of the fraction is lower than the preset recovery standard, the fraction enters the waste liquid tank via the fifth or sixth branch.

[0028] Furthermore, an output end of the target pure product tank is connected to a post-processing tank; the concentrate is crystallized or freeze-dried in the post-processing tank to obtain a target pure polypeptide; an output end of the impure product recovery tank is connected to a sample tank, and the concentrated fraction enters the sample tank for further purification.

[0029] Furthermore, the present invention also includes an online solvent recovery and online liquid preparation system, which can be used in conjunction with the above-mentioned chromatography automation system, or can be used offline alone; its core includes: a solvent recovery buffer tank, a solvent mixing tank and a solvent tank group. The solvent recovery buffer tank is respectively connected to the target pure product tank, the impure product recovery tank, the waste liquid tank and the solvent recovery tank; the connecting pipelines are respectively provided with vacuum distillation devices, and the solvents in the above four tanks are subjected to vacuum distillation and cooled and collected before entering the solvent recovery buffer tank. A high-precision mass flowmeter is provided on the pipeline connecting the solvent recovery buffer tank and the solvent mixing tank. The ratio of the solvent components entering the solution mixing tank can be obtained by measurement and calculation by the flowmeter. According to the difference between the ratio of the solvent components in the solution mixing tank and the ratio of the solvent components in the preset eluent tank, the solvent components and mass of the solvent tank group entering the mixing tank are adjusted and controlled.

[0030] Compared with the existing chromatographic purification system and the application method of purifying organic molecules, the advantages and beneficial effects of the present invention are as follows:

[0031] 1. The closed-loop fully automatic chromatographic purification system, which integrates high-pressure chromatographic separation equipment, high-performance liquid chromatography online detection and fraction automatic collection equipment, online solvent automatic recovery and liquid preparation equipment, not only improves the purification yield of the target product, improves the purification operation efficiency, reduces the operation space, reduces the personnel cost and solvent loss cost; and because the waiting process for the fraction separation line analysis and the fraction merging process are eliminated, the workload is further reduced, and the loss caused by the deterioration of the target product and the residue during merging is reduced, and the environmental and safety risks caused by the discharge of solvents in the above process are also reduced. Moreover, the human error factor is eliminated through full automation, making the whole process more stable and reliable.

[0032] Specific:

[0033] (1) For the first time, an accurate and fast online detection device including a buffer tank switch and an online ultra-high performance liquid chromatograph is created to ensure that the purity of the fraction after purification by the chromatographic column can be detected in a timely and accurate manner, and thereby control the automatic sample collection device to achieve automatic collection; due to the accurate and fast detection of the online ultra-high performance liquid chromatograph, the interval between each sampling detection can be compressed to within 1-5 minutes, and no more than 10 minutes at most, thereby not only ensuring the accuracy of the purity of the collected product, but also reducing the yield loss caused by the collection volume of a single fraction to a minimum, thereby improving the purification yield of the target product; the online ultra-high performance liquid chromatograph of the present invention uses a core-shell silica gel medium filler, thereby greatly shortening the analysis and detection time of each fraction, ensuring the subdivision of each fraction time period, thereby achieving the optimization of the yield.

[0034] (2) The present invention detects the purity of each fraction online and directly controls the fractions to enter the target pure product collection tank, the impure product collection tank (for secondary purification) and the waste liquid tank respectively. This not only reduces the space, but also eliminates the waiting process for the fraction separation line analysis and the fraction merging process, further reducing the workload, reducing the loss caused by the deterioration of the target product and the residue during merging, and reducing the environmental and safety risks caused by the emission of solvents in the above process. Furthermore, human error factors are eliminated through full automation.

[0035] (3) Through the online intelligent solvent recovery and liquid preparation system, the solvent in the fraction is recovered and utilized to the greatest extent possible. The solvent without chromatographic peaks is automatically recovered, and the solvent with chromatographic peaks is automatically proportioned and recovered as needed, thereby reducing solvent waste. The use of intelligent automatic solvent recovery devices reduces labor costs and equipment usage, shortens recovery time, improves efficiency, and reduces waste liquid treatment costs.

[0036] (4) A precisely controlled eluent infusion pump is used to ensure that the flow error is within 1.5%; and a high-precision mass flow meter is used to accurately measure the flow online. When the flow fluctuates, the control cabinet is used to accurately adjust the flow in real time, thereby further achieving accurate and stable separation effects and improving the yield of the target product.

[0037] (5) The present invention creatively integrates 1) UV-visible online monitoring, 2) UHPLC online detection, 3) fraction collection automatic control, 4) solvent recovery control, and 5) online liquid preparation based on precise density monitoring to achieve closed-loop, precise intelligent control, thereby achieving precision, efficiency, and stability in chromatographic purification of organic compounds. The closed-loop system of the present invention enables the entire purification process to be operated continuously, including solvent recycling, which not only improves efficiency, but also reduces the pollution that may be introduced in the offline recovery process, and reduces the losses caused by offline recovery, air pollution, and safety risks.

[0038] 2. For polypeptides with a molecular weight of more than 2000D, the target substance has high similarity with impurities, is difficult to separate, and is prone to tailing and self-aggregation. In order to achieve accurate separation of large molecular weight polypeptides (2000D-6000D), improve sample loading, resolution and yield, the present invention also adopts the following key innovations in chromatographic columns and chromatographic fillers:

[0039] (1) The present invention further uses two or more silanes for mixed bonding, taking advantage of the difference in the effects of different silanes on the target and impurities, that is, the effects of polar silanes on the target and impurities are different from those of non-polar silanes on the target and impurities. The sum of the effects will improve the separation effect and selectivity, and further enhance the separation of chromatographic filler stationary phases for large molecular peptides and other target and impurity mixtures with very similar compositions and structures. The silanes described herein do not include tailing agents with no more than 5 carbon atoms used for tailing.

[0040] (2) The present invention also effectively reduces tailing and improves peak shape and separation effect by controlling the consistency and accuracy of the chromatographic column temperature and the eluent temperature (the temperature can be selected between 10°C and 30°C, and the error is within ±3°C).

[0041] (3) The injection end is designed to allow sample solution and pure water to enter alternately, reducing sample diffusion. At the same time, a guard column filled with large particles is used to retain strongly adsorbed impurities, colloids in the solution, and a small amount of solids that may precipitate in the guard column, avoiding excessive column pressure and contamination of the filler of the separation and purification chromatographic column.

[0042] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0044] Figure 1 It is a traditional preparative liquid chromatography system;

[0045] Figure 2 A schematic diagram of a fully automatic chromatography purification system according to an embodiment of the present invention;

[0046] Figure 3 UV online detection spectrum for peptide separation;

[0047] Figure 4 It is a schematic diagram of oil-free cylinder type chromatography column;

[0048] Figure 5This is the AA cross-section of the oil-free cylinder type chromatographic column;

[0049] Figure 6 Chromatograms prepared for the separation of hydrophobic hexapeptides using BS AQ C18 10 μm 120 Å for comparative example 3;

[0050] Figure 7 Chromatograms prepared for the separation of hydrophobic hexapeptides using BS AQ C18 / C8 10 μm 120 Å for comparative example 1;

[0051] Figure 8 Chromatogram of the separation of crude hydrophobic decapeptide prepared by BS AQ C8 10 μm 120 Å for comparative example 2;

[0052] Fig. 9 This is the chromatogram of the preparation of the crude hydrophobic decapeptide separated by BS AQ C8 / C12 (C12 accounts for 12.7%) 10μm 120Å in Example 2.

[0053] Among them: 1. Eluent tank; 2. Sample tank; 3. Infusion pump; 4. Sample loading pump; 5. Chromatographic column; 6. UV detection; 7. Fraction collection; 8. Waste liquid; 9. Online detection device; 10. Target pure product tank; 11. Non-pure product recovery tank; 12. Solvent mixing tank; 13-1. Valve 1; 13-2. Valve 2; 13-3. Valve 3; 13-4. Valve 4; 13-5. Valve 5; 14. Full wavelength UV-visible detector; 15. First collection buffer tank; 16. Second collection buffer tank; 17. Solvent recovery tank; 18 , online ultra-high performance liquid chromatograph; 19, post-processing tank; 20, waste liquid tank; 21, solvent tank group; 22-1, high-precision flow control valve 1; 22-2, high-precision flow control valve 2; 22-4, high-precision flow control valve 4; 22-5, high-precision flow control valve 5; 23-1, multi-stage variable frequency centrifugal pump 1; 23-2, multi-stage variable frequency centrifugal pump 2; 23-3, multi-stage variable frequency centrifugal pump 3; 23-4, multi-stage variable frequency centrifugal pump 4; 23-5, multi-stage variable frequency centrifugal pump 5; 24-1, high-precision mass flowmeter. 25, solvent buffer tank. DETAILED DESCRIPTION

[0054] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and should not be construed as limiting the present invention.

[0055] As a basic embodiment of the present invention, this embodiment provides a fully automatic chromatography purification system, such as Figure 2As shown, it includes: a chromatography system main device, an online detection device, an automatic sample collection device and a control system. The control system is connected to the above devices, and the connection includes a strong current connection and a weak current connection.

[0056] The control system consists of a remote control center and a control cabinet to realize data transmission and electrical control; the remote control center communicates with the control cabinet to realize data transmission and command transmission with the control cabinet.

[0057] The main device of the chromatography system includes a sample tank 2, a sample pump 4, an eluent tank 1, an infusion pump 3, and a chromatography column 5. The sample tank 2 and the eluent tank 1 are respectively connected to the liquid inlet of the chromatography column 5 through pipelines, and the liquid outlet of the chromatography column 5 is connected to the online detection device and the automatic sample collection device. The fully automatic chromatography purification system is controlled by a control system;

[0058] The online detection device includes: an online ultra-high performance liquid chromatograph (UHPLC), an automatic sampling device and a data processing and data transmission device, wherein the automatic sampling device includes a first collection buffer tank 15, a second collection buffer tank 16, an analytical sample sampler and an online quantitative dilution and a plurality of control valves;

[0059] The outlet of the chromatographic column 5 is provided with a main line and two branches formed by the ends of the main line, which are respectively connected to the first collection buffer tank 15 and the second collection buffer tank 16, and both branches are provided with valves; the eluent alternately enters the first collection buffer tank 15 and the second collection buffer tank 16 through the valves;

[0060] The first collection buffer tank 15 and the second collection buffer tank 16 are also connected to the analytical sample sampler through a pipeline, and the other end of the analytical sample sampler is connected to the online ultra-high performance liquid chromatograph 18, which is used to detect the purity of the target object in the fractions of the first collection buffer tank 15 and the second collection buffer tank 16; the online detection device is also electrically connected to the control cabinet to provide the detection data to the control system; online quantitative sampling and quantitative dilution can be achieved in the following manner: a valve three 13-3 is installed on the lower end pipeline of an electrically controlled injection pump, and the valve three 13-3 is respectively connected to the first collection buffer tank 15, the second collection buffer tank 16, the diluent tank, and the injection loop, and is connected to the analytical chromatographic column of the online ultra-high performance liquid chromatograph 18 through the injection loop; a certain amount of sample fraction and a certain amount of diluent are quantitatively extracted and mixed in the injection loop through the injection pump and valve switching; after switching to the passage connected to the analytical chromatographic column, the diluted sample solution in the injection loop is sent to the column head of the analytical chromatographic column.

[0061] The automatic sample collection device includes: a switching valve at the lower outlet of the first collection buffer tank 15 and the second collection buffer tank 16, a target pure product tank 10, an impure product recovery tank 11 and a waste liquid tank 20; the control system controls the valve 4 13-4 at the lower end of the first collection buffer tank 15 and the valve 5 13-5 at the lower end of the second collection buffer tank 16 according to the detected target purity data in the first collection buffer tank 15 and the second collection buffer tank 16, and respectively sends the qualified target pure product, unqualified product and waste liquid to the corresponding target pure product tank 10, impure product recovery tank 11 and waste liquid tank 20;

[0062] Specifically: the control system controls the valve switches of the first branch, the second branch, the third branch, the fourth branch, the fifth branch and the sixth branch according to the detection results of the online ultra-high performance liquid chromatograph 18, and the fractions automatically collected include the target pure product, the impure product that can be purified twice and the collection of waste liquid;

[0063] The first branch is a connecting pipeline between the first collecting buffer tank 15 and the target pure product tank 10, the second branch is a connecting pipeline between the first collecting buffer tank 15 and the impure product recovery tank 11, the third branch is a connecting pipeline between the second collecting buffer tank 16 and the target pure product tank 10, the fourth branch is a connecting pipeline between the second collecting buffer tank 16 and the impure product recovery tank 11, the fifth branch is a connecting pipeline between the first collecting buffer tank 15 and the waste liquid tank 20, and the sixth branch is a connecting pipeline between the second collecting buffer tank 16 and the waste liquid tank 20.

[0064] It can be understood that it includes a sample tank 2, an eluent tank 1, a chromatographic column 5, a first collecting buffer tank 15, a second collecting buffer tank 16, an online ultra-high performance liquid chromatograph 18, a target pure product tank 10, an impure product recovery tank 11, a solvent mixing tank 12, a waste liquid tank 20 and a solvent tank group 21; the sample tank 2 and the eluent tank 1 are respectively connected to one end of the chromatographic column 5 through pipelines, and a pump is arranged on the pipeline; a main pipeline is arranged at the outlet of the chromatographic column 5 for fractions, and two branches are formed by the ends of the main pipeline, the two branches are respectively connected to the first collecting buffer tank 15 and the second collecting buffer tank 16, and valves are arranged on both branches.

[0065] The target pure product tank 10, the impure product recovery tank 11 and the waste liquid tank 20 are respectively connected to the first collecting buffer tank 15 and the second collecting buffer tank 16 through pipelines, and valves are provided on the connecting pipelines. The first collecting buffer tank 15 and the second collecting buffer tank 16 are also connected to the analytical sample sampler and the online ultra-high performance liquid chromatograph 18 in sequence; the other ends of the target pure product tank 10, the impure product recovery tank 11 and the waste liquid tank 20 are all connected to the solvent mixing tank 12 by pipeline, and the solvent mixing tank 12 is connected to the eluent tank 1 by pipeline.

[0066] The online detection device 9 is also electrically connected to the control cabinet to control the opening and closing of the plurality of valves.

[0067] The control cabinet includes a signal receiver and a signal transmitter, and controls the functions of related equipment according to the signals fed back by external equipment. The principles of the above signal reception and control belong to the prior art. All control cabinets that can realize related functions can be used as the control cabinet of this embodiment, which will not be elaborated here.

[0068] It can be understood that the sample tank 2 is used to place the crude solution to be purified, and the crude solution flows into the chromatographic column 5 during the purification process. During the purification process, the crude solution in the sample tank 2 and the eluent in the eluent tank 1 are pumped into the chromatographic column 5 by pumps respectively. After the fraction flowing out of the chromatographic column 5 is tested for purity by the online detection device 9, the control cabinet controls the opening and closing of the valves on different pipelines according to the purity of the fraction, so that the fraction with the purity reaching the preset standard enters the target pure product tank 10, and the fraction with the purity not reaching the preset standard enters the impure product recovery tank 11; the fractions in the target pure product tank 10 and the impure product recovery tank 11 are subjected to vacuum volatilization and cooling in a closed system to separate the solvent from the fraction and introduce it into the solvent mixing tank 12, and then enter the eluent tank 1.

[0069] In this embodiment, the chromatographic column 5 is a dynamic axial pressure chromatographic column (DAC), and the body of the chromatographic column is made of stainless steel.

[0070] The column filling length is 40 cm.

[0071] The eluent is connected to the dynamic axial pressure chromatography column (DAC) via a constant flow mode infusion pump 3 .

[0072] The temperature of the eluent tank 1, the chromatographic column 5 and other parts of the closed-loop fully automatic chromatographic purification system is controlled by a fluid coil, and the temperature range is 20° C. to 23° C.

[0073] like Figure 2 As shown, the online detection device is an online ultra-high performance liquid chromatograph 18, which is connected to the first collection buffer tank 15 and the second collection buffer tank 16, and is used to detect the purity of the target substance in the fractions of the first collection buffer tank 15 and the second collection buffer tank 16.

[0074] Furthermore, the online ultra high performance liquid chromatograph 18 is electrically connected to the control cabinet, and the control cabinet is configured to control the switching of the valves of the first branch, the second branch, the third branch, the fourth branch, the fifth branch and the sixth branch of the first collection buffer tank 15 and the second collection buffer tank 16 according to the results of the online ultra high performance liquid chromatograph 18.

[0075] Among them, the first branch is the connecting pipeline between the first collecting buffer tank 15 and the target pure product tank 10; the second branch is the connecting pipeline between the first collecting buffer tank 15 and the impure product recovery tank 11; the third branch is the connecting pipeline between the second collecting buffer tank 16 and the target pure product tank 10; the fourth branch is the connecting pipeline between the second collecting buffer tank 16 and the impure product recovery tank 11, the fifth branch is the connecting pipeline between the first collecting buffer tank 15 and the waste liquid tank 20, and the sixth branch is the connecting pipeline between the second collecting buffer tank 16 and the waste liquid tank 20.

[0076] The online ultra-high performance liquid chromatograph 18 can realize online and on-site process analysis of the fractions and provide real-time data, so that the control cabinet can control the flow direction of the fractions at different stages in real time.

[0077] The online ultra-high performance liquid chromatograph 18 detects the purity of the target substance in the fraction of the first collection buffer tank 15 or the second collection buffer tank 16 by online sampling. The online ultra-high performance liquid chromatograph 18 is electrically connected to the control cabinet so that the control cabinet controls the branch valve according to the detection result of the online ultra-high performance liquid chromatograph 18.

[0078] The online sampling method is an online ultra-high performance liquid chromatograph 18, where the analytical sample sampler alternately extracts 10 μl fractions from the first collection buffer tank 15 and the second collection buffer tank 16, and after online dilution, enters the online ultra-high performance liquid chromatograph 18 for target purity detection. The target purity can be set to 98.0%-99.9% as needed.

[0079] When the crude polypeptide is purified by the fully automatic chromatography purification system of this embodiment, the following steps are included:

[0080] S1. A chromatographic column 5 filled with spherical mesoporous silica matrix reverse phase chromatography filler; the column filling height is 40 cm, the total content of other metals in the filler except silicon does not exceed 100 μg / g, the average pore size ranges from 90Å to 150Å, the porosity is 0.8-1.0 cm3 / g, and the specific surface area is 280-380m 2 / g, the average particle size range is 10 microns; the proportion of micropores with a pore size range below 70Å is less than 15%, and the proportion of micropores below 40Å is less than 5%;

[0081] S2. The crude polypeptide solution to be purified is placed in sample tank 2, and the eluent is prepared and placed in eluent tank 1 (preferably an aqueous solution containing acetonitrile);

[0082] S3. Quantitatively pump the sample solution into the chromatographic column 5; then pump the mobile phase solution in the eluent tank 1 into the chromatographic column 5; the eluent and the chromatographic column 5 are temperature-controlled by the liquid coil outside the column and the tank, and the temperature range is 20°C to 23°C; the temperature difference between the eluent and the chromatographic column 5 is within ±3°C.

[0083] S4. Collect the fractions and detect the fractions using an online detection device, and make the fractions enter different tanks according to the test results of the fractions;

[0084] Specifically:

[0085] S411, after the valve of the first collection buffer tank 15 is opened, the distillate enters the first collection buffer tank 15, and the first collection buffer tank 15 collects the distillate, and at this time the second collection buffer tank 16 is in an empty state. After the distillate collected in the first collection buffer tank 15 reaches a preset volume or after a preset time, the distillate is controlled by the valve and switched to flow into the second collection buffer tank 16.

[0086] Of course, the distillate may also flow into the second collection buffer tank 16 first. For this embodiment, after the valve of the second collection buffer tank 16 is opened, the distillate enters the second collection buffer tank 16, and the second collection buffer tank 16 collects the distillate. At this time, the first collection buffer tank 15 is in an empty state. After the distillate collected in the second collection buffer tank 16 reaches a preset volume or after a preset time, the distillate is controlled by the valve and switched to flow into the first collection buffer tank 15.

[0087] S412, the first collection buffer tank 15 and the second collection buffer tank 16 cyclically perform fraction collection and fraction detection work until the sample purification is completed.

[0088] S42. Automatically sample and detect the purity of the target substance from the first collection buffer tank 15 or the second collection buffer tank 16 using an online ultra-high performance liquid chromatograph 18. According to the test results, the fraction whose purity of the target substance reaches the preset standard is transferred to the target pure product tank 10; the fraction whose purity of the target substance does not reach the preset standard is transferred to the non-pure product recovery tank 11;

[0089] S5. The concentrated solution in the target pure product tank 10 is transferred to the post-processing tank 19 for crystallization or freeze-drying to obtain a pure target polypeptide, and the concentrated solution is obtained by evaporating the fraction in the target pure product tank 10 by vacuum evaporation;

[0090] Further, as another embodiment, the online detection device further includes a full-wavelength ultraviolet-visible detector 14 (such as Figure 2 The portion shown by the yellow dotted line) is a full-wavelength UV-visible detector 14 on the main pipe at the outlet of the chromatographic column 5, and is used to detect whether the eluent in the main pipe contains a chromatographic peak of the target object. In addition to the two branches mentioned above, a seventh branch is formed at the end of the main pipe at the outlet of the chromatographic column 5, and the seventh branch is connected to the solvent recovery tank 17.

[0091] As an implementation example, when purifying a polypeptide sample in the fully automatic chromatography purification system of this embodiment, acetonitrile-water is used as the eluent, such as Figure 3As shown, the absorption threshold of the full-wavelength UV-visible detector 14 is set to 35mAU. After the sample is injected, the absorption value is lower than 35mAU at 0-4.7min and remains at a stable absorption value without any chromatographic peak. During this period, the branch of the control cabinet control valve 13-1 leading to the solvent recovery tank 17 is opened, and the fraction enters the solvent recovery tank 17; between 7.5 and 17.5 min, the chromatographic peak detected by the full-wavelength UV-visible detector 14 contains the polypeptide target, so the branch of the control cabinet control valve 13-1 leading to the first collection buffer tank 15 and the second collection buffer tank 16 is opened, and the fraction enters the first collection buffer tank 15 or the second collection buffer tank 16, that is, the fully automatic chromatographic purification system enters the sample UHPLC online detection and fraction automatic collection mode;

[0092] During the processing, the control cabinet control valve 2 13 - 2 opens the valve leading to the first collection buffer tank 15 branch or the second collection buffer tank 16 branch, and the first collection buffer tank 15 or the second collection buffer tank 16 collects the fractions with chromatographic peaks.

[0093] The fractions with chromatographic peaks are collected by the first collection buffer tank 15 and the second collection buffer tank 16; due to the large injection volume of preparative chromatographic separation, the full-wavelength UV-visible detector 14 cannot distinguish the content or concentration of the target and impurities. Therefore, the fractions entering the first collection buffer tank 15 and the second collection buffer tank 16 may be fractions whose target purity reaches the preset standard or fractions whose target purity does not reach the preset standard; fractions whose target purity reaches the preset standard need to be collected using the target pure product tank 10, while fractions whose target purity does not reach the preset standard need to be collected using the impure product recovery tank 11; fractions with lower purity and content enter the waste liquid tank 20. Therefore, the output ends of the first collection buffer tank 15 and the second collection buffer tank 16 need to be connected to the target pure product tank 10, the impure product recovery tank 11 and the waste liquid tank 20, so that the corresponding fractions are collected using the corresponding tanks according to the purity.

[0094] The specific method is: first, after the valve of the first collecting buffer tank 15 is opened, the distillate enters the first collecting buffer tank 15, and the first collecting buffer tank 15 collects the distillate. At this time, the second collecting buffer tank 16 is empty; after the collection of the first collecting buffer tank 15 is completed, the distillate is controlled by the valve and switched to flow into the second collecting buffer tank 16.

[0095] Of course, the distillate can also flow into the second collecting buffer tank 16 first, and the second collecting buffer tank 16 collects the distillate. At this time, the first collecting buffer tank 15 is empty; after the second collecting buffer tank 16 completes the collection, the distillate is controlled by the valve and switched to flow into the first collecting buffer tank 15.

[0096] It can be understood that after the operation is stable, when the first collection buffer tank 15 collects the distillate, the online ultra-high performance liquid chromatograph 18 performs a target purity detection on the distillate of the second collection buffer tank 16. When the purity of the target in the distillate reaches the preset standard, the control cabinet controls the third branch valve to open, and the distillate flows to the target pure product tank 10; when the purity of the target in the distillate does not reach the preset standard, the control cabinet controls the fourth branch valve to open, and the distillate flows to the non-pure product recovery tank 11; when the purity of the target in the distillate is lower (less than 50%), the distillate enters the waste liquid tank 20.

[0097] When the second collection buffer tank 16 collects the distillate, the online ultra-high performance liquid chromatograph 18 performs a target purity detection on the distillate of the first collection buffer tank 15. When the purity of the target in the distillate reaches the preset standard, the control cabinet controls the first branch valve to open, and the distillate flows to the target pure product tank 10; when the purity of the target in the distillate does not reach the preset standard, the control cabinet controls the second branch valve to open, and the distillate flows to the non-pure product recovery tank 11; when the purity of the target in the distillate is lower (less than 50%), the distillate enters the waste liquid tank 20.

[0098] Therefore, the first collection buffer tank 15 and the second collection buffer tank 16 are used to collect and detect fractions in a cycle, and only three tanks are used to collect fractions containing chromatographic peaks of the target substance according to the set purity. The target pure product tank 10 collects fractions whose purity reaches the preset standard of the target substance, and after further concentration, a solid product is obtained by freeze drying or other methods; the impure product recovery tank 11 collects fractions whose purity does not reach the preset standard of the target substance, so as to further recover and purify again; the waste liquid tank 20 collects fractions with lower purity that do not need to be recovered and purified again.

[0099] Further, another embodiment of the present invention also includes an automatic solvent recovery device (such as Figure 2The front end of the automatic solvent recovery device is connected to the solvent recovery tank 17, the target pure product tank 10, the impure product recovery tank 11 and the waste liquid tank 20 respectively, and the rear end is connected to the eluent tank 1. The automatic solvent recovery device includes: a solvent mixing tank 12, a solvent buffer tank 25 and a solvent tank group 21, as well as matching switching valves and pumps, and also includes a reduced pressure distillation and cooling collection device arranged between the solvent buffer tank 25 and the solvent recovery tank 17, the target pure product tank 10, the impure product recovery tank 11 and the waste liquid tank 20; the input end of the solvent buffer tank 25 is connected to the target pure product tank 10, the impure product recovery tank 11 and the waste liquid tank 20 through a pipeline, and a pump and a valve are arranged on the pipeline; the output end of the solvent buffer tank 25 is connected to the first input end of the solvent mixing tank 12 through a pipeline, and a high-precision flow meter is arranged on the pipeline; the solvent tank group 21 is connected to the second input end of the solvent mixing tank 12 through a pipeline, and a pump and a valve are arranged on the pipeline; the solvent mixing tank 12 is also connected to the eluent tank 1 through a pipeline.

[0100] It can be understood that, by using the full-wavelength UV-visible detector 14 and the online ultra-high performance liquid chromatograph 18 simultaneously, the fraction flowing out of the chromatographic column 5 can be intelligently divided into four parts: a fraction not containing the target substance (the fraction entering the solvent recovery tank 17), a target pure product fraction (i.e., the fraction flowing into the target pure product tank 10), an impure product fraction (i.e., the fraction flowing into the impure product recovery tank 11) and a waste liquid (i.e., the fraction flowing into the waste liquid tank 20). Then, the solvent in the fraction can be recovered to the maximum extent through a vacuum distillation device. Then, the component ratio of the recovered solvent can be detected by a high-precision mass flow meter between the solvent buffer tank 25 and the solvent mixing tank 12. Finally, according to needs, the solvent tank group 21 is used to provide the corresponding solvent component to achieve online automatic liquid preparation to obtain the same component ratio as the eluent.

[0101] As an example, when purifying a crude polypeptide product using the closed-loop fully automatic chromatography purification system of this embodiment, acetonitrile-water is used as the eluent, such as Figure 3 As shown, the absorption threshold of the full-wavelength UV-visible detector 14 is set to 35mAU. After the sample is injected, the absorption value is lower than 35mAU at 0-4.7min and remains at a stable absorption value without any chromatographic peak. During this period, the branch of the control cabinet control valve 13-1 leading to the solvent recovery tank 17 is opened, and the distillate enters the solvent recovery tank 17, and then enters the eluent tank 1 through the solvent recovery tank 17 to realize the direct circulation of the solvent; between 4.7-7.5min and 17.5-30min, the full-wavelength UV-visible detector 14 detects a chromatographic peak that does not contain the target substance, and the distillate also enters the solvent recovery tank 17, and then the clean solvent is recovered by distillation and cooling through reduced pressure distillation, and then enters the solvent buffer tank 25.

[0102] Between 7.5 and 17.5 minutes, the chromatographic peak detected by the full-wavelength UV-visible detector 14 contains the polypeptide target, so the branch of the control cabinet control valve 13-1 leading to the first collection buffer tank 15 and the second collection buffer tank 16 is opened, and the fraction enters the first collection buffer tank 15 or the second collection buffer tank 16, that is, the closed-loop fully automatic chromatography purification system enters the sample UHPLC online detection and fraction automatic collection mode. The separation cycle is calculated as 35.5 minutes, and the proportion of recovered acetonitrile is close to 90%; of which about 15% is directly applied without vacuum distillation and mixing for the fraction containing the chromatographic peak containing the target.

[0103] During the processing, the control cabinet control valve 2 13 - 2 opens the valve leading to the first collection buffer tank 15 branch or the second collection buffer tank 16 branch, and the first collection buffer tank 15 or the second collection buffer tank 16 collects the fractions with chromatographic peaks.

[0104] The fractions with chromatographic peaks are collected by the first collection buffer tank 15 and the second collection buffer tank 16; due to the large injection volume of preparative chromatographic separation, the full-wavelength UV-visible detector 14 cannot distinguish the content or concentration of the target and impurities. Therefore, the fractions entering the first collection buffer tank 15 and the second collection buffer tank 16 may be fractions whose target purity reaches the preset standard or fractions whose target purity does not reach the preset standard; fractions whose target purity reaches the preset standard need to be collected using the target pure product tank 10, while fractions whose target purity does not reach the preset standard need to be collected using the impure product recovery tank 11; fractions with lower purity and content enter the waste liquid tank 20. Therefore, the output ends of the first collection buffer tank 15 and the second collection buffer tank 16 need to be connected to the target pure product tank 10, the impure product recovery tank 11 and the waste liquid tank 20, so that the corresponding fractions are collected using the corresponding tanks according to the purity.

[0105] It can be understood that after the operation is stable, when the first collection buffer tank 15 collects the distillate, the online ultra-high performance liquid chromatograph 18 performs a target purity detection on the distillate of the second collection buffer tank 16. When the purity of the target in the distillate reaches the preset standard, the control cabinet controls the third branch valve to open, and the distillate flows to the target pure product tank 10; when the purity of the target in the distillate does not reach the preset standard, the control cabinet controls the fourth branch valve to open, and the distillate flows to the non-pure product recovery tank 11; when the purity of the target in the distillate is lower (less than 50%), the distillate enters the waste liquid tank 20.

[0106] When the second collection buffer tank 16 collects the distillate, the online ultra-high performance liquid chromatograph 18 performs a target purity detection on the distillate of the first collection buffer tank 15. When the purity of the target in the distillate reaches the preset standard, the control cabinet controls the first branch valve to open, and the distillate flows to the target pure product tank 10; when the purity of the target in the distillate does not reach the preset standard, the control cabinet controls the second branch valve to open, and the distillate flows to the non-pure product recovery tank 11; when the purity of the target in the distillate is lower (less than 50%), the distillate enters the waste liquid tank 20.

[0107] Therefore, the first collection buffer tank 15 and the second collection buffer tank 16 are used to collect and detect fractions in a cycle, and only three tanks are used to collect fractions containing chromatographic peaks of the target substance according to the set purity. The target pure product tank 10 collects fractions whose purity reaches the preset standard of the target substance, and after further concentration, a solid product is obtained by freeze drying or other methods; the impure product recovery tank 11 collects fractions whose purity does not reach the preset standard of the target substance, so as to further recover and purify again; the waste liquid tank 20 collects fractions with lower purity that do not need to be recovered and purified again.

[0108] Traditional liquid chromatography needs to collect the fractions of the entire flat peak. In order to improve the accuracy of fraction collection, small-volume fraction collection tubes are needed. In other words, the higher the accuracy, the smaller the volume of fractions collected by each fraction collection tube, the more fraction collection tubes are needed, and the more workbench space is needed; subsequently, the target content and concentration in the fractions of each collection tube must be tested offline to determine the qualified range of the collected fractions for combined tube processing.

[0109] The solvent in the fraction of the target pure product tank 10 is evaporated in a vacuum in a closed system and then cooled and then drained into the solvent mixing tank 12 .

[0110] It is understandable that when the vacuum volatilization and cooling operation is performed, the target pure product tank 10 is a concentration kettle. The concentration kettle has a double-layer structure, which is heated by heat-conducting oil, and is stirred by a mechanical pump to make the heating uniform; and the vacuum pump is connected to the reaction kettle to reduce the vacuum degree in the kettle; the reduction of the vacuum degree can reduce the boiling point of the solvent, and the solvent can be evaporated at a lower heating temperature, thereby improving the concentration efficiency of the target polypeptide. The distilled solvent is condensed by a glass condenser to convert it from a gaseous state to a liquid state, and is drained into the solvent buffer tank 25 to complete the recovery and reuse of the solvent.

[0111] The other output end of the target pure product tank 10 is connected to the post-processing tank 19; after the distillate of the target pure product tank 10 is vacuum evaporated in a closed system to remove the solvent therein, the remaining concentrated liquid remains in the target pure product tank 10 and finally enters the post-processing tank 19 through a pipeline.

[0112] Further, such as Figure 2As shown, the concentrated solution is crystallized or freeze-dried in the post-processing tank 19 to obtain the target pure polypeptide.

[0113] It is understood that when the crystallization operation is performed, the post-processing tank 19 is a reactor with high and low temperatures, and the crystallization is directly performed in the reactor. Specifically, the sample is dissolved in a hot solvent such as 60°C ethanol, and then gradually cooled to room temperature. The target substance is supersaturated and precipitated due to the difference in solubility; the impurity content is low and will remain in the solution.

[0114] It is understandable that when freeze drying is performed, the post-processing tank 19 is a freeze drying bottle or a freeze drying plate. Specifically, after the sample is subjected to vacuum rotary evaporation to remove the organic solvent, the remaining is an aqueous solution of the sample; then the sample aqueous solution is placed in a freeze drying bottle or a freeze drying plate, frozen into ice by a refrigerator or liquid nitrogen, and then the freeze drying bottle is connected to the freeze drying machine pipeline through the bottle mouth or the freeze drying plate is placed inside the freeze drying machine for freeze drying. The freeze dryer is a high vacuum environment, so water is continuously reduced by sublimation until it is completely removed, and the remaining solid is the purified target polypeptide.

[0115] The solvent in the fractions of the impure product recovery tank 11 and the waste liquid tank 20 also enters the solvent buffer tank 25 after a vacuum volatilization and cooling process similar to the above in a closed system.

[0116] After the fraction in the impure product recovery tank 11 is evaporated in a closed system to remove the solvent, the remaining concentrated liquid remains in the impure product recovery tank 11 and enters the sample tank 2 under the control of the control cabinet to be purified again through the chromatographic column 5 as a sample.

[0117] The residues produced by the distillation in the waste liquid tank 20 must be cleaned regularly. The cleaning method is that the residues after concentration in the waste liquid tank 20 will be cleaned and discharged at regular intervals.

[0118] Through the above embodiment, since the solvents in the target pure product tank 10, the impure product recovery tank 11 and the fractions in the waste liquid tank 20 are subjected to vacuum volatilization and then cooling operations, and the solvents obtained after the operations are drained into the solvent buffer tank 25, the solvents collected in the solvent buffer tank 25 contain both the solvent volatilized from the purifiable fraction and the solvent volatilized from the impure fraction, thereby providing the original solvent for the subsequent automatic liquid preparation procedure.

[0119] Further, such as Figure 2 As shown, the first output end of the target pure product tank 10 is connected to the solvent buffer tank 25 through a pipeline, and a high-precision flow control valve 22-1 and a multi-stage variable frequency centrifugal pump 23-1 are arranged on the pipeline;

[0120] The first output end of the impure product recovery tank 11 is connected to the solvent buffer tank 25 through a pipeline, and a high-precision flow control valve 22-2 and a multi-stage variable frequency centrifugal pump 23-2 are arranged on the pipeline.

[0121] As a possible implementation, Figure 2 As shown, the waste liquid tank 20 is connected to the solvent buffer tank 25 through a pipeline, and a high-precision flow control valve 5 22-5 and a multi-stage variable frequency centrifugal pump 5 23-5 are arranged on the pipeline.

[0122] It is understandable that the multi-stage variable frequency centrifugal pump is used to transport the recovered solvent to the solvent buffer tank 25, and the high-precision flow control valve can accurately control the volume of the solvent flowing through the pipeline. A high-precision mass flow meter 24-1 is installed on the pipeline between the solvent buffer tank 25 and the solvent mixing tank 12, and the high-precision mass flow meter can detect the density of the recovered solvent, thereby calculating the solvent component ratio, and feeding back the detection result to the control cabinet, which controls the opening and closing of the corresponding solvent tank valve of the solvent tank group 21 according to the detection result to transport the corresponding solvent to mix and obtain the same component ratio of the eluent.

[0123] like Figure 2 As shown, the input end of the solvent mixing tank 12 is connected to the solvent tank group 21. The solvent tank group 21 can be a combination of several solvent tanks according to the ratio of the eluent, for example, it can be set to 1-3; wherein each solvent tank can store a suitable solvent according to the needs, which can be a water-soluble acid, a water-soluble base or a water-soluble salt, or an organic solvent. The solvent in the solvent tank group 21 can be pumped into the solvent mixing tank 12 according to the modulation requirements.

[0124] Furthermore, the recovered solvent is measured and calculated by a mass flow meter to obtain a corresponding component ratio, and the ratio is automatically adjusted to the ratio of the eluent in the mixing tank.

[0125] It can be understood that a high-precision mass flow meter and a solvent mixing tank 12 are introduced into the chromatographic purification system. The high-precision mass flow meter is used to detect the density of the recovered solvent and compare it with the density of the eluent. The required solvent volume can be calculated based on the comparison result of the density of the required eluent and the density of the recovered solvent. Then, the control cabinet adjusts the solvent tank group 21 to add a certain volume of the corresponding solvent to the solvent mixing tank 12, and adjusts the proportion of the recovered solvent in the solvent mixing tank 12 to the set proportion of the eluent.

[0126] Specifically, the online liquid preparation device automatically detects the density of the recovered solvent through a high-precision mass flow meter and compares it with the density of the eluent; based on the density difference and the total volume, the compensation solvent volume is calculated, and after the online liquid preparation automatically replenishes the corresponding solvent, the density comparison and calculation are performed again, and the corresponding solvent is further replenished until the density of the solvent after liquid preparation is consistent with the density of the eluent, which means that the ratio of the recovered solvent after liquid preparation is now completely consistent with the ratio of the eluent, and can be recycled as an eluent.

[0127] Further, such as Figure 2 As shown, the output end of the solvent mixing tank 12 is connected to the eluent tank 1, and the prepared solvent is input into the eluent tank 1 to realize the recycling of the solvent, thereby improving the utilization rate of the solvent and reducing the cost of waste liquid treatment.

[0128] Further, such as Figure 2 As shown, the first output end of the solvent recovery tank 17 is connected to the pipeline of the eluent tank 1 ; the second output end of the solvent recovery tank 17 is connected to the solvent buffer tank 25 .

[0129] After detection by the full-wavelength UV-visible detector 14, the solvent without chromatographic peak in the solvent recovery tank 17 can enter the eluent tank 1; the solvent with chromatographic peak, after vacuum distillation, is cooled and recovered as pure solvent and enters the solvent buffer tank 25. The solvent in the solvent buffer tank 25 is automatically detected by the high-precision flow meter, and the solvent is compensated and mixed in the mixing tank until it meets the eluent solvent ratio and then drained into the eluent tank 1.

[0130] As a possible implementation, the chromatographic column 5 is preferably a dynamic axial pressure chromatographic column (DAC).

[0131] As a possible implementation, the filler filled in the chromatographic column 5 is a spherical chromatographic filler based on silica gel.

[0132] Furthermore, in addition to the tailing agent, the silica gel surface is bonded and modified with two or more silanes, and the proportion of any one of them is not less than 10%; one of the two silanes is a polar silane and the other is a non-polar silane, or the two are different non-polar silanes.

[0133] As a possible implementation, the eluent and the chromatographic column 5 are temperature controlled within a temperature range of 10°C to 30°C.

[0134] Furthermore, there are two eluent tanks 1, each containing an eluent of different density. A high-pressure constant-current pump is provided on the connecting pipeline between the eluent tank 1 and the chromatographic column 5. By controlling the flow ratio of the two high-pressure constant-current pumps respectively, online gradient mixing of the eluent is achieved, and gradient elution of the sample is performed.

[0135] Furthermore, it is used for reverse phase purification of organic substances such as peptides, macrolides, and cyclosporine.

[0136] The fully automatic chromatography purification system of the present invention realizes the full automation of the entire chromatography purification cycle process for the first time, which not only improves the purification efficiency, reduces human mistakes and errors, saves workbench space, reduces losses in solvent recycling, but also reduces safety risks and environmental pollution.

[0137] The automatic liquid dispensing system can automatically mix and dispense the initial solvent and the recycled solvents collected in each section according to the set ratio, and adopts multi-stage variable frequency centrifugal pumps, high-precision mass flow meters, high-precision flow control valves and other devices to achieve automatic liquid dispensing under the calculation, correction and control of the liquid dispensing workstation, eliminating manual offline operation, accurate and reliable, and highly automated;

[0138] The coordinated use of detectors and tanks during fraction collection, solvent recovery, and automatic liquid preparation lays the foundation for full automation of the purification process.

[0139] Furthermore, the chromatographic column filler is a spherical mesoporous silica matrix reverse phase chromatographic filler, the column filling height is 20-100 cm, preferably, 20-40 cm; the total content of other metals in the filler except silicon does not exceed 50 μg / g, the average pore size ranges from 90Å to 150Å, and the porosity is 0.8-1.0m 3 / g, specific surface area is 280-380m 2 / g, with an average particle size ranging from 5 microns to 30 microns;

[0140] The proportion of micropores with a pore size below 70Å is less than 15%, and the proportion of micropores below 40Å is less than 5%;

[0141] The eluent used for separation is an aqueous solution containing a water-soluble acid, base or salt, or an organic solvent soluble in water.

[0142] Furthermore, the chromatographic filler is bonded with two or more silane groups, wherein the silane groups that do not contain the above-mentioned polar groups contain not less than 5 carbon atoms, and the proportion of the silane group with the lowest content is not less than 10%.

[0143] Furthermore, the silyl group refers to an alkylsiloxy group containing a polar group or not, including a siloxy group modified with a polar group; it also includes: octadecyldimethylsiloxy, octadecyldiisopropylsiloxy, octadecyldiisobutylsiloxy, hexadecyldimethylsiloxy, dodecyldimethylsiloxy, decyldimethylsiloxy, octyldimethylsiloxy, hexyldimethylsiloxy, butyldimethylsiloxy, propyldimethylsiloxy, isopropyldimethylsiloxy, isobutyldimethylsiloxy, aminopropyldimethylsiloxy, and aminoethyldimethylsiloxy.

[0144] Furthermore, a high-precision mass flow meter monitors the flow rate accurately in real time and feeds back to the control cabinet; the control cabinet instantly adjusts the flow rate to the set value to ensure the flow rate accuracy; and a high-precision mass flow meter is used to monitor the density of the recovered solvent in real time and feed back to the control cabinet. The control cabinet controls the solvent tank to add the required volume of the corresponding solvent according to the density of the recovered solvent, so that the density of the mixed solvent reaches the set value of the eluent density.

[0145] It can be understood that after the density of the recovered solvent is measured by a high-precision mass flow meter, it is compared with the density of the eluent and fed back to the control cabinet. The control cabinet calculates the required volume of the solvent based on the comparison result, and the control cabinet controls the valve of the corresponding solvent tank of the solvent tank group 21 to open, and injects the required volume of the corresponding solvent into the solvent mixing tank 12, adjusts the proportion of the recovered solvent to the set proportion of the eluent, and then transfers it to the eluent tank 1, thereby realizing the recovery and reuse of the solvent.

[0146] Example 1: Comparison between high-precision flowmeter and ordinary flowmeter in insulin purification and separation

[0147]

[0148] The introduction of high-precision flow meters improves the stability of the preparation and purification process, resulting in higher purity and yield of purification, and reduces variables introduced into the process.

[0149] Furthermore, by precisely controlling the temperature of the chromatographic column and the eluent and making them consistent, the peak shape and separation effect can be effectively improved, as shown in Example 2.

[0150] Furthermore, the temperature of the chromatographic column and the eluent is controlled between 10°C and 30°C, and the chromatographic column and the eluent are kept constant and the temperature is consistent, and the temperature difference between the eluent and the chromatographic column is within ±3°C.

[0151]

[0152] From Example 2 ( Fig. 9 ), Comparative Example 1 ( Figure 7 )、Comparative Example 2( Figure 8 ) and Comparative Example 3 ( Figure 6) It can be seen that changes in column temperature will lead to increased peak broadening. Mobile phase and column temperature control need to be precisely controlled within a certain range to obtain the best separation; increasing the temperature of the column or eluent will weaken the retention of the main peak, and the impurities before the peak cannot be effectively separated; decreasing the temperature of the column or eluent will increase the retention time, prolong the separation time, and deteriorate the separation from the subsequent impurities; if there is no proper column thermostat, a radial temperature gradient will form in the column, affecting the separation efficiency (theoretical plate number, N) and analyte retention. When there is a large temperature difference between the eluent and the chromatographic column itself, the peak will be deformed and the separation will decrease.

[0153] In Example 2, when the chromatographic column and the eluent were both maintained at 20° C., the main peak was separated from both the front impurities and the back impurities.

[0154] In comparative example 1, when the temperature of the chromatographic column and the eluent were both lowered to 10° C., the sample retention time increased, and the front impurities were separated and the rear impurities were encapsulated.

[0155] In comparative example 2, when the temperature of the chromatographic column and the eluent were both raised to 40°C, the sample retention time decreased, and the main peak and the front and rear impurities were not separated.

[0156] In comparative example 3, the eluent temperature was lowered to 5°C, the chromatographic column was maintained at 20°C, the sample retention time increased, but the separation of the impurities before and after became worse.

[0157] Further, such as Figure 4-7 As shown, the dynamic axial pressurization chromatographic column adopts an oil-free cylinder type chromatographic column.

[0158] It is understandable that the purpose of separation and purification is to obtain a target polypeptide with high purity, and the oil-free cylinder chromatographic column can effectively avoid contamination of the separated sample caused by oil leakage.

[0159] Furthermore, when the mixture on the chromatographic column is eluted with an eluent for column separation, the eluent is used as a reference for monitoring with a full-wavelength ultraviolet-visible detector 14. When no target polypeptide appears, the collected solution enters the eluent recovery tank; when the target polypeptide is found to appear, the fraction is collected and enters the first collection buffer tank 15 or the second collection buffer tank 16; an online ultra-high performance liquid chromatograph 18 is used to automatically sample from the first collection buffer tank 15 or the second collection buffer tank 16 to detect the purity of the target. According to the test results, the fraction whose target purity reaches the preset standard is transferred to the target pure product tank 10; the fraction whose target purity does not reach the preset standard but exceeds the preset "recoverable fraction purity" is transferred to the impure product recovery tank 11, and the fraction in the impure product recovery tank 11 is concentrated and enters the sample tank 2 to be used as a sample for secondary purification; for the fraction whose target is even lower than the "recoverable fraction purity", it is transferred to the waste liquid tank 20.

[0160] Furthermore, the fractions are collected and detected by an online detection device, and the fractions are placed into different tanks according to the detection results of the fractions; the online detection device includes a full-wavelength ultraviolet-visible detector 14 and an online ultra-high performance liquid chromatograph 18; the specific steps include:

[0161] S41. Scan the fractions prepared by high-performance chromatography with a full-wavelength UV-visible detector 14 to detect whether there are chromatographic peaks in the fractions. The fractions without chromatographic peaks are collected into the solvent recovery tank 17, and the fractions with chromatographic peaks are collected into the first collection buffer tank 15 or the second collection buffer tank 16; wherein,

[0162] S411, after the valve of the first collecting buffer tank 15 is opened, the distillate enters the first collecting buffer tank 15, and the first collecting buffer tank 15 collects the distillate, and at this time the second collecting buffer tank 16 is in an empty state; after the distillate collected in the first collecting buffer tank 15 reaches a preset volume or after a preset time, the distillate is controlled by the valve and switched to flow into the second collecting buffer tank 16; or

[0163] The distillate first flows into the second collection buffer tank 16. After the valve of the second collection buffer tank 16 is opened, the distillate enters the second collection buffer tank 16. The second collection buffer tank 16 collects the distillate. At this time, the first collection buffer tank 15 is empty. After the distillate collected by the second collection buffer tank 16 reaches a preset volume or after a preset time, the distillate is controlled by the valve and switched to flow into the first collection buffer tank 15.

[0164] S412, the first collection buffer tank 15 and the second collection buffer tank 16 are cyclically used to collect fractions and detect the purity of the fractions by UHPLC; when the purity reaches the preset standard, the fraction is collected to the "target pure product tank 10" through valve control; for the fractions that do not reach the purity standard but exceed the "recoverable fraction purity", they are collected to the "non-pure product collection tank" for further purification; for the fractions that are even lower than the "recoverable fraction purity", they are collected to the "waste liquid tank 20"; the cycle is repeated until the target content is lower than the set value.

[0165] It can be understood that the chromatographic peak in S41 can also be characterized by the target object, that is, the fractions prepared by high performance chromatography are scanned with a full-wavelength UV-visible detector 14 to detect whether the target object exists in the fractions. The fractions without the target object are collected and entered into the solvent recovery tank 17, and the fractions with the target object are collected and entered into the first collection buffer tank 15 or the second collection buffer tank 16.

[0166] It is understandable that the fraction without chromatographic peaks can be basically considered as the eluent that has not been mixed with impurities in the sample, and this part of the fraction can be recycled and reused. During the flow of this part of the fraction, the control cabinet controls the branch valve of the solvent recovery tank 17 to open, so that this part of the fraction enters the solvent recovery tank 17.

[0167] For the fractions with chromatographic peaks, it can be considered that there may be target substances to be purified, and these fractions need further processing. During the processing, the control cabinet control valve 2 13-2 opens the valve leading to the first collection buffer tank 15 branch or the second collection buffer tank 16 branch, and the first collection buffer tank 15 or the second collection buffer tank 16 collects the fractions with chromatographic peaks.

[0168] An online ultra-high performance liquid chromatograph 18 is used to automatically take samples from the first collection buffer tank 15 or the second collection buffer tank 16 to detect the purity of the target object. According to the detection result, the fraction whose purity of the target object reaches the preset standard is transferred to the target pure product tank 10; the fraction whose purity of the target object does not reach the preset standard is transferred to the non-pure product recovery tank 11.

[0169] Furthermore, the preset volume is 3-10 L, and the preset time is 1-10 min.

[0170] The target object is collected in a segmented manner, which uses the preset volume of the collection buffer tank for segmented collection. This preset volume can be determined according to needs and can be 3-10L. The smaller the preset volume, the shorter the segment interval of fraction collection. Under the premise of ensuring purity, it can reduce losses and improve yields; however, the shorter the segment interval of fraction collection, the higher the frequency of pump valve opening and closing, which will lead to an increase in the failure rate of the device and increase the maintenance cost of the device.

[0171] If the interval between fraction collection is too long, the amount of fraction collected that has the target substance content reaching the preset standard will be insufficient or even absent.

[0172] In addition, the selection of the preset time is determined according to the properties of the purified material, and different times are used for different polypeptides. For polypeptide purification in this embodiment, 5L is preferred in combination with the maintenance issues of the device and the content requirements of the target product.

[0173] In addition, based on a fixed flow rate, time can be used to monitor whether the preset volume has been reached. The collection time can be set to 1-10 minutes according to the preset volume. Using time to characterize whether the preset volume has been reached can simplify equipment settings and avoid the risk of human error, thereby ensuring the quality of the fraction.

[0174] Further, the solvent recovery step is included, specifically including:

[0175] The solvent recovery and utilization includes two paths. The solvent recovery and utilization of the first path is the solvent recovery and utilization of the fraction in the solvent recovery tank 17 , and the solvent recovery and utilization of the second path is the solvent recovery and utilization of the fraction in the target pure product tank 10 , the impure product recovery tank 11 and the waste liquid tank 20 .

[0176] The first path is the solvent recovery and utilization of the fraction in the solvent recovery tank 17, specifically: after the fraction in the solvent recovery tank 17 is confirmed in proportion, for the fraction that meets the set ratio of the eluent, the valve of the pipeline connecting the solvent recovery tank 17 and the eluent tank 1 is opened, and the fraction enters the eluent tank 1, so as to realize the recovery and reuse of the solvent in the fraction;

[0177] For the fractions that do not meet the set ratio of the eluent, the density is measured by a high-precision mass flow meter and compared with the density of the eluent, and the required volume of the solvent is calculated based on the comparison result. The control cabinet controls the valve of the corresponding solvent tank of the solvent tank group 21 to open, and injects the required volume of the corresponding solvent into the solvent mixing tank 12. The proportion of the recovered fraction is adjusted to the set ratio of the eluent and then transferred to the eluent tank 1, so as to realize the recovery and reuse of the solvent in the fraction.

[0178] The second path of solvent recovery includes the following steps:

[0179] S61. The fractions of the target pure product tank 10 and the impure product recovery tank 11 are evaporated in a closed system under vacuum, and the solvent in the fractions is separated by vacuum evaporation, and then condensed into a liquid solvent mixture after cooling;

[0180] S62. After the density of the solvent mixture condensed into a liquid is measured by a high-precision mass flow meter, it is compared with the density of the eluent, and the required volume of the solvent is calculated based on the comparison result. The control cabinet controls the valve of the corresponding solvent tank of the solvent tank group 21 to open, and injects the required volume of the corresponding solvent into the solvent mixing tank 12. The proportion of the recovered solvent mixture is adjusted to the set proportion of the eluent and then transferred to the eluent tank 1, so as to realize the recovery and reuse of the solvent in the fraction.

[0181] Furthermore, a saturated solution of the crude polypeptide to be purified is prepared by dissolving an eluent, and the prepared eluent is placed in an eluent tank 1. The eluent is a homogeneous solution containing a volatile organic solvent and water, and the organic solvent is methanol, ethanol, acetonitrile, acetone or tetrahydrofuran.

[0182] Furthermore, the eluent is pure water or an aqueous solution, to which an acid, a base or a salt is added, and the acid, a base or a salt is formic acid, acetic acid, trifluoroacetic acid, ammonia water, triethylamine, ammonium acetate, ammonium carbonate or triethylammonium acetate.

[0183] Furthermore, the control system implements linkage control on the injection pump, the infusion pump and the switching valve in front of the column to achieve rapid alternating entry of the sample and the eluent. The loading device also includes a short column filled with large-particle chromatographic filler, the particle size of the chromatographic filler is 20μm-100μm, the short column is arranged between the axial pressure column and the injection pump, and the front and rear ends of the short column have multi-channel switching valves, the switching valve is connected to the control device through an electric control line, the switching valve and the control device have data transmission, and the switching valve is controlled by the control software through the control device. The length of the short column is between 1-10 cm.

[0184] Furthermore, after separation, the target fraction with qualified purity enters the target pure product tank 10; the fraction containing impurities exceeding the recovery standard enters the impure product recovery tank 11; the fraction in the target pure product tank 10 is subjected to vacuum freeze drying to remove the solvent to obtain a solid.

[0185] It is understood that when freeze drying is performed, after the sample is subjected to vacuum rotary evaporation to remove the organic solvent, the remaining sample is an aqueous solution; the sample aqueous solution is then placed in a freeze drying bottle or freeze drying tray, frozen into ice by a refrigerator or liquid nitrogen, and then the freeze drying bottle is connected to the freeze drying machine pipeline through the bottle mouth or the freeze drying tray is placed inside the freeze drying machine for freeze drying. The freeze drying machine is a high vacuum environment, so water is continuously reduced by sublimation until it is completely removed, and the remaining solid is the purified target polypeptide.

[0186] The proportion of micropores with a pore size range below 40Å is less than 5%.

[0187] Furthermore, a high-precision mass flow meter is used to confirm the solvent ratio in the solvent recovery tank 17. The high-precision mass flow meter is arranged on the pipeline between the solvent recovery tank 17 and the mixing tank.

[0188] It is understandable that, by using the full-wavelength UV-visible detector 14 and the online ultra-high performance liquid chromatograph 18 simultaneously, the fraction flowing out of the chromatographic column 5 can be intelligently divided into two parts: the fraction that can directly recover the solvent (i.e., the fraction that meets the set ratio of the eluent flowing into the solvent recovery tank 17) and the fraction that can be recovered after the liquid is prepared (i.e., the fraction that does not meet the set ratio of the eluent flowing into the solvent recovery tank 17, the fraction that flows into the target pure product tank 10, the fraction that flows into the impure product recovery tank 11, and the fraction that flows into the waste liquid tank 20), so as to realize the maximum recovery and utilization of the solvent in the fraction, thereby reducing the waste of solvent and reducing labor costs. It can also reduce the number of collection tanks and save work surface space.

[0189] By recovering the solvent in the solvent recovery tank 17, the target pure product tank 10, the impure product recovery tank 11, and the waste liquid tank 20, the solvent recovery efficiency can be improved, the solvent loss can be reduced, the existing solvent storage can be reduced, the equipment investment and the floor space can be effectively reduced, and the safety and environmental risks can be reduced. Moreover, since the solvent post-processing volume is reduced, the processing cost of the waste solvent is reduced.

[0190] In order to achieve accurate separation of large molecular weight polypeptides (2000D-6000D), the present invention adopts the following key innovations in the purification device:

[0191] 1. For the first time, an accurate and fast online detection system including buffer tank switching and UHPLC was created to ensure that the purity of the fractions purified by the chromatographic column can be detected in a timely and accurate manner and to achieve automated collection; due to the accurate and fast detection of online UHPLC, the interval between each sampling detection can be compressed to within 1 minute, which not only ensures the accuracy of the purity of the collected products, but also minimizes the yield loss caused by the collection volume of a single fraction;

[0192] 2. Use a precision-controlled eluent infusion pump to ensure that the flow error is within 1.5%; and use a high-precision mass flow meter to accurately measure the flow online, and when the flow fluctuates, the control cabinet can be used to accurately adjust the flow in real time;

[0193] 3. The present invention also effectively reduces tailing, improves peak shape and separation effect by controlling the consistency and accuracy of the chromatographic column temperature and the eluent temperature (the temperature can be selected between 10°C and 30°C, and the temperature difference between the eluent and the chromatographic column is within ±3°C);

[0194] 4. The present invention adopts an axial pressure column with a cylinder-free design, which completely eliminates the risk of contaminating the purified sample due to oil leakage;

[0195] 5. The present invention creatively integrates 1) UV-visible online monitoring, 2) UHPLC online detection, 3) fraction collection automatic control, 4) solvent recovery control, and 5) online liquid preparation based on precise density monitoring into a system, and realizes closed-loop, precise intelligent control, thereby achieving accuracy, efficiency and stability in polypeptide purification.

[0196] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0197] It is not difficult for those skilled in the art to understand that the present invention includes any combination of the invention content and specific implementation methods of the above specification and the various parts shown in the drawings. Due to the limited space and to make the specification concise, the various schemes composed of these combinations are not described one by one. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

[0198] Although the embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and cannot be understood as limiting the present invention. Those skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and purpose of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A fully automatic chromatography purification system, comprising: The main device of the chromatography system, the online detection device, the automatic sample collection device and the control system, the control system is connected with the above devices, the connection includes strong current connection and weak current connection, and is characterized in that: The control system consists of a remote control center and a control cabinet to realize data transmission and electrical control; the remote control center communicates with the control cabinet to realize data transmission and command transmission with the control cabinet; The main device of the chromatography system includes a sample tank, a sample loading pump, an eluent tank, an infusion pump, and a chromatography column, wherein the sample tank and the eluent tank are respectively connected to the liquid inlet of the chromatography column through pipelines, and the liquid outlet of the chromatography column is connected to the online detection device and the automatic sample collection device. The fully automatic chromatography purification system is controlled by a control system; The online detection device comprises: an online ultra-high performance liquid chromatograph, an automatic sampling device and a data processing and data transmission device, wherein the automatic sampling device comprises a first collection buffer tank, a second collection buffer tank, an analytical sample sampler and a plurality of control valves; A main pipeline is provided at the outlet of the chromatographic column, and two branches formed by the end of the main pipeline are respectively connected to the first collection buffer tank and the second collection buffer tank, and valves are provided on both branches; the eluent enters the first collection buffer tank and the second collection buffer tank alternately through the valve; The first collection buffer tank and the second collection buffer tank are also connected to the analytical sample sampler through a pipeline, and the other end of the analytical sample sampler is connected to an online ultra-high performance liquid chromatograph, and the online ultra-high performance liquid chromatograph is used to detect the purity of the target substance in the fractions of the first collection buffer tank and the second collection buffer tank; the online detection device is also electrically connected to the control cabinet to provide the detection data to the control system; The automatic sample collection device comprises: a first collection buffer tank and a second collection buffer tank, a target pure product tank, an impure product recovery tank and a waste liquid tank; the control system controls the valves at the lower ends of the first collection buffer tank and the second collection buffer tank according to the detected target purity data in the first collection buffer tank and the second collection buffer tank, and respectively sends the qualified target pure product, the unqualified product and the waste liquid to the corresponding target pure product tank, the impure product recovery tank and the waste liquid tank; Specifically: the control system controls the valve switches of the first branch, the second branch, the third branch, the fourth branch, the fifth branch and the sixth branch according to the detection results of the online ultra-high performance liquid chromatograph, and the fractions automatically collected include the target pure product, the impure product that can be purified twice and the collection of waste liquid; The first branch is a connecting pipeline between the first collecting buffer tank and the target pure product tank, the second branch is a connecting pipeline between the first collecting buffer tank and the impure product recovery tank, the third branch is a connecting pipeline between the second collecting buffer tank and the target pure product tank, the fourth branch is a connecting pipeline between the second collecting buffer tank and the impure product recovery tank, the fifth branch is a connecting pipeline between the first collecting buffer tank and the waste liquid tank, and the sixth branch is a connecting pipeline between the second collecting buffer tank and the waste liquid tank.

2. The fully automatic chromatography purification system according to claim 1, characterized in that: The online detection device also includes a full-wavelength UV-visible detector, which is located on the main pipe of the chromatographic column eluent outlet and is used to detect whether the eluent in the main pipe contains a chromatographic peak of the target object. In addition to the two branches mentioned above, a seventh branch is formed at the end of the main pipe of the chromatographic column eluent outlet. The seventh branch is connected to a solvent recovery tank, and the other end of the solvent recovery tank is connected to a solvent buffer tank through a pipeline.

3. The fully automatic chromatography purification system according to claim 1, characterized in that: It also includes an automatic solvent recovery device, thereby forming a complete fully automatic continuous circulation purification system, which is characterized in that: the front end of the automatic solvent recovery device is connected to the target pure product tank, the impure product recovery tank and the waste liquid tank respectively, and the rear end is connected to the eluent tank, and the automatic solvent recovery device includes: a solvent mixing tank, a solvent buffer tank and a solvent tank group; the input end of the solvent buffer tank is connected to the target pure product tank, the impure product recovery tank and the waste liquid tank through a pipeline, and a pump, a valve and a reduced pressure distillation device are provided on the pipeline; the output end of the solvent buffer tank is connected to the first input end of the solvent mixing tank through a pipeline, and a high-precision flow meter is provided on the pipeline; the solvent tank group is connected to the second input end of the solvent mixing tank through a pipeline, and a pump and a valve are provided on the pipeline; the solvent mixing tank is also connected to the eluent tank through a pipeline.

4. The fully automatic chromatography purification system according to claim 3, characterized in that: The output end of the target pure product tank is connected to the solvent buffer tank through a pipeline, and the other output end of the target pure product tank is connected to the post-processing tank.

5. The fully automatic chromatography purification system according to claim 4, characterized in that: There is also a vacuum distillation device between the target pure product tank and the solvent buffer tank.

6. The fully automatic chromatography purification system according to claim 4, characterized in that: The concentrated solution is crystallized or freeze-dried in a post-processing tank to obtain a pure target product.

7. The fully automatic chromatography purification system according to claim 3, characterized in that: The first output end of the impure product recovery tank is connected to the solvent buffer tank through a pipeline, and the second output end is connected to the sample tank through a pipeline.

8. The fully automatic chromatography purification system according to claim 7, characterized in that: There is also a vacuum distillation device between the impure product recovery tank and the solvent mixing tank.

9. The fully automatic chromatography purification system according to claim 3, characterized in that: The input end of the solvent mixing tank is connected to a solvent tank group, and the solvent tank group is a combination of several solvent tanks according to the proportion of the eluent.

10. The fully automatic chromatography purification system according to claim 3, characterized in that: The recovered solvent is measured and calculated by a mass flow meter to obtain the corresponding component ratio, and the ratio is automatically adjusted to the ratio of the eluent in the mixing tank.

11. The fully automatic chromatography purification system according to claim 3, characterized in that: The first output end of the solvent recovery tank is connected to the eluent tank pipeline; the second output end of the solvent recovery tank is connected to the solvent buffer tank.

12. The fully automatic chromatography purification system according to claim 1, characterized in that: The chromatographic column is a dynamic axial pressurized chromatographic column.

13. The fully automatic chromatography purification system according to claim 1, characterized in that: The filler filled in the chromatographic column is a spherical chromatographic filler with a silica gel matrix; the filling length of the chromatographic column is 20cm-100cm.

14. The fully automatic chromatography purification system according to claim 13, characterized in that: In addition to the tail-sealing agent, the surface of the silica gel is bonded and modified with two or more silanes, one of which accounts for no less than 10%; one of the two silanes is a polar silane and the other is a non-polar silane, or the two are different non-polar silanes.

15. The fully automatic chromatography purification system according to claim 1, characterized in that: The eluent and the chromatographic column are temperature controlled within a temperature range of 10°C to 30°C; the temperature difference between the chromatographic column and the eluent is within ±3°C.

16. The fully automatic chromatography purification system according to claim 1, characterized in that: There are two eluent tanks, each containing eluents of different components or different component ratios. A high-pressure constant-current pump is provided on the connecting pipeline between the eluent tank and the chromatographic column. By controlling the flow ratio of the two high-pressure constant-current pumps respectively, online gradient mixing of the eluent is achieved, and gradient elution of the sample is performed.

17. The fully automatic chromatography purification system according to claim 1, characterized in that: Used for reverse phase purification of peptides, macrolides, and cyclosporine organic compounds.

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