Method for regulating and controlling concentration band width of target component in double-column circulation chromatography system

Abstract

The invention discloses a method for regulating and controlling the width of a concentration band of a target component in a double-column circulating chromatography system. The system adopted by the method comprises a first chromatographic column, a second chromatographic column and a valve assembly, after the raw materials are added into the system, an eluent flows in from an inlet of the first chromatographic column, after a certain time, the valve assembly is switched to enable the eluent to flow in from an inlet of the second chromatographic column, and after a certain time, the valve assembly is switched to enable the eluent to flow in from the inlet of the first chromatographic column, so that the adding position of the eluent is changed; one switching period is formed by changing the position once; the chromatographic column into which an eluent flows is called as an upstream column, and the other chromatographic column is called as a downstream column; in the operation process, a modifier with the elution capacity weaker than that of the eluent is added from the downstream column in a part of time, and a diluent with the elution capacity not weaker than that of the eluent is added from an inlet of the downstream column in another part of time, or neither the modifier nor the diluent is added; the method has the advantage that the concentration band width of the target component in the double-column circulating chromatographic system can be flexibly regulated and controlled.

Description

Technical Field

[0001] The present application relates to the technical field of chromatographic separation, and in particular to a method for regulating the concentration band width of a target component in a double-column circulating chromatographic system. Background Art

[0002] Double column circulation chromatography is an efficient chromatographic separation method. Its basic principle is as follows Figure 1-2 As shown, two chromatographic columns, namely the first chromatographic column 1' and the second chromatographic column 2', are connected by a valve assembly 3'. By switching the valves, the upstream and downstream positions of the chromatographic columns are changed, so that the concentration bands of the components to be separated circulate in a closed loop formed by the two columns: Figure 1 As shown, the raw material liquid first enters the first chromatographic column 1', and when the target component concentration spectrum band all leaves the first chromatographic column 1' and enters the second chromatographic column 2', the valve is switched, as shown in the attached Figure 2 As shown, at this time, the concentration band will pass through the second chromatographic column 2' and re-enter the first chromatographic column 1'; when the target component concentration band completely leaves the second chromatographic column 2' and enters the first chromatographic column 1', the column is switched again, and the cycle is repeated to ensure that the target component concentration band always circulates in the two columns, which is equivalent to extending the chromatographic column, and the system pressure will not increase. Therefore, the separation ability of the double-column circulation chromatography is very strong, and even for some separation systems with very small separation factors, the circulation chromatography can well separate them. However, when the target component circulates in the chromatographic column, its concentration band will continue to expand spontaneously due to the influence of factors such as axial diffusion, mass transfer resistance and nonlinear adsorption, which can easily lead to the target component not being completely separated, but its concentration band has occupied the entire separation space. At this time, the front edge of the target component concentration band catches up with its trailing edge, resulting in the unsustainable circulation operation. Although this dilemma can be solved by increasing the column length or reducing the processing volume, the separation efficiency is reduced.

[0003] In summary, the key to improving the efficiency of circulating chromatography separation is to prevent or slow down the band expansion. For example, Patent ZL201811024239.7 proposes a new type of dual-column circulating chromatography separation system and method. This technology reduces the elution capacity of the mobile phase inside the downstream column by always introducing a modifier between the two chromatographic columns. In the process of the target component concentration band migrating from the upstream chromatographic column to the downstream chromatographic column, the migration rate of the concentration band front entering the downstream chromatographic column first will decrease, and the concentration band will shrink as a result. This special concentration band shrinkage effect can counteract the spontaneous expansion of the concentration band.

[0004] However, in certain separation situations, the composition of the mobile phase is very sensitive to the elution ability. If the content of solvents with weak elution ability in the mobile phase is slightly reduced, the migration rate of the concentration band will drop sharply. At this time, if the technical solution of patent ZL201811024239.7 is still used, it will easily lead to excessive contraction of the concentration band of the target component, reducing its width and increasing its height, that is, the concentration of the target component in the column increases, thereby increasing the column pressure (when the pressure is too high, it may also cause the infusion line to rupture), and finally the target component is analyzed out and blocks the column. For example, when using reverse phase chromatography to separate and purify biological products such as peptides, proteins and nucleic acids, the water content in the mobile phase is very sensitive to the migration rate of the concentration band of the target. If water is used as a modifier and is continuously added to the system from the connection between the two columns during the entire separation process, that is, the flow state of the medium is like Figure 3 As shown, the water content in the downstream is higher than that in the upstream (X 下 >X 上 ), the difference in migration rate between the front and back edges of the band will be very large, and the shrinkage effect will be much stronger than the spontaneous expansion of the band, resulting in the shrinkage of the target band and the increase of the target concentration in the column, which can easily lead to an increase in system pressure and even the precipitation of the target component due to excessive concentration. Although the difference in water content between the upstream and downstream can be reduced by reducing the flow rate of the modifier, thereby reducing the difference in migration rate between the front and back edges of the band, the reduction of the shrinkage effect of the target component band is limited, and the column pressure and product precipitation may still be increased due to continuous shrinkage. Therefore, in the case where the composition of the mobile phase significantly affects its elution ability, it is necessary to rationally utilize the shrinkage effect to more flexibly regulate the concentration band width of the components to be separated. Summary of the invention

[0005] In view of the deficiencies of the prior art, the present application provides a method for flexibly regulating the concentration band width of a target component in a dual-column circulating chromatography system.

[0006] In order to solve the above technical problems, the technical solution provided by the present application is implemented as follows: a method for regulating the concentration band width of a target component in a dual-column circulating chromatography system, the system used in the method includes a first chromatographic column, a second chromatographic column and a valve assembly; after the raw material is added to the system, the eluent flows into the system from the inlet of the first chromatographic column, after a certain period of time, the valve assembly is switched to allow the eluent to flow into the system from the inlet of the second chromatographic column, and after a certain period of time, the valve assembly is switched to allow the eluent to flow into the system from the inlet of the first chromatographic column, and the eluent addition position is changed reciprocally in this way; each change of position is called a switching cycle; the chromatographic column into which the eluent flows is called the upstream column, and the other chromatographic column is called the downstream column; during the operation, a modifier with an elution ability weaker than that of the eluent is added from the inlet of the downstream column for a part of the time, and a diluent with an elution ability not weaker than that of the eluent is added from the inlet of the downstream column for another part of the time, or neither the modifier nor the diluent is added.

[0007] Furthermore, in order to make the elution ability of the modifier weaker than that of the eluent, the volume percentage of the solvent with weak elution ability in the modifier can be higher than the volume percentage of the solvent with weak elution ability in the eluent.

[0008] Furthermore, in order to make the elution ability of the diluent not weaker than that of the eluent, the volume percentage of the solvent with weak elution ability in the diluent is not higher than the volume percentage of the solvent with weak elution ability in the eluent.

[0009] Furthermore, in order to add a modifier having an elution ability weaker than that of the eluent from the downstream column inlet during a part of the time, and not add a modifier but add a diluent having an elution ability not weaker than that of the eluent from the downstream column inlet during another part of the time, or neither add a modifier nor a diluent, the following method can be adopted: in a part of the switching cycle, add the modifier from the downstream column inlet; and in another part of the switching cycle, do not add the modifier but add the diluent from the downstream column inlet, or neither add the modifier nor the diluent.

[0010] Furthermore, in order to add a modifier having an elution ability weaker than that of the eluent from the downstream column inlet during a part of the time, and not add a modifier but add a diluent having an elution ability not weaker than that of the eluent from the downstream column inlet during another part of the time, or to add neither a modifier nor a diluent, the following method can be adopted: in a part of the switching cycle, the modifier is added from the downstream column inlet; and in another part of the switching cycle, the diluent is added instead of the modifier from the downstream column inlet.

[0011] Furthermore, in order to add a modifier having an elution ability weaker than that of the eluent from the downstream column inlet during a part of the time, and not add a modifier but add a diluent having an elution ability not weaker than that of the eluent from the downstream column inlet during another part of the time, or to add neither a modifier nor a diluent, the following method can be adopted: in a part of the switching cycle, the modifier is added from the downstream column inlet; and in another part of the switching cycle, neither the modifier nor the diluent is added from the downstream column inlet.

[0012] Furthermore, in order to add a modifier having an elution ability weaker than that of the eluent from the downstream column inlet during a part of the time, and not add a modifier but a diluent having an elution ability not weaker than that of the eluent from the downstream column inlet during another part of the time, or to add neither the modifier nor the diluent, the following method can also be adopted: during a part of each switching cycle, the modifier is added from the downstream column inlet; and during another part of each switching cycle, no modifier is added but a diluent is added from the downstream column inlet, or to add neither the modifier nor the diluent.

[0013] Furthermore, in order to add a modifier having an elution ability weaker than that of the eluent from the downstream column inlet during a part of the time, and not add a modifier but add a diluent having an elution ability not weaker than that of the eluent from the downstream column inlet during another part of the time, or to add neither the modifier nor the diluent, the following method can also be adopted: during a part of each switching cycle, the modifier is added from the downstream column inlet; and during another part of each switching cycle, the diluent is added instead of the modifier from the downstream column inlet.

[0014] Furthermore, in order to add a modifier having an elution ability weaker than that of the eluent from the downstream column inlet during a part of the time, and not add a modifier but add a diluent having an elution ability not weaker than that of the eluent from the downstream column inlet during another part of the time, or to add neither a modifier nor a diluent, the following method can also be adopted: during a part of each switching cycle, the modifier is added from the downstream column inlet; and during another part of each switching cycle, neither the modifier nor the diluent is added from the downstream column inlet.

[0015] Furthermore, the valve assembly is a four-way valve, a six-way valve or more.

[0016] Furthermore, the inlet and outlet of the upstream column and the downstream column are provided with medium delivery pipelines, and the modifier, diluent, raw material liquid and eluent are respectively delivered to the corresponding chromatographic columns through the medium delivery pipelines by corresponding delivery pumps.

[0017] Advantages and beneficial effects of this application:

[0018] 1. In dual-column circulation chromatography, the concentration band of the separated components circulates in a closed loop formed by two columns. During the migration of the band, the concentration band will continue to expand spontaneously due to the influence of factors such as axial diffusion, mass transfer resistance and nonlinear adsorption. This phenomenon may be called the band expansion effect. If the elution ability of the mobile phase inside the upstream column is stronger than the elution ability of the mobile phase inside the downstream column, when the concentration band of the component to be separated migrates from the upstream column to the downstream column, the migration rate of the trailing edge of the band is faster than the migration rate of its leading edge, and the band will shrink. This phenomenon may be called the band shrinkage effect. The shrinkage effect and the expansion effect counteract each other and control the width of the component concentration band. The more sensitive the mobile phase composition is to the elution ability, the greater the difference in migration rate between the front and rear edges of the band, and the stronger the shrinkage effect. When the shrinkage effect is much stronger than the expansion effect, the concentration band will become narrower and higher, and the separated component will be concentrated and finally precipitated. Since the expansion effect is spontaneous and difficult to control, the key is how to adjust the contraction effect, so as to more effectively control the band width to avoid the increase of column pressure and the analysis of the separated components. The existing patent ZL201811024239.7 only provides a technical solution by always introducing a modifier between the upstream and downstream chromatographic columns. Although the intensity of the contraction effect can be adjusted by changing the flow rate of the modifier, this method is relatively single and limited. It is difficult to cope with some extreme cases where the mobile phase composition is sensitive to the elution ability, and thus cannot solve the problem that the reduction of the contraction effect of the target component band is limited, and the contraction effect may still increase due to continuous contraction. The technical problems of adding column pressure and precipitating products; based on this, the technical solution of the present application proposes two new ideas to control the width of the concentration band, namely, adjusting the action time of the shrinkage effect (i.e., the state without adding diluent as described below), and alternately introducing a modifier and a diluent into the downstream column, and controlling the elution ability of the diluent to be not weaker than that of the eluent. Therefore, after the diluent is introduced between the two columns, when the concentration band moves from the upstream chromatographic column to the downstream chromatographic column, the migration rate of the band front will increase, thereby making the band quickly widen and shorten, which is equivalent to introducing a band dilution effect, which can prevent the band from excessive shrinkage more effectively than the expansion effect; specifically, it is achieved through the following 4 measures:

[0019] (1) In one part of the switching cycle, a modifier with a weaker elution capacity than the eluent is added from the downstream column inlet, while in another part of the switching cycle, no modifier is added from the downstream column inlet but a diluent with an elution capacity not weaker than the eluent is added. Figure 6 Therefore, in a part of the switching cycle, the volume percentage of the solvent with weak elution ability in the mobile phase of the downstream chromatographic column is higher than the volume percentage of the solvent with weak elution ability in the mobile phase of the upstream chromatographic column (i.e., X 下 >X 上), resulting in a band shrinkage effect; in the other part of the switching cycle, the volume percentage of the solvent with weak elution ability in the mobile phase of the downstream chromatographic column is not higher than the volume percentage of the solvent with weak elution ability in the mobile phase of the upstream chromatographic column (i.e., X 下 ≤X 上 ), resulting in a band dilution effect.

[0020] (2) A modifier with an elution capacity weaker than that of the eluent is added from the downstream column inlet during a portion of the switching cycle, while neither a modifier nor a diluent with an elution capacity not weaker than that of the eluent is added from the downstream column inlet during another portion of the switching cycle. Figure 7 Therefore, in a part of the switching cycle, the volume percentage of the solvent with weak elution ability in the mobile phase of the downstream chromatographic column is higher than the volume percentage of the solvent with weak elution ability in the mobile phase of the upstream chromatographic column (i.e., X 下 >X 上 ), resulting in a band shrinkage effect; in the other part of the switching cycle, the volume percentage of the solvent with weak elution ability in the mobile phase in the downstream chromatographic column is equal to the volume percentage of the solvent with weak elution ability in the mobile phase in the upstream chromatographic column (i.e. X 下 =X 上 ), so no band shrinkage effect occurs.

[0021] (3) During a portion of each switching cycle, a modifier having an elution capacity weaker than that of the eluent is added from the downstream column inlet, and during another portion of each switching cycle, no modifier is added but a diluent having an elution capacity not weaker than that of the eluent is added from the downstream column inlet. Figure 8 Therefore, during a portion of each switching cycle, the volume percentage of the solvent with weak elution ability in the mobile phase of the downstream chromatographic column is higher than the volume percentage of the solvent with weak elution ability in the mobile phase of the upstream chromatographic column (i.e., X 下 >X 上 ), resulting in a band shrinkage effect; and in another part of each switching cycle, the volume percentage of the solvent with weak elution ability in the mobile phase of the downstream chromatographic column is not higher than the volume percentage of the solvent with weak elution ability in the mobile phase of the upstream chromatographic column (i.e., X 下 ≤X 上 ), resulting in a band dilution effect.

[0022] (4) During a portion of each switching cycle, a modifier having an elution capacity weaker than that of the eluent is added from the downstream column inlet, while during another portion of each switching cycle, neither the modifier nor the diluent having an elution capacity not weaker than that of the eluent is added from the downstream column inlet. Fig. 9Therefore, during a portion of each switching cycle, the volume percentage of the solvent with weak elution ability in the mobile phase of the downstream chromatographic column is higher than the volume percentage of the solvent with weak elution ability in the mobile phase of the upstream chromatographic column (i.e., X 下 >X 上 ), resulting in a band shrinkage effect; and in the other part of each switching cycle, the volume percentage of the solvent with weak elution ability in the mobile phase of the downstream chromatographic column is equal to the volume percentage of the solvent with weak elution ability in the mobile phase of the upstream chromatographic column (i.e. X 下 =X 上 ), so no band shrinkage effect occurs.

[0023] 2. This application is the first to use a two-column cyclic separation process. The composition of the mobile phase is very sensitive to the elution ability. If the content of the solvent with weak elution ability in the mobile phase is slightly reduced, the migration rate of the concentration band will drop sharply. At this time, if the technical solution of patent ZL201811024239.7 is still used, the concentration band of the target component will easily shrink excessively, reducing its width and increasing its height, that is, the concentration of the separated component in the column increases, thereby increasing the column pressure, and finally the separated group is analyzed out and the column is blocked from the technical problem perspective. By providing a method for regulating the width of the concentration band of the target component in the double-column cyclic chromatography, the width of the concentration band of the target component can be more effectively adjusted from two aspects: adjusting the action time of the band shrinkage effect and introducing the dilution effect. Therefore, while giving full play to the advantages of the existing patent technology, it can also avoid the problems of excessive shrinkage of the concentration band of the target component causing increased column pressure and analysis of the target group, effectively making up for the shortcomings of the prior art, and at the same time solving the technical problem of excessive shrinkage of the concentration band of the target component. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a structural schematic diagram of the first state of the double-column circulation chromatography system in the prior art.

[0025] Figure 2 It is a structural schematic diagram of the second state of the double-column circulation chromatography system in the prior art.

[0026] As shown in the attached drawings: 1'. First chromatographic column, 2'. Second chromatographic column, 3'. Valve assembly.

[0027] Figure 3 The figure is a graph showing the relationship between the volume percentage of the solvent with weak elution ability in the mobile phase of the downstream chromatographic column of the prior art and time; wherein the ordinate is the volume percentage of the solvent with weak elution ability in the mobile phase, the abscissa t only refers to a time, and X 下 The corresponding dotted line is the water content of the downstream column, X 上 The corresponding dot-dashed line is the upstream water content.

[0028] Figure 4 This is a schematic structural diagram of the first state of the double-column circulation chromatography system of the present application.

[0029] Figure 5 This is a schematic structural diagram of the second state of the double-column circulation chromatography system of the present application.

[0030] As shown in the attached figure: 1. the first chromatographic column, 2. the second chromatographic column, 3. the first valve assembly, 4. the second valve assembly, 5. the raw material liquid pump, 6. the eluent pump, 7. the modifier pump, 8. the diluent pump, 9. the detector, 10. the medium delivery pipeline.

[0031] Figure 6 : is a graph showing the change in volume percentage of the solvent with weak elution ability in the mobile phase of the downstream chromatographic column of the present application over time (the proportion of the mobile phase of the downstream column switched during the entire cycle); wherein the ordinate is the volume percentage of the solvent with weak elution ability in the mobile phase, the abscissa t only refers to a time, and X 下 The corresponding dotted line is the volume percentage of the solvent with weak elution ability in the downstream mobile phase, X 上 The corresponding dotted line is the volume percentage of the solvent with weak elution ability in the upstream mobile phase; X 下 The whole cycle adjustment is shown, in which the modifier pump is turned on and the diluent pump is turned off in the 1st, 3rd, 5th and 7th switching cycles, so X 下 >X 上 , and in the 2nd, 4th, 6th and 8th switching cycles, the modifier pump is turned off and the diluent pump is turned on. Therefore, in these 4 switching cycles, X 下 ≤X 上 .

[0032] Figure 7 : is a graph showing the change in volume percentage of the solvent with weak elution ability in the mobile phase of the downstream chromatographic column of the present application over time (the proportion of the mobile phase of the downstream column switched during the entire cycle); wherein the ordinate is the volume percentage of the solvent with weak elution ability in the mobile phase, the abscissa t only refers to a time, and X 下 The corresponding dotted line is the volume percentage of the solvent with weak elution ability in the downstream mobile phase, X 上 The corresponding dotted line is the volume percentage of the solvent with weak elution ability in the upstream mobile phase; X 下 The whole cycle adjustment is shown, in which the modifier pump is turned on and the diluent pump is turned off in the 1st, 3rd, 5th and 7th switching cycles, so X 下 >X 上 , and the modifier pump and diluent pump are turned off in the 2nd, 4th, 6th and 8th switching cycles, so X 下 =X上 .

[0033] Figure 8 This is a graph showing the change in the volume percentage of the solvent with weak elution ability in the mobile phase of the downstream chromatographic column of the present application over time (the ratio of the downstream column mobile phase switched at different time periods within a cycle). The ordinate is the volume percentage of the solvent with weak elution ability in the mobile phase, the abscissa t only refers to a time, and X 下 The corresponding dotted line is the volume percentage of the solvent with weak elution ability in the downstream mobile phase, X 上 The corresponding dotted line is the volume percentage of the solvent with weak elution ability in the upstream mobile phase; during a part of each switching cycle, the modifier pump is turned on and the diluent pump is turned off, so X 下 >X 上 , and during the other part of each switching cycle, the modifier pump is turned off and the diluent pump is turned on, so X 下 ≤X 上 .

[0034] Fig. 9 This is a graph showing the change in the volume percentage of the solvent with weak elution ability in the mobile phase of the downstream chromatographic column of the present application over time (the ratio of the downstream column mobile phase switched at different time periods within a cycle). The ordinate is the volume percentage of the solvent with weak elution ability in the mobile phase, the abscissa t only refers to a time, and X 下 The corresponding dotted line is the volume percentage of the solvent with weak elution ability in the downstream mobile phase, X 上 The corresponding dotted line is the volume percentage of the solvent with weak elution ability in the upstream mobile phase; during a part of each switching cycle, the modifier pump is turned on and the diluent pump is turned off, so X 下 >X 上 , and the modifier pump and diluent pump are turned off during another part of each switching cycle, so X 下 =X 上 .

[0035] Fig.10 This is a schematic structural diagram of the first state of the double-column circulation chromatography system used in Comparative Example 1-2.

[0036] Fig.11 This is a schematic structural diagram of the second state of the double-column circulation chromatography system used in Comparative Example 1-2. DETAILED DESCRIPTION

[0037] The present application is further described in detail below through examples, but the present application is not limited to the following examples.

[0038] The following uses the separation and purification of semaglutide as an example to explain in detail the technical solution of the present application. The semaglutide raw material is provided by Tianjin Aonuo Technology Co., Ltd. On a carbon octadecyl bonded silica gel column, acetonitrile / water / trifluoroacetic acid (500 / 500 / 1, v / v / v) is used as the mobile phase. Before and after the main peak of semaglutide, there are several front and rear impurity peaks, and the area percentage of each impurity peak is higher than 0.5%. The present application provides a method for regulating the width of the concentration band of the target component in a double-column circulating chromatography. While giving full play to the advantages of the existing patented technology, it avoids problems such as excessive shrinkage of the concentration band of the target component, resulting in increased column pressure and analysis of the target group.

[0039] 1. Double column switching circulation chromatography separation system

[0040] The components of the double-column circulating chromatographic separation system of this application are provided by Tianjin Aonuo Technology Co., Ltd., including: two C18 chromatographic columns (filler particle size 10μm; column inner diameter 1cm, column length 15cm), two two-position four-way valve assemblies, two detectors, and four infusion pumps. Figure 4-5 As shown, two chromatographic columns, a valve assembly infusion pump and a detector are connected as shown in the figure, wherein the two chromatographic columns are set as a first chromatographic column 1 and a second chromatographic column 2, and the two chromatographic columns are called the upstream chromatographic column or the downstream chromatographic column in turn according to the switching result; wherein the four-way valve between the raw liquid inlet and the two chromatographic columns is set as a first valve assembly 3, and the other is a second valve assembly 4, and four delivery pumps are defined as a raw liquid pump 5, an eluent pump 6, a modifier pump 7 and a diluent pump 8 according to the medium to be delivered; where medium flow is required between various components, a medium delivery pipeline 10 is set to achieve connection between each other and delivery of the medium; and a detector 9 is also set on the medium delivery pipeline between the first chromatographic column 1 and the second valve assembly 4, and between the second chromatographic column 2 and the second valve assembly 4, respectively, for detecting the development and changes of the separation component spectrum band, so as to effectively monitor the effective operation of the separation system formed by the method of the present application.

[0041] In the specific operation process, the upstream and downstream positions of the two chromatographic columns can be changed alternately by switching two four-way valves. In each switching cycle, the chromatographic column corresponding to the eluent added from the inlet is called the upstream column, and the other chromatographic column is the downstream column, that is, the chromatographic column into which the newly added eluent flows first is the upstream column, and the other is the downstream column; the two detectors 9 at the tail of the two columns can monitor the development and changes of the separated component bands; four pumps are used to transport raw material liquid, eluent, modifier and diluent respectively. The eluent is acetonitrile / water / trifluoroacetic acid (volume ratio: 500 / 500 / 1, v / v / v), the modifier is water / trifluoroacetic acid (volume ratio: 500 / 1, v / v), and the diluent is acetonitrile / water / trifluoroacetic acid (volume ratio: 550 / 500 / 1, v / v / v). The semaglutide raw material was dissolved in acetonitrile / water / trifluoroacetic acid (500 / 500 / 1, v / v / v) to prepare a raw material solution with a total concentration of 25 mg / mL.

[0042] 2. Finished product inspection

[0043] The purity of the product was analyzed by high performance liquid chromatography. The high performance liquid chromatography system was provided by Beijing Aonuo Technology Co., Ltd. Liquid chromatography analysis conditions: the chromatographic column was an American Phenomenex Knitex XB-18 column (4.6×150 mm, 5 μm), the mobile phase was acetonitrile / water, gradient elution, flow rate was 1.0 mL / min, the detection wavelength was 210 nm, and the column temperature was 60°C.

[0044] Example 1 - Separation of semaglutide using a dual-column circulating chromatography separation system

[0045] At room temperature, add 60mL of raw material liquid into the system through the raw material liquid pump; then, start the eluent pump at the same time and add the eluent into the system at a flow rate of 4mL / min. After 10 minutes, the two two-position four-way valves are switched at the same time, so that the two chromatographic columns exchange upstream and downstream positions, and the eluent still flows into the system at a flow rate of 4mL / min. This cycle is repeated every 10 minutes, for a total of 8 switches, and a total of 8 switching cycles. In the first 5 minutes of each switching cycle, turn on the modifier pump (turn off the diluent pump) with a flow rate of 0.2mL / min, and turn off the modifier pump (turn off the diluent pump) in the next 5 minutes. For details, please refer to the attached Fig. 9 As shown, the volume percentage of the solvent with strong elution ability in the mobile phase of the downstream chromatographic column changes with time. During the separation process, the system pressure did not change much and was maintained at 5MPa. After running 8 switching cycles, the collected semaglutide had a purity of >98% and a concentration of 26mg / mL.

[0046] Example 2 - Separation of semaglutide using a dual-column circulating chromatography separation system

[0047] At room temperature, add 60mL of raw material liquid into the system through the raw material liquid pump; then, start the eluent pump at the same time and add the eluent into the system at a flow rate of 4mL / min. After 10 minutes, the two two-position four-way valves are switched at the same time, so that the two chromatographic columns exchange upstream and downstream positions, and the eluent still flows into the system at a flow rate of 4m / min. This cycle is repeated every 10 minutes, for a total of 8 switches, and a total of 8 switching cycles. In the first 8 minutes of each switching cycle, turn on the modifier pump (turn off the diluent pump) with a flow rate of 0.2mL / min, and in the next 2 minutes, turn off the modifier pump and turn on the diluent pump with a flow rate of 0.1mL / min; refer to the attached for details. Figure 8 As shown, the volume percentage of the solvent with strong elution ability in the mobile phase of the downstream chromatographic column changes with time. During the separation process, the system pressure did not change much and was maintained at 4.8-4.9MPa. After running 8 switching cycles, the collected semaglutide had a purity of >98% and a concentration of 25mg / mL.

[0048] Example 3 - Separation of semaglutide using a dual-column circulating chromatography separation system

[0049] At room temperature, 60 mL of raw material liquid is added to the system through the raw material liquid pump; then, the eluent pump is started at the same time to add the eluent to the system at a flow rate of 4 mL / min. After 10 minutes, the two two-position four-way valves are switched at the same time, so that the two chromatographic columns exchange upstream and downstream positions, and the eluent still flows into the system at a flow rate of 4 m / min. This cycle is repeated every 10 minutes, for a total of 8 times, and a total of 8 switching cycles. Specifically, as shown in the attached Figure 7 As shown, the modifier pump was turned on (the diluent pump was turned off) in the 1st, 3rd, 5th and 7th switching cycles, with a flow rate of 0.2 mL / min; the modifier pump was turned off (the diluent pump was turned off) in the 2nd, 4th, 6th and 8th switching cycles. During the separation process, the system pressure did not change much and was maintained at 5.1 MPa. After running 8 switching cycles, the collected semaglutide had a purity of >98% and a concentration of 25 mg / mL.

[0050] Example 4 - Separation of semaglutide using a dual-column circulating chromatography separation system

[0051] At room temperature, 60 mL of raw material liquid is added to the system through the raw material liquid pump; then, the eluent pump is started at the same time to add the eluent to the system at a flow rate of 4 mL / min. After 10 minutes, the two two-position four-way valves are switched at the same time, so that the two chromatographic columns exchange upstream and downstream positions, and the eluent still flows into the system at a flow rate of 4 m / min. This cycle is repeated every 10 minutes, for a total of 8 times, and a total of 8 switching cycles. Specifically, as shown in the attached Figure 6As shown, the modifier pump was turned on (the diluent pump was turned off) in the 1st, 3rd, 5th and 7th switching cycles, with a flow rate of 0.2 mL / min; the modifier pump was turned off in the 2nd, 4th, 6th and 8th switching cycles, and the diluent pump was turned on with a flow rate of 0.1 mL / min. During the separation process, the system pressure did not change much and was maintained at 5.1 MPa. After running 5 switching cycles, the collected semaglutide had a purity of >98% and a concentration of 24 mg / mL.

[0052] Comparative Example 1 - Separation of semaglutide using a dual-column circulating chromatography separation system (modifier is always introduced)

[0053] At room temperature, 60 mL of raw material solution was added to the system via the raw material solution pump; then, the eluent pump and the modifier pump were started at the same time, and the eluent and modifier were added to the system at a flow rate of 4 mL / min and 0.2 mL / min. Fig.10 and 11 As shown, it includes a first chromatographic column 1' and a second chromatographic column 2', a valve assembly 3', a raw liquid pump 5' and an eluent pump 6'. The modifier is always introduced through the modifier pump 7' on the connecting section of the first chromatographic column 1' and the second chromatographic column 2'. After 10 minutes, the two two-position four-way valves are switched at the same time, so that the two chromatographic columns exchange upstream and downstream positions with each other, and the eluent and modifier still flow into the system at a flow rate of 4m / min and 0.2mL / min. This cycle is repeated every 10 minutes. During the separation process, the system pressure gradually increased. Finally, when the fifth cycle was running to 8.2 minutes, the system pressure exceeded 13MPa. In order to prevent the pipeline from rupturing, the test was terminated in advance. The purity of the semaglutide finally collected was 94% and the concentration was 45mg / mL. Comparative Example 2 - Separation of semaglutide using a dual-column circulating chromatography separation system (reducing the amount of raw liquid and always introducing the modifier)

[0054] At room temperature, 30 mL of raw material solution was added to the system via the raw material solution pump; then, the eluent pump and the modifier pump were started at the same time, and the eluent and modifier were added to the system at a flow rate of 4 mL / min and 0.2 mL / min. Fig.10 and 11As shown, it includes a first chromatographic column 1' and a second chromatographic column 2', a valve assembly 3', a raw liquid pump 5' and an eluent pump 6', and a modifier is always introduced into the connecting section of the first chromatographic column 1' and the second chromatographic column 2' through a modifier pump 7'; after 10 minutes, two two-position four-way valves are switched at the same time, so that the two chromatographic columns exchange upstream and downstream positions, and the eluent and modifier still flow into the system at a flow rate of 4m / min and 0.2mL / min. This cycle is repeated, switching once every 10 minutes, a total of 8 times, and a total of 8 switching cycles. During the separation process, the system pressure gradually increased, and when it finally ran to the 8th cycle, the system pressure exceeded 8MPa. The purity of the semaglutide finally collected was 98.2%, and the concentration was 30mg / mL.

[0055] Through the results of the above-mentioned embodiments and comparative embodiments, it can be concluded that the method for regulating the band width of the target component proposed in the present application can effectively avoid the increase of column pressure due to excessive shrinkage of the target component while maintaining the high separation ability of the cyclic chromatography (the column pressure of the present application is about 5.0 MPa, while that of the comparative example can even be as high as 13 MPa, and basically it must be above 8.0 MPa), and even cause the target component to be analyzed out and block the column.

[0056] The above embodiments are intended to illustrate and further explain the present application, rather than to limit the present application. Any modifications made within the spirit and scope of protection of the present application shall fall within the protection scope of the present application.

Claims

1. A method for regulating the concentration band width of a target component in a dual-column circulation chromatography system, characterized in that: The system adopted by the method includes a first chromatographic column, a second chromatographic column and a valve assembly; after the raw material is added to the system, the eluent flows into the system from the inlet of the first chromatographic column, after a certain period of time, the valve assembly is switched to allow the eluent to flow into the system from the inlet of the second chromatographic column, and after another certain period of time, the valve assembly is switched to allow the eluent to flow into the system from the inlet of the first chromatographic column, and the position of adding the eluent is changed repeatedly in this way; each change of position is called a completion of a switching cycle; the chromatographic column into which the eluent flows is called an upstream column, and the other chromatographic column is called a downstream column; during the operation, a modifier with an elution ability weaker than that of the eluent is added from the inlet of the downstream column for a part of the time, and a diluent with an elution ability not weaker than that of the eluent is added from the inlet of the downstream column for another part of the time, and no modifier is added from the inlet of the downstream column but a diluent with an elution ability not weaker than that of the eluent is added, or neither the modifier nor the diluent is added.

2. The method for regulating the concentration band width of a target component in a dual-column circulation chromatography system according to claim 1, characterized in that: In order to make the elution ability of the modifier weaker than that of the eluent, the volume percentage of the solvent with weak elution ability in the modifier is higher than the volume percentage of the solvent with weak elution ability in the eluent.

3. The method for regulating the concentration band width of a target component in a dual-column circulation chromatography system according to claim 1, characterized in that: In order to make the elution ability of the diluent not weaker than that of the eluent, the volume percentage of the solvent with weak elution ability in the diluent is not higher than the volume percentage of the solvent with weak elution ability in the eluent.

4. The method for regulating the concentration band width of a target component in a dual-column circulation chromatography system according to claim 1, characterized in that: In order to allow a modifier having an elution ability weaker than that of the eluent to be added from the downstream column inlet during a part of the time, while no modifier is added from the downstream column inlet during another part of the time but a diluent having an elution ability not weaker than that of the eluent is added, or neither the modifier nor the diluent is added, the following method is adopted: in a part of the switching cycle, the modifier is added from the downstream column inlet; and in another part of the switching cycle, no modifier is added but a diluent is added, or neither the modifier nor the diluent is added, from the downstream column inlet.

5. The method for regulating the concentration band width of a target component in a dual-column circulation chromatography system according to claim 4, characterized in that: In order to add a modifier whose elution ability is weaker than that of the eluent from the downstream column inlet during a part of the time, and add a diluent whose elution ability is not weaker than that of the eluent from the downstream column inlet during another part of the time without adding the modifier, or to add neither the modifier nor the diluent, the following method is adopted: in a part of the switching cycle, the modifier is added from the downstream column inlet; and in another part of the switching cycle, the diluent is added from the downstream column inlet without adding the modifier.

6. The method for regulating the concentration band width of a target component in a dual-column circulation chromatography system according to claim 4, characterized in that: In order to allow a modifier having an elution ability weaker than that of the eluent to be added from the downstream column inlet during a part of the time, while no modifier is added from the downstream column inlet during another part of the time but a diluent having an elution ability not weaker than that of the eluent is added, or neither the modifier nor the diluent is added, the following method is adopted: in a part of the switching cycle, the modifier is added from the downstream column inlet; and in another part of the switching cycle, no modifier and no diluent are added from the downstream column inlet.

7. The method for regulating the concentration band width of a target component in a dual-column circulation chromatography system according to claim 1, characterized in that: In order to add a modifier having an elution ability weaker than that of the eluent from the downstream column inlet during a part of the time, and add a diluent having an elution ability not weaker than that of the eluent from the downstream column inlet in another part of the time without adding the modifier, or add neither the modifier nor the diluent, the following method is adopted: during a part of the time of each switching cycle, the modifier is added from the downstream column inlet; and during the other part of the time of each switching cycle, the diluent is added instead of the modifier, or neither the modifier nor the diluent is added from the downstream column inlet.

8. The method for regulating the concentration band width of a target component in a dual-column circulation chromatography system according to claim 7, characterized in that: In order to add a modifier having an elution ability weaker than that of the eluent from the downstream column inlet during a part of the time, and add a diluent having an elution ability not weaker than that of the eluent from the downstream column inlet during another part of the time without adding the modifier, or to add neither the modifier nor the diluent, the following method is adopted: during a part of each switching cycle, the modifier is added from the downstream column inlet; and during another part of each switching cycle, the diluent is added from the downstream column inlet without adding the modifier.

9. The method for regulating the concentration band width of a target component in a dual-column circulation chromatography system according to claim 7, characterized in that: In order to allow a modifier having an elution ability weaker than that of the eluent to be added from the downstream column inlet during a part of the time, while no modifier is added from the downstream column inlet during another part of the time but a diluent having an elution ability not weaker than that of the eluent is added, or neither the modifier nor the diluent is added, the following method is adopted: during a part of each switching cycle, the modifier is added from the downstream column inlet; and during another part of each switching cycle, no modifier and no diluent are added from the downstream column inlet.

10. The method for regulating the concentration band width of a target component in a dual-column circulation chromatography system according to claim 1, characterized in that: The valve assembly is a four-way valve, a six-way valve or more; the inlet and outlet of the upstream column and the downstream column are provided with medium delivery pipelines, and the modifier, diluent, raw material liquid and eluent are respectively delivered to the corresponding chromatographic column through the corresponding delivery pumps and the medium delivery pipelines.

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