Double-column switching circulating chromatographic separation method

Abstract

A double-column switching circulating chromatographic separation method comprises the following steps: adding a raw material solution into a system from a first chromatographic column, enabling an eluent to flow into the system from an inlet of the first chromatographic column for a certain period of time, switching a valve assembly to enable the eluent to flow into the system from an inlet of a second chromatographic column, and separating the eluent from the second chromatographic column after a certain period of time. The valve assembly is switched, so that the eluent flows into the system from the inlet of the first chromatographic column, and the adding position of the eluent is changed in a circulating manner; one switching period is completed when the position is changed once; after running to the Nth period and N is greater than or equal to 2 switching periods, the flushing system recovers target components; in the process, a chromatographic column into which an eluent flows is called as an upstream column, and the other corresponding chromatographic column is called as a downstream column; the method has the advantage that all the components can be separated more sufficiently.

Description

Technical Field

[0001] The present application relates to the technical field of chromatographic separation, and specifically to a method for separating target components from raw materials using a dual-column switching circulating chromatographic separation system, namely, a dual-column switching circulating chromatographic separation method. Background Art

[0002] Double column circulation chromatography is an efficient chromatographic separation method, in which the eluent and the raw material liquid to be separated are introduced into the system. The basic principle of the traditional double column circulation chromatography is as follows: Figure 1 As shown: two chromatographic columns, namely the first chromatographic column 1' and the second chromatographic column 2', are connected in series through two sets of valve components 3'; during operation, the valve components 3' are switched to change the upstream and downstream positions of the chromatographic columns, so that the concentration bands of the components to be separated circulate in the two columns: as shown in the attached figure Figure 1 As shown, it is position A, the raw material liquid first enters the first chromatographic column 1' through the raw material pump 4', and then the target component concentration spectrum band moves from the first chromatographic column 1' to the second chromatographic column 2' under the continuous elution of the eluent through the eluent pump 5'; when the target component concentration spectrum band completely leaves the first chromatographic column 1', the valve is switched, and at this time, as shown in the attached figure, the target component concentration spectrum band moves from the first chromatographic column 1' to the second chromatographic column 2'. Figure 2 As shown, it is position B, and the concentration band will move from the second chromatographic column 2' back to the first chromatographic column 1'; when the concentration band of the target component completely leaves the second chromatographic column 2', the column is switched again; Figure 1-Figure 2 The above process is repeated by switching valves between these two positions, ensuring that the target component concentration band always circulates within the two columns. This operation is equivalent to extending the chromatographic columns without increasing the pressure within the system. At the same time, the leading and trailing impurities in the separated components move to the front and back ends of the target component concentration band, respectively, and gradually flow out of the system, gradually reducing the area of overlap between the impurity and target component concentration bands until satisfactory separation is achieved.

[0003] The core of this dual-column cyclic chromatography separation lies in the fact that within each switching cycle, the target component concentration band consistently elutes from the upstream column to the downstream column. However, during the separation process, the target component concentration band gradually broadens due to factors such as nonlinear adsorption, mass transfer resistance, and axial dispersion. This can easily lead to a phenomenon where the target component concentration band occupies more than twice the column length before satisfactory separation is achieved from non-target components. To ensure the purity of the target component, either the switching cycle must be continued or the switching cycle must be terminated prematurely and fraction cutting implemented. Either approach reduces the target component yield. Therefore, the technical bottleneck of dual-column cyclic chromatography lies in band broadening.

[0004] Patent ZL201811024239.7 discloses a technique for preventing band broadening. The technique involves adding a modifier pumped in by a modification pump 6' to the middle connecting pipe of the two columns that are always connected in series in a double-column circulation chromatography. Figure 3-Figure 4 As shown in FIG), and the elution ability of the modifier is set to be weaker than the elution ability of the eluent, when the concentration band of the target component moves from the first chromatographic column 1' to the second chromatographic column 2' ( Figure 3 ie position A), or move from the second chromatographic column 2' to the first chromatographic column 1' ( Figure 4 At position B), the velocity of the leading edge of the band entering the downstream column slows down, becoming slower than the trailing edge still in the upstream column, resulting in a band contraction effect. This contraction effect counteracts band broadening, thereby improving the separation in dual-column circulation chromatography.

[0005] This patented technical solution is very clever, but because it also emphasizes that the two chromatographic columns must always maintain fluid flow communication, it also brings some shortcomings, as follows:

[0006] First, when separating and purifying biological products such as peptides and nucleic acids, the mobile phase composition is highly sensitive to the separation process. Gradient elution is more robust and reliable than isocratic elution, and it also compresses the spectral band, preventing band broadening. However, if this patented technology performs gradient elution during every switching cycle, the mobile phase composition within the system will be disrupted due to column switching, leading to separation failure. For example, when separating peptides in reversed-phase chromatography mode, if gradient elution is implemented, at the end of the nth switching cycle, assuming that the first chromatographic column is located upstream, the mobile phase in the column must have a higher organic solvent content than the mobile phase in the second chromatographic column located downstream (the higher the organic solvent content in the mobile phase, the stronger its elution ability). When the system switches to the n+1th cycle, the positions of the upstream and downstream columns are interchanged. At this time, the first chromatographic column is located downstream and the second chromatographic column is located upstream. The organic solvent content in the mobile phase in the first chromatographic column is significantly higher than the organic solvent content in the mobile phase in the upstream second chromatographic column, which will affect the migration of the target component and thus affect the robustness of the separation process. After the target component is eluted to the downstream in the nth cycle, if the upstream first chromatographic column can be flushed separately and the composition of the mobile phase inside it can be adjusted to the initial composition, this problem can be well solved.

[0007] Secondly, in many separation scenarios, in addition to impurities with a relatively close retention profile to the target component (weak impurities), the raw material also contains some particularly strongly retained impurities (strong impurities). Therefore, after the target component is eluted from the upstream column to the downstream column, assuming the first column is located upstream, most of the strong impurities and a small number of weak impurities will be retained in the upstream first column, but the weak impurities will be closer to the tail of the upstream first column. When the switching system enters the next cycle, the impurities follow the column to the downstream. These weak impurities will continue to flow out of the downstream first column, while the strong impurities will not completely flow out of the downstream first column. As a result, the target component band front catches up with these strong impurities. This obviously causes the target component band front and the strong impurities to overlap, which is detrimental to separation. However, if the upstream first column can be flushed separately after the target component is eluted downstream in the nth cycle, this problem can be effectively solved.

[0008] Finally, in some separation situations where the content of precursor impurities is much higher than that of the target component, the target component will move from the upstream column to the downstream column along with the precursor impurities. At this time, the tag-along effect caused by nonlinear adsorption will increase the difficulty of separating the target component. However, if the target component has not yet eluted from the upstream column in the nth cycle (the precursor impurities elute from the upstream column earlier than the target component, for example, in a switching cycle of 10 minutes, only the precursor impurities elute from the upstream column in 0-3 minutes, the precursor impurities and the target product elute from 3-6 minutes, only the target product elutes from 6-7 minutes, the latter impurities and the target product elute from 7-8 minutes, and the latter impurities elute from 8-10 minutes; 1) At the beginning of the switching, only the precursor impurities elute from the upstream column, and the target product has not yet eluted; 2) At the end of the switching, the target product has all flowed from the upstream column into the downstream column, and thereafter only the latter impurities elute. During these two time periods, the effluent from the upstream column may not flow into the downstream column, but may be directly discharged from the system), being able to flush the first upstream chromatographic column separately and directly discharge the precursor impurities from the system can effectively solve this problem. Summary of the Invention

[0009] In view of the above-mentioned deficiencies in the prior art, the present application provides a dual-column switching circulation chromatography separation method that can more fully achieve the separation of various components.

[0010] In order to solve the above technical problems, the technical solution provided by the present invention is implemented as follows: a dual-column switching cyclic chromatography separation method, the separation system adopted in the method includes a first chromatographic column, a second chromatographic column and a valve assembly; the operation process is: first, the raw material liquid is added to the system from the first chromatographic column, and then 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. 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 then 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 repeatedly in this way; each change of position is called a switching cycle; after running to the Nth cycle and N≥2 switching cycles, the system is flushed to recover the target component; the chromatographic column into which the eluent flows in this process is called the upstream column, and the corresponding other chromatographic column is called the downstream column.

[0011] Furthermore, the composition of the eluent in each switching cycle can be constant for isocratic elution or varied for gradient elution.

[0012] Furthermore, the eluent includes eluent A and eluent B. When the respective flow rates of the eluent A and eluent B entering the system are constant, it corresponds to isocratic elution; when the respective flow rates of the eluent A and eluent B entering the system change in real time and the total flow rate of the two is constant, it corresponds to gradient elution.

[0013] Furthermore, each change in position refers to a change in the eluent inlet position; the eluent always enters the system from the chromatographic column located upstream.

[0014] Furthermore, detectors are installed at the tails of the first chromatographic column and the second chromatographic column respectively.

[0015] Furthermore, the above-mentioned dual-column switching cycle chromatography separation method has a more specific separation step including:

[0016] (1) Feeding: Add the raw material liquid into the system from the inlet of the first chromatographic column;

[0017] (2) Purification: including multiple switching cycles, each switching cycle includes three stages; the first switching cycle: stage 1 (removal of front impurities): the eluent is added from the inlet of the first chromatographic column, and the outlet effluent of the first chromatographic column is discharged from the system; stage 2: the eluent flows from the inlet of the first chromatographic column, the outlet effluent of the first chromatographic column is combined with the modifier and flows into the inlet of the second chromatographic column, and the outlet effluent of the second chromatographic column is discharged from the system; stage 3 (removal of strongly retained rear impurities): the eluent is added from the inlet of the first chromatographic column, and the outlet effluent of the first chromatographic column is discharged from the system;

[0018] After a certain period of time, the system switches and enters the second switching cycle: Phase 1 (removal of front impurities): eluent is added from the inlet of the second chromatographic column, and the outlet effluent of the second chromatographic column is discharged from the system; Phase 2: eluent flows into the inlet of the second chromatographic column, the outlet effluent of the second chromatographic column is combined with the modifier and flows into the inlet of the first chromatographic column, and the outlet effluent of the first chromatographic column is discharged from the system; Phase 3 (removal of strongly retained rear impurities): eluent is added from the inlet of the second chromatographic column, and the outlet effluent of the second chromatographic column is discharged from the system; After a certain period of time, the system switches and enters the third switching cycle: Phase 1 (removal of front impurities): eluent is added from the inlet of the first chromatographic column, and the effluent of the first chromatographic column is discharged from the system ; Stage 2: eluent flows into the inlet of the first chromatographic column, the effluent from the outlet of the first chromatographic column is combined with the modifier and flows into the inlet of the second chromatographic column, and the effluent from the outlet of the second chromatographic column is discharged from the system; Stage 3 (removal of strongly retained impurities): eluent is added from the inlet of the first chromatographic column, and the effluent from the outlet of the first chromatographic column is discharged from the system; after a certain period of time, the fourth switching cycle is entered, and the double columns are switched back and forth in this way; non-target components are gradually discharged from the system in stages 1 and 3, while target components flow from the upstream column into the downstream column in stage 2, and then switch to the upstream, and flow from the upstream into the downstream again in stage 2 of the next switching cycle, thereby separating the target components from the non-target components.

[0019] Furthermore, the above-mentioned stages 1 to 3 are completed in sequence within one switching cycle.

[0020] Furthermore, in each switching cycle, the eluent always keeps flowing into the upstream chromatographic column.

[0021] Furthermore, when the system uses a modifier, the elution capacity of the eluent is always greater than the elution capacity of the modifier.

[0022] Furthermore, after at least one switching cycle has passed and the strongly retained impurities have been completely removed, the third stage (removal of the strongly retained impurities) of each subsequent switching cycle can be omitted.

[0023] Furthermore, when the content of the impurities in the raw material is less than the target component content, the first stage (eliminating the impurities) in each switching cycle can be completely omitted.

[0024] Furthermore, when the content of the impurities in the raw material liquid is greater than the target component content, after at least one switching cycle, the first stage (eliminating the impurities) of each subsequent switching cycle can be omitted.

[0025] Furthermore, the composition of the eluent in each switching cycle can be constant for isocratic elution or variable for gradient elution; that is, both isocratic elution and gradient elution can be performed on the upstream column; for example, gradient elution is suitable for separating large molecules such as peptides and proteins, while isocratic elution can be used for small molecules.

[0026] Furthermore, the eluent described in the present application includes eluent A and eluent B. When the respective flow rates of the eluent A and eluent B entering the system are constant, it corresponds to isocratic elution; when the respective flow rates of the eluent A and eluent B entering the system change in real time and the total flow rate of the two is constant, it corresponds to gradient elution; for example, when the flow rate of eluent A increases and the flow rate of eluent B decreases, the effect of gradient elution is achieved.

[0027] Advantages and beneficial effects of the present invention:

[0028] The present invention provides a method for separating a target component from a raw material by utilizing dual-column switching circulation chromatography. In each switching cycle, the fluid flow paths of the two columns are connected only when the target component moves from the upstream column to the downstream. Before the leading edge of the target component spectral band begins to flow out of the upstream column or after the trailing edge of the target component spectral band basically completely flows out of the upstream column, the fluid flow paths of the two columns are not connected, and both the front impurities and the rear impurities are discharged from the upstream column to the system. In order to completely elute the strong rear impurities, an eluent with stronger elution ability can be used. After the strong rear impurities are eluted, the composition of the mobile phase in the upstream column can be adjusted to prepare for the next switching cycle. This solution comprehensively solves the three shortcomings of the prior art and can separate and purify the target component from the raw material. The dual-column switching circulation chromatography separation method provided by the present invention is easy to operate, can implement gradient elution, and improve the robustness of the separation process. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Schematic diagram of the structure of a double-column circulating chromatography system in the prior art (position A, six-way valve).

[0030] Figure 2 Schematic diagram of the structure of a double-column circulating chromatography system in the prior art (position B, six-way valve).

[0031] Figure 3 This is a schematic diagram of the structure of the double-column circulating chromatography system of ZL201811024239.7 (position A, six-way valve).

[0032] Figure 4 This is a schematic diagram of the structure of the dual-column circulating chromatography system of ZL201811024239.7 (position B, six-way valve).

[0033] As attached Figure 1-4Shown: 1'. First chromatographic column, 2'. Second chromatographic column, 3'. Valve assembly, 4'. Raw liquid pump, 5'. Eluent pump, 6'. Modification pump.

[0034] Figure 5 This is a structural schematic diagram of the double-column circulating chromatography system separation method of this application when the effluent from the first chromatography column flows into the second chromatography column.

[0035] Figure 6 This is a structural schematic diagram of the double-column circulating chromatography system separation method of this application when the effluent from the second chromatography column flows into the first chromatography column.

[0036] Figure 7 This is a structural schematic diagram of the state in which the first chromatographic column of the dual-column circulating chromatography system separation method of the present application serves as the upstream column for direct discharge.

[0037] Figure 8 This is a schematic structural diagram of the state diagram of the dual-column circulating chromatography system separation method of this application, in which the second chromatography column acts as the upstream column for direct discharge.

[0038] As shown in the attached figure: 1. first chromatographic column, 2. second chromatographic column, 3. first valve assembly, 4. second valve assembly, 5. raw liquid pump, 6. eluent A pump, 7. eluent B pump, 8. modifier pump, 9. detector, 10. medium delivery pipeline.

[0039] Figure 9 HPLC chromatogram of the raw material solution.

[0040] Figure 10 This is the spectrum of tilpotide collected in Example 1 analyzed by high performance liquid chromatography.

[0041] Figure 11 This is the spectrum of tilpotide collected in Example 2 analyzed by high performance liquid chromatography.

[0042] Figure 12 This is the spectrum of tilpotide collected in Example 3 analyzed by high performance liquid chromatography.

[0043] Figure 13 This is the spectrum of tilpotide collected in Example 4 analyzed by high performance liquid chromatography. DETAILED DESCRIPTION

[0044] The present application is further described in detail below through examples, but the present application is not limited to the following examples.

[0045] The following takes the separation and purification of tilpotide as an example to describe the technical solution of the present application in detail.

[0046] 1. Dual-column switching circulation chromatography separation system

[0047] The double-column circulating chromatographic separation system of this application includes: two C8 chromatographic columns (filler particle size 10 μm; column inner diameter 1 cm, column length 15 cm), two two-position four-way valve assemblies, two detectors, and four infusion pumps. Figure 5-8 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 named the first chromatographic column 1 and the second chromatographic column 2, respectively, 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 the first valve assembly 3, the other is the second valve assembly 4, and the four delivery pumps are defined as the raw liquid pump 5, the eluent A pump 6, the eluent B pump 7, and the modifier pump 8 according to the medium to be delivered; where medium flow is required between each component, 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 10 between the first chromatographic column 1 and the second valve assembly 4, the second chromatographic column 2 and the second valve assembly 4, respectively, whose function is to detect the development and changes of the separation component spectrum band and the component content of the corresponding port effluent, etc., so as to effectively monitor the effective operation of the separation system formed by this method of the present application.

[0048] During operation, the upstream and downstream positions of the two chromatographic columns can be alternated by switching two four-way valves. During each switching cycle, the column into which the eluent flows first becomes the upstream column, and the other column becomes the downstream column. Two detectors 9 at the tails of the two columns monitor the development and changes in the separated component bands and the component content of the effluent from the corresponding ports. Four pumps are used to transport the raw material solution, eluent A / eluent B, and modifier, respectively. In this example, eluent A is acetonitrile / water / trifluoroacetic acid (volume ratio of the three components: 30 / 70 / 0.1, v / v / v), eluent B is acetonitrile / water / trifluoroacetic acid (volume ratio of the three components: 40 / 60 / 0.1, v / v / v), and the modifier is water. The teilpotide raw material is dissolved in acetonitrile / water / trifluoroacetic acid (500 / 500 / 1, v / v / v) to prepare a raw material solution with a concentration of 25 mg / mL. The front impurities in Examples 1-4 of the present application are impurities that are less retained than the target substance but elute earlier, and the back impurities are impurities that are more retained than the target substance but elute later.

[0049] 2. Sample analysis and testing

[0050] The purity of the samples was analyzed by high performance liquid chromatography. Liquid chromatography analysis conditions: the chromatographic column was a Phenomenex Knitex XB-18 column (4.6×150 mm, 5 μm) from the United States, the mobile phase was acetonitrile / water / trifluoroacetic acid (500 / 500 / 1, v / v / v), and the detection wavelength was 210 nm. Figure 9This is the HPLC chromatogram of the raw material solution. From the chromatogram, it can be seen that there are several front and back impurity peaks before and after the main peak of tilpotide, and the area percentage of each impurity peak is higher than 0.5%.

[0051] Example 1—Separation and purification of tepote using a dual-column circulating chromatography system [strong after-effects were not completely eluted]

[0052] At room temperature, 40 mL of raw material liquid is added to the system from the inlet of the first chromatographic column through the raw material liquid pump; then, the first switching cycle is started. After 15 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. This cycle is repeated, switching once every 15 minutes, a total of 8 times, and a total of 8 switching cycles. In each switching cycle, the flow rate of the eluent A pump decreases linearly from 2 mL / min to 0 mL / min, and the flow rate of the eluent B pump increases linearly from 0 mL / min to 2 mL / min; the sum of the flow rates of the eluent A pump and the eluent B pump is constant at 2 mL / min. During the 4th to 10th minute of each switching cycle, the flow paths of the two columns are connected, and the modifier pump 8 (corresponding to the attached) is turned on. Figure 5-6 The flow rate is 4 mL / min. During the rest of the time (0-4 min and 10-15 min), the effluent from the upstream column outlet is directly discharged from the system (corresponding to the attached Figure 7 and Figure 8 After running 8 switching cycles, the collected telpotide was analyzed by high performance liquid chromatography. Figure 10 As shown, the purity by peak area normalization method is 85%, with strong contamination by secondary impurities, and the yield is 80%.

[0053] Example 2—Separation and purification of tepote using a dual-column circulating chromatography system [sufficient elution of strong impurities, and unequilibration of the upstream column before switching]

[0054] At room temperature, 40mL of raw material liquid is added to the system from the inlet of the first chromatographic column through the raw material liquid pump; then, the first switching cycle is started, and the eluent A pump and the eluent B pump are started at the same time, with gradient elution and a total flow rate of 2mL / min. After 20 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 gradient elution is performed again with a total flow rate of 2m / min. This cycle is repeated, switching every 20 minutes, for a total of 6 times, and a total of 6 switching cycles. From the beginning to the 15th minute of each switching cycle, the flow rate of the eluent A pump decreases linearly from 2mL / min to 0mL / min, and the flow rate of the eluent B pump increases linearly from 0mL / min to 2mL / min. During the 4th to 10th minute of each switching cycle, the flow paths of the two columns are connected, and the modifier pump is turned on with a flow rate of 4mL / min (corresponding to the attached Figure 5-Figure 6During the rest of the time (0-4 min and 10-20 min), the effluent from the upstream column outlet is directly discharged from the system (corresponding to the attached Figure 7-Figure 8 The system is shown in the state), wherein the flow rate of the eluent A pump is 0 during the 15-20 min period, and the flow rate of the eluent B pump is 2 mL / min. After running 6 switching cycles, the collected telportide was analyzed by high performance liquid chromatography, as shown in the attached Figure 11 As shown, the purity by peak area normalization method was >98.5%, and the yield was 75%.

[0055] Example 3 - Isolation and purification of tepote using a dual-column circulating chromatography system [elution of strong impurities and equilibration of the upstream column before switching]

[0056] At room temperature, 40mL of raw material liquid is added to the system from the inlet of the first chromatographic column through the raw material liquid pump; then, the first switching cycle is started, and the eluent A pump and the eluent B pump are started at the same time, with gradient elution and a total flow rate of 2mL / min. After 22 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 gradient elution is performed again with a total flow rate of 2m / min. This cycle is repeated, switching every 22 minutes, for a total of 6 times, and a total of 6 switching cycles. From the beginning to the 15th minute of each switching cycle, the flow rate of the eluent A pump decreases linearly from 2mL / min to 0mL / min, and the flow rate of the eluent B pump increases linearly from 0mL / min to 2mL / min. During the 4th to 10th minute of each switching cycle, the flow paths of the two columns are connected, and the modifier pump is turned on with a flow rate of 4mL / min (corresponding to the attached Figure 5 and Figure 6 During the rest of the time (0-4 min and 10-22 min), the effluent from the upstream column outlet is directly discharged from the system (corresponding to the attached Figure 7-Figure 8 The system is shown in the state), wherein during the 15-20 min period, the strong and impurities are discharged, the eluent A pump flow rate is 0, and the eluent B pump flow rate is 2 mL / min. During the 20-22 min period, the upstream column is equilibrated, the eluent A pump flow rate is 2 mL / min, and the eluent B pump flow rate is 0. After running 6 switching cycles, the collected telportide was analyzed by high performance liquid chromatography, as shown in the attached figure. Figure 12 As shown, the purity by peak area normalization method was >98.5%, and the yield was 82%.

[0057] Example 4—Separation and purification of tepote using a dual-column circulating chromatography system [isocratic elution, sufficient elution of strong impurities, and equilibration of the upstream column before switching]

[0058] At room temperature, 40 mL of raw material liquid is added to the system from the inlet of the first chromatographic column through the raw material liquid pump; then, the first switching cycle is started. After 24 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. This cycle is repeated, switching once every 24 minutes, for a total of 6 times, and a total of 6 switching cycles. From the beginning to the 20th minute of each switching cycle, the flow rate of the eluent A pump is 1.2 mL / min, and the flow rate of the eluent B pump is 0.8 mL / min. During the 4th to 10th minute of each switching cycle, the flow paths of the two columns are connected, and the modifier pump is turned on with a flow rate of 1 mL / min (corresponding to the auxiliary Figure 5-Figure 6 During the rest of the time (0-4 min and 10-24 min), the effluent from the upstream column outlet is directly discharged from the system (corresponding to the attached Figure 7-Figure 8 The system is shown in the state), wherein during the 10-20 min period, the strong and impurities are discharged, the eluent A pump flow rate is 0, and the eluent B pump flow rate is 2 mL / min. During the 20-24 min period, the upstream column is equilibrated, the eluent A pump flow rate is 1.2 mL / min, and the eluent B pump flow rate is 0.8 mL / min. After running 6 switching cycles, the collected telportide was analyzed by high performance liquid chromatography, as shown in the attached figure. Figure 13 As shown, the purity by peak area normalization method was >98% and the yield was 84%.

[0059] It can be seen from the above embodiments that the separation method of the present application can effectively solve three types of problems existing in the background technology of the prior art by adjusting the timing of disconnection and connection of the first chromatographic column and the second chromatographic column, and controlling the gradient or isocratic elution of the eluent: that is, after eluting the target component to the downstream in the nth cycle, the upstream first chromatographic column can be flushed separately to adjust the composition of the mobile phase inside it to the initial composition, thereby solving the technical problem that "the organic solvent content in the mobile phase inside the first chromatographic column is significantly higher than the organic solvent content in the mobile phase inside the upstream second chromatographic column, which will affect the migration of the target component and thus affect the robustness of the separation process"; and can also solve the technical problem that "some The particularly strong post-impurities (strong post-impurities) are retained, and the post-impurities follow the column switching to the downstream. Those weak post-impurities will continue to flow out of the downstream first chromatographic column, while the strong post-impurities will not all flow out of the downstream first chromatographic column, so that the spectral leading edge of the target component catches up with these strong post-impurities. This obviously causes the target component and the strong post-impurities to overlap, which is not conducive to separation. The technical problem can also be solved by flushing the upstream first chromatographic column separately before the target component flows out of the upstream column in the nth cycle, and directly discharging the pre-impurities from the system, so as to solve the technical problem of "in some separation situations where the content of pre-impurities is much higher than that of the target component, the target component will move from the upstream column to the downstream column along with the pre-impurities. At this time, the nonlinear adsorption will cause a tag-along effect, which increases the difficulty of separating the target component."

[0060] 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 scope of protection of the present application.

Claims

1. A dual-column switching cyclic chromatography separation method, wherein the separation system used in the method comprises a first chromatographic column, a second chromatographic column and a valve assembly, characterized in that: The operation process of this method is as follows: first, the raw material liquid is added to the system from the first chromatographic column, and then 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. 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. The eluent addition position is changed repeatedly in this way; each change of position is called a switching cycle; after running to the Nth cycle and N≥2 switching cycles, the system is flushed to recover the target component; the chromatographic column into which the eluent flows in this process is called the upstream column, and the corresponding other chromatographic column is called the downstream column.

2. The dual-column switching circulation chromatography separation method according to claim 1, characterized in that: Each change in position refers to a change in the position of the eluent inlet, and the eluent always enters the system from the chromatographic column located upstream.

3. The dual-column switching circulation chromatography separation method according to claim 1, characterized in that: Detectors are respectively installed at the tails of the first chromatographic column and the second chromatographic column.

4. The dual-column switching circulation chromatography separation method according to claim 1, characterized in that: The composition of the eluent in each switching cycle can be constant for isocratic elution or change for gradient elution; the eluent includes eluent A and eluent B. When the respective flow rates of the eluent A and eluent B entering the system are constant, it corresponds to isocratic elution; when the respective flow rates of the eluent A and eluent B entering the system change in real time and the total flow rate of the two is constant, it corresponds to gradient elution.

5. The dual-column switching circulation chromatography separation method according to claim 1, characterized in that: The specific separation steps include: (1) Feeding: Add the raw material liquid into the system from the inlet of the first chromatographic column; (2) Purification: including multiple switching cycles, each switching cycle includes three stages; the first switching cycle: stage 1: the eluent is added from the inlet of the first chromatographic column, and the outlet effluent of the first chromatographic column is discharged from the system; stage 2: the eluent flows from the inlet of the first chromatographic column, the outlet effluent of the first chromatographic column is combined with the modifier and flows into the inlet of the second chromatographic column, and the outlet effluent of the second chromatographic column is discharged from the system; stage 3: the eluent is added from the inlet of the first chromatographic column, and the outlet effluent of the first chromatographic column is discharged from the system; After a certain period of time, the system switches and enters the three stages of the second switching cycle: Stage 1: Eluent is added from the inlet of the second chromatographic column, and the effluent from the outlet of the second chromatographic column is discharged from the system; Stage 2: Eluent flows from the inlet of the second chromatographic column, the effluent from the outlet of the second chromatographic column is combined with the modifier and flows into the inlet of the first chromatographic column, and the effluent from the outlet of the first chromatographic column is discharged from the system; Stage 3: Eluent is added from the inlet of the second chromatographic column, and the effluent from the outlet of the second chromatographic column is discharged from the system; After a certain period of time, the system switches and enters the three stages of the third switching cycle: Stage 1: eluent is added from the inlet of the first chromatographic column, and the effluent from the first chromatographic column is discharged from the system; Stage 2: eluent flows into the inlet of the first chromatographic column, the effluent from the outlet of the first chromatographic column is combined with the modifier and flows into the inlet of the second chromatographic column, and the effluent from the outlet of the second chromatographic column is discharged from the system; Stage 3: eluent is added from the inlet of the first chromatographic column, and the effluent from the outlet of the first chromatographic column is discharged from the system; After a certain period of time, it enters the fourth switching cycle, and the dual columns are switched back and forth in this way; non-target components are gradually discharged from the system in stages 1 and 3, while target components flow from the upstream column into the downstream column in stage 2, and then switch to the upstream, and flow from the upstream into the downstream again in stage 2 of the next switching cycle, thereby separating the target components from the non-target components.

6. The dual-column switching circulation chromatography separation method according to claim 5, characterized in that: The first to third stages are completed sequentially in one switching cycle; in each switching cycle, the eluent always keeps flowing into the upstream chromatographic column.

7. The dual-column switching circulation chromatography separation method according to claim 5, characterized in that: When the system uses a modifier, the elution capacity of the eluent is always greater than the elution capacity of the modifier.

8. The dual-column switching circulation chromatography separation method according to claim 5, characterized in that: After at least one switching cycle and the complete removal of strongly retained impurities, the third stage of each subsequent switching cycle can be omitted; when the content of front impurities in the raw material is less than the target component content, the first stage of each switching cycle can be omitted; when the content of front impurities in the raw material liquid is greater than the target component content, after at least one switching cycle, the first stage of each subsequent switching cycle can be omitted.

9. The dual-column switching circulation chromatography separation method according to claim 5, characterized in that: The composition of the eluent in each switching cycle can be constant for isocratic elution or varied for gradient elution.

10. The dual-column switching circulation chromatography separation method according to claim 9, characterized in that: The eluent includes eluent A and eluent B. When the respective flow rates of eluent A and eluent B entering the system are constant, it corresponds to isocratic elution; when the respective flow rates of eluent A and eluent B entering the system change in real time and the total flow rate of the two is constant, it corresponds to gradient elution.

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