Simulated moving-bed chromatographic separation method and simulated moving-bed chromatographic separation system

Abstract

In this simulated moving-bed chromatographic separation method for separating each component in a stock solution by a difference in adsorption force to an adsorbent by using a circulation system in which a plurality of unit packed towers filled with the adsorbent are connected in series and endlessly via piping, the piping of the circulation system including a stock solution supply port, an eluent supply port, an extraction port X of a fraction containing a component to be purified, and an extraction port Y of a fraction containing a component other than the component to be purified, a stock solution supply cycle includes: a step (i) of supplying the stock solution from the stock solution supply port: and a step (ii) of stopping the supply of the stock solution following the step (i). In the stock solution supply cycle, the amount x of the component to be purified recovered from the extraction port X through the step (i) and the step (ii) is measured, and in the next stock solution supply cycle, the stock solution containing the same amount of the component to be purified as the amount x of the component to be purified is supplied to the stock solution supply port.

Description

Simulated moving bed chromatographic separation method and simulated moving bed chromatographic separation system

[0001] The present invention relates to a simulated moving bed chromatographic separation method and a simulated moving bed chromatographic separation system.

[0002] In simulated moving bed (SMB) chromatographic separation, multiple unit packed towers (hereinafter simply referred to as "packed towers" or "columns") packed with an adsorbent capable of selectively adsorbing specific components among multiple components contained in a stock solution are connected in series via piping, and the most downstream packed tower is connected to the most upstream packed tower to form an endless circulation system. A stock solution and an eluent are supplied to this circulation system, and a fast-moving fraction (weakly adsorbing fraction) and a slow-moving fraction (strongly adsorbing fraction) are extracted from different positions within the circulation system. The stock solution supply position, eluent supply position, weakly adsorbing fraction extraction position, and strongly adsorbing fraction extraction position are then intermittently moved in the direction of fluid circulation in the circulation system while maintaining a constant positional relationship. Repeating this operation simulates a moving bed process, allowing for continuous stock solution supply. Various operating methods have been proposed for simulated moving bed chromatographic separation methods depending on the purpose, and efforts are being made to put them into practical use. For example, Patent Document 1 discloses a chromatographic separation method for separating and purifying components in a stock solution by a simulated moving bed method using a circulation system in which a plurality of unit packed towers filled with an adsorbent are connected in series and endlessly via piping, wherein the circulation system has a stock solution supply port F, a weakly adsorptive fraction outlet A, an eluent supply port D, and a strongly adsorptive fraction outlet C in the piping, in this order, facing the direction of fluid flow, and at least one unit packed tower is disposed between the stock solution supply port F and the weakly adsorptive fraction outlet A, between the weakly adsorptive fraction outlet A and the eluent supply port D, between the eluent supply port D and the strongly adsorptive fraction outlet C, and between the strongly adsorptive fraction outlet C and the stock solution supply port F, and the chromatographic separation method includes sequentially repeating two steps that satisfy specific conditions.

[0003] Because simulated moving bed chromatographic separation allows for continuous, highly purified purification of target substances, its application in the medical field is also being considered. For example, in the production of antibody drugs, extracts and culture media from antibody-producing cultured cells contain not only the target antibody, but also fragments that do not fully function as antibodies, resulting from cleavage of the antibody, and large aggregates formed by aggregation of the antibody. Generally, these fragments have few interaction sites with the adsorbent and are weakly adsorbent. Conversely, aggregates are highly adsorbent. Therefore, when applying simulated moving bed chromatographic separation to the purification of antibody drugs, the target antibody must be separated as a moderately adsorbent fraction, which exhibits intermediate adsorption to the adsorbent. On the other hand, both the weakly adsorbent fraction and the strongly adsorbent fraction must be sufficiently removed with high removal rates. For example, Patent Document 2 describes a simulated moving bed chromatographic separation method using a circulation system in which a plurality of unit packed columns packed with an adsorbent are connected in series and endlessly via piping to separate, using two or more eluents, components contained in a stock solution that are weakly adsorbent, strongly adsorbent, and intermediately adsorbent components with intermediate adsorption properties to the adsorbent, wherein the piping of the circulation system is provided with a stock solution supply port F, two or more eluent supply ports D corresponding to each of the two or more eluents, an outlet A for a weakly adsorbent fraction containing the weakly adsorbent, an outlet B for a medium adsorbent fraction containing the medium adsorbent, and an outlet C for a strong adsorbent fraction containing the strongly adsorbent, and the stock solution supply port F, the outlet A, the outlet B, and the outlet C are positioned in a specific positional relationship. The method described in Patent Document 2 enables the intermediately adsorbent component, which is the component to be purified in the stock solution, to be separated and isolated with high purity using a smaller amount of adsorbent.

[0004] JP 2019-32284 A JP 2020-85881 A

[0005] In simulated moving bed chromatographic separation, when the concentration of each component in the circulation system is high and exceeds the column (adsorbent) load capacity, the concentration distribution of each component in the circulation system becomes broad, resulting in an earlier elution start time and a so-called tailing-like peak shape with a broader base and a trailing tail. This is thought to be because the high-concentration components in the circulation system also act as eluents, pushing out some of the components trapped at the tip of the column. Figure 1 is a graph schematically illustrating an example of separation and purification of two components (a strongly adsorbent component and a weakly adsorbent component) in a simulated moving bed chromatographic separation system with four columns. Note that Figure 1 is merely a schematic diagram illustrating the supply of raw solution and eluent and the extraction of each component, and does not accurately represent actual operation or the behavior of each component. In the graph shown in Figure 1, the strongly adsorbent component is shown as the component to be purified. The open arrows indicate the flow direction of the liquid phase, and the filled inverted triangles indicate the feed and desorbent feed positions, as well as the extraction positions of fractions containing strongly and weakly adsorbable components (labeled "Extract" and "Raffinate," respectively). As shown in Figure 1(a), when the concentration of each component is low (i.e., when the sample volume per column is small), the peak shape of each component in the circulation system is approximately a normal distribution mountain shape. On the other hand, as shown in Figure 1(c), when the concentration of each component is high and the sample volume per column is overloaded, the peak shape of each component in the circulation system becomes broad, and the elution start time and elution peak time are earlier (hereinafter, this concentration-dependent change in peak shape is also referred to as the "extrusion tailing phenomenon").

[0006] In the early stages of a cycle, the system is unstable, and the recovery rate of the target component is generally significantly lower than 100%, resulting in the accumulation of unrecovered target components in the circulation system. In a typical SMB separation method, the amount of raw solution supplied to the circulation system is constant for each cycle, resulting in the accumulation of unrecovered target components in the circulation system. Even in such a case, if the concentration of each component in the circulation system is sufficiently low relative to the column's processing capacity, the peaks of each component will shift upward, but the component concentrations will not overload the column, and the extrusion tailing phenomenon described above will not occur as the cycle progresses (Figure 1(b)). If extrusion tailing does not occur, the recovery rate of the target component will increase as the cycle progresses, and the supply rate of raw solution and the recovery rate of the target component will be balanced, ultimately stabilizing the system (steady state). Therefore, even with conventional SMB separation methods, the target component can be separated with high purity and high recovery rate as desired. On the other hand, when a high concentration of sample is supplied, the absolute amount of the target components that are not fully recovered in each cycle accumulates (accumulates) in the circulation system, which can eventually lead to an overload of the column. When the component concentrations become overloaded, the concentration distribution of each component becomes broad, as shown in Figure 1(c), resulting in the extrusion tailing phenomenon. For example, even if the components are separated as shown in Figure 1(b) until midway through the operation, the amount of the target components in the circulation system may exceed the column's load capacity before stabilization as the cycle progresses. When this extrusion tailing phenomenon occurs, the elution time fluctuates over time, making it difficult to continuously separate the target components with high purity and high recovery. (For example, in Figure 1(c), the concentration distribution of the strongly adsorptive components is significantly tilted diagonally rightward, so the recovery rate from the extraction point (Extract) remains low and does not increase as the cycle progresses.) However, conventional simulated moving bed chromatographic separation methods do not take into account the accumulation of the unrecovered target components. In order to prevent the occurrence of the extrusion tailing phenomenon described above, it is conceivable to increase the amount of adsorbent in the column, but there is a limit to the amount of adsorbent that can be packed into the column.In addition, in the past, the concentration of the raw solution was low, and the load on the adsorbent was limited even when the cycle was repeated, making the extrusion tailing phenomenon difficult to manifest. However, recently, from the perspective of productivity, there has been an increasing demand to recover the target component of purification at a high concentration (i.e., to supply a raw solution with a high concentration), and the problem of the extrusion tailing phenomenon tends to become more apparent.

[0007] The present invention aims to provide a chromatographic separation method using a simulated moving bed system that enables stable separation of a high-purity target component with a high recovery rate, and a chromatographic separation system suitable for carrying out the chromatographic separation method.

[0008] In view of the above problems, the present inventors have conducted extensive research and have found that in a chromatographic separation method using a simulated moving bed system, stable separation and purification is possible even when the concentration of each component in the raw solution is high, by controlling the amount of the component to be purified recovered to be equal to the amount of the component to be purified in the raw solution supplied in the next or subsequent step, so that the concentration of each component in the circulation system in each cycle is constant.The present invention was completed through further research based on these findings.

[0009] The above-mentioned object of the present invention has been achieved by the following means: [1] A simulated moving bed chromatographic separation method using a circulation system in which a plurality of unit packed towers packed with an adsorbent are connected in series and endlessly via piping to separate components in a stock solution based on differences in their adsorptivity to the adsorbent, wherein the piping of the circulation system comprises a stock solution supply port, an eluent supply port, an outlet X for a fraction containing a component to be purified, and an outlet Y for a fraction containing components other than the component to be purified, the simulated moving bed chromatographic separation method comprising a stock solution supply cycle consisting of a step (i) of supplying the stock solution from the stock solution supply port and a step (ii) of stopping the supply of the stock solution following the step (i), wherein an amount x of the component to be purified recovered from the outlet X through the steps (i) and (ii) is measured, and a stock solution containing an amount of the component to be purified equal to the amount x of the component to be purified is supplied to the stock solution supply port in the next stock solution supply cycle. [2] The simulated moving bed chromatographic separation method according to [1] above, wherein the simulated moving bed is a gradient simulated moving bed. [3] The simulated moving bed chromatographic separation method according to [1] or [2] above, wherein the component to be purified is a component that is strongly or moderately adsorbent to the adsorbent. [4] The simulated moving bed chromatographic separation method according to any of [1] to [3] above, wherein the amount of stock solution supplied in the second or subsequent stock solution supply cycles is controlled based on the amount x of the component to be purified recovered in the first stock solution supply cycle. [5] The simulated moving bed chromatographic separation method according to any of [1] to [3] above, wherein the amount x of the component to be purified recovered in each stock solution supply cycle is monitored, and the amount of stock solution supplied in the next stock solution supply cycle is controlled based on the monitoring results.[6] A simulated moving bed chromatographic separation system that separates components in a stock solution based on differences in adsorption force to the adsorbent, using a circulation system in which a plurality of unit packed towers filled with an adsorbent are connected in series and endlessly via piping, wherein the piping of the circulation system is provided with a stock solution supply port, an eluent supply port, an outlet X for a fraction containing a component to be purified, and an outlet Y for a fraction containing components other than the component to be purified, and the simulated moving bed chromatographic separation system has a stock solution supply cycle consisting of step (i) in which the stock solution is supplied from the stock solution supply port and step (ii) in which the supply of the stock solution is stopped following step (i), a means for measuring an amount x of the component to be purified recovered from the outlet X through steps (i) and (ii), and a means for supplying a stock solution containing the component to be purified in an amount equal to the amount x of the component to be purified to the stock solution supply port in the next stock solution supply cycle.

[0010] According to the simulated moving bed chromatographic separation method of the present invention, a highly pure target component can be stably separated and collected with a high recovery rate. Furthermore, the simulated moving bed chromatographic separation system of the present invention can be suitably used for carrying out the simulated moving bed chromatographic separation method of the present invention.

[0011] Figure 1 is a graph schematically showing the separation of two components in a simulated moving bed chromatographic separation system. Figure 2 is a graph schematically showing the change in the recovery amount of a component to be purified depending on whether or not the extrusion tailing phenomenon occurs in a simulated moving bed chromatographic separation system. Figure 3 is a system diagram showing an example of a simulated moving bed chromatographic separation system of the present invention. Figure 4 is a flow diagram showing each substep constituting step (A) in the first cycle in the operating process of the simulated moving bed chromatographic separation system in this embodiment. Figure 5 is a flow diagram showing each substep constituting step (A) in the second cycle in the operating process of the simulated moving bed chromatographic separation system in this embodiment.

[0012] A preferred embodiment of the present invention will be described below, but the present invention is not limited to the following embodiment except as defined by the present invention.

[0013] [Simulated Moving Bed Chromatographic Separation Method] The simulated moving bed chromatographic separation method of the present invention (hereinafter also simply referred to as the "method of the present invention") is a method for separating components in a stock solution based on differences in their adsorption strength to the adsorbent, using a circulation system in which a plurality of unit packed columns packed with an adsorbent are connected in series and endlessly via piping. The piping of the circulation system includes a stock solution inlet for supplying the stock solution, an eluent inlet for supplying an eluent, an outlet X for a fraction containing the component to be purified, and an outlet Y for a fraction containing components other than the component to be purified. The piping of the circulation system may have one eluent inlet, or may have two or more eluent inlets for supplying two or more eluents. Furthermore, the piping of the circulation system may have one outlet Y for a fraction containing a component other than the component to be purified, or may have two or more outlets Y for withdrawing two or more components other than the component to be purified that have different adsorption properties. The component to be purified is usually one type, and therefore there is usually only one outlet X for the fraction containing the component to be purified. From the perspective of supplying and stopping the stock solution, the method of the present invention involves repeating a stock solution supply cycle, in which step (i) of supplying the stock solution from the stock solution supply port and step (ii) of stopping the supply of the stock solution following step (i) are each performed once. After a stock solution supply cycle A is completed, the amount of the component to be purified contained in the stock solution supplied to the stock solution supply port in the next stock solution supply cycle B is controlled to be the same as the amount x of the component to be purified recovered from outlet X in stock solution supply cycle A. In the present invention, the term "same amount" means substantially the same amount, and the amounts may vary slightly as long as the effects of the present invention are achieved. The term "substantially the same amount" preferably means that the ratio of [amount of the component to be purified contained in the raw solution] / [amount x of the component to be purified recovered from outlet X] is in the range of 0.9 / 1.0 to 1.1 / 1.0, and more preferably in the range of 0.95 / 1.0 to 1.05 / 1.0.

[0014] In the present invention and this specification, the components contained in the raw solution may be grouped into two groups (strongly adsorbent and weakly adsorbent) or three groups (strongly adsorbent, medium adsorbent, and weakly adsorbent) based on the difference in their adsorption strength to the adsorbent, or may be grouped into more groups as necessary. In the method of the present invention, the components to be purified can be appropriately set according to the above grouping. That is, in the above example, any of a strong adsorbent, a medium adsorbent, a weakly adsorbent, etc. may be the components to be purified. A "strongly adsorbent component" means a component that has a strong adsorption strength to the adsorbent among the multiple components contained in the raw solution, a "weakly adsorbent component" means a component that has a weak adsorption strength to the adsorbent (or a component that has no adsorption strength) among the multiple components contained in the raw solution, and a "medium adsorbent component" means a component that has a weaker adsorption strength to the adsorbent than the strong adsorbent component but a stronger adsorption strength to the adsorbent than the weakly adsorbent component. In other words, the terms "strongly adsorbent," "moderately adsorbent," and "weakly adsorbent" refer to the relative strength of adsorption when comparing the adsorption strength of each component contained in the raw solution to the adsorbent. The above-mentioned "strongly adsorbent component," "moderately adsorbent component," and "weakly adsorbent component" may each consist of a single component or multiple components. Furthermore, the multiple components may have the same or different adsorption strengths. The grouping of each component in the raw solution can be appropriately set depending on the purpose. For example, if the raw solution contains four components, the component with the strongest adsorption strength to the adsorbent can be classified as the strong adsorbent, and the components with the second to fourth strongest adsorption strengths to the adsorbent can be classified as the weak adsorbent. In a similar case, two components in descending order of their adsorption strength to the adsorbent can be classified as the strong adsorbent, the component with the third strongest adsorption strength to the adsorbent can be classified as the medium adsorbent, and the component with the weakest adsorption strength to the adsorbent can be classified as the weak adsorbent. Similarly, when the raw solution contains five or more components, separation and purification based on various groupings can be performed.

[0015] In the present invention and this specification, the "component to be purified" separated by the method of the present invention may be a component contained in the stock solution that does not have adsorption capacity to the adsorbent, or a component that has adsorption capacity to the adsorbent. The "component to be purified" is preferably a strongly or moderately adsorbent component. That is, in the various groupings described above, the strongly adsorbent component can be the "component to be purified." Furthermore, when grouping components into three, it is also preferable to use the strongly or moderately adsorbent component in the various groupings described above as the "component to be purified." As described above, in the method of the present invention, the component to be purified may be one type or two or more types. Specific examples of components to be purified include sucrose, starch sugar, sugar alcohols, antibodies, oligonucleotides, proteins, viral vectors, etc. For example, antibodies separated and purified to high purity by the method of the present invention can be used as antibody pharmaceuticals. That is, by applying the method of the present invention to separate antibodies contained in the stock solution, a method for producing antibody pharmaceuticals can be provided. More specifically, according to the method of the present invention, an antibody drug can be obtained by using a culture medium of antibody-producing cells and / or an extract of antibody-producing cells as a stock solution and isolating the antibodies contained therein. In the present invention, the terms "culture medium of antibody-producing cells" and "extract of antibody-producing cells" encompass those that have been fractionated or purified to a certain extent by subjecting the culture medium of antibody-producing cells or the extract of antibody-producing cells to various treatments such as centrifugation or chromatographic separation.

[0016] The change in recovery rate of a target component in a typical simulated moving bed chromatographic separation system depending on whether or not extrusion tailing occurs is explained using the schematic diagram shown in Figure 2. The peak shapes and separation values ​​of the target component shown in Figure 2 are shown as examples to facilitate understanding of the present invention, and the present invention is not limited to the configuration shown in Figure 2 except as specified in the present invention. Also, "%" indicates mass %. Figure 2(a) shows the concentration distribution of the target component in the first cycle (first cycle) and the next cycle (second cycle) of a raw material supply cycle in a simulated moving bed chromatographic separation system in which extrusion tailing does not occur. Each system shown in Figure 2 is operated using a step gradient method, and in the first cycle, the target component that has migrated through columns 2 to 4 is extracted from the end of column 4. In the first cycle, 80% of the target component present in the circulation system is present in columns 2 to 4, and the remaining 20% ​​is present in column 5. The target component in column 5 that was not recovered in the first cycle remains in the circulation system and is again separated in the circulation system together with the new target component supplied in the second cycle. Because the SMB chromatographic separation system shown in Figure 2(a) does not exhibit extrusion tailing, the 20% of the target component remaining in the first cycle is separated again in the following second cycle, with, for example, 19% remaining in columns 3-5 and the remaining 1% remaining in column 1. As a result, when the target component that has migrated through columns 3-5 is extracted from the end of column 5 in the second cycle, the recovery amount of the target component in the second cycle is greater than the recovery amount of the target component in the first cycle. Thus, in a separation system that does not exhibit extrusion tailing, the target component can be stably separated with a high recovery rate by repeating the cycle. In contrast, Figure 2(b) shows the concentration distribution of the target component in the first and second cycles in a SMB chromatographic separation system exhibiting extrusion tailing. In the first cycle of FIG. 2(b), the amount of the component to be purified supplied exceeds the capacity of the column, so extrusion tailing has already occurred, causing the peak to appear earlier.In the first cycle, as in FIG. 2(a), the target component that has migrated through columns 2-4 is extracted from the end of column 4. Therefore, the target component that was not recovered in the first cycle in column 5 remains in the circulation system and is separated again in the circulation system together with the new target component that is supplied in the second cycle. On the other hand, in the example of FIG. 2(b), the peak is advanced due to the occurrence of extrusion tailing, and 20% of the target component that accumulated in the first cycle is separated in the subsequent second cycle so that, for example, 1% is present in columns 3-5 and the remaining 19% is present in columns 1 and 2. As a result, the amount of target component recovered in the second cycle is not significantly different from the amount recovered in the first cycle, and a large amount of target component accumulates in the circulation system. Further repetition of the cycle makes it impossible to maintain the performance of the separation system. In the method of the present invention, the amount of the component to be purified contained in the stock solution supplied to the stock solution supply port in the next stock solution supply cycle B (e.g., the second cycle) is controlled to be the same as the amount x of the component to be purified recovered from the withdrawal port X in the stock solution supply cycle A (e.g., the first cycle), so that the high-purity component to be purified can be stably separated at a high recovery rate regardless of whether or not the extrusion tailing phenomenon occurs, and the component to be purified does not accumulate excessively in the circulation system, so that the performance of the separation system can be maintained. The method of the present invention is particularly preferably applied to a system in which the extrusion tailing phenomenon occurs (or a system in which the amount of the component to be purified in the circulation system exceeds the allowable load of the column when the stock solution supply cycle is operated repeatedly the desired number of times).

[0017] <Circulation System> In the method of the present invention, except for the control of the feed rate of the raw solution in the raw solution supply cycle, known circulation systems and operating methods used in simulated moving bed systems can be widely applied. For example, reference can be made to JP 2009-36536 A, JP 4606092 A, JP 4771460 A, JP 4938728 A, JP 5330499 A, JP 5661209 A, JP 5133872 A, JP 6732575 A, JP 6912320 A, JP 7225024 A, JP 7367206 A, JP 3590088 A, JP 3611343 A, JP 6013639 A, and the like. An example of a circulation system applicable to the method of the present invention will be described below with reference to the drawings, but the present invention is not limited to these embodiments except as specified in the present invention. The drawings referred to below are explanatory diagrams for facilitating understanding of the present invention, and the sizes and relative relationships of the components may be changed for the sake of explanation and do not directly represent the actual relationships. Furthermore, matters other than those specified in the present invention are not limited to the shapes, relative positional relationships, etc. shown in these drawings. Furthermore, conditions other than those specified in the present invention, such as the capacity of the unit packed tower, the cross-sectional area and length of the piping, and the flow rate of the liquid supplied to the circulation system, can be appropriately set depending on the purpose.

[0018] A preferred embodiment of the circulation system used in the method of the present invention is shown in Figure 3. The circulation system shown in Figure 3 is suitable for separating three components (a strongly adsorbable component, a moderately adsorbable component, and a weakly adsorbable component), but the number of components to be separated is not particularly limited in the circulation system used in the method of the present invention. The circulation system 100 shown in Figure 3 includes four unit packed towers (columns) (unit packed towers 10a, 10b, 10c, and 10d) packed with adsorbent Ab. The outlet of each unit packed tower is connected to the inlet of the adjacent unit packed tower via pipe 1, so that the unit packed towers are connected in series as a whole. The outlet of the rearmost unit packed tower (e.g., unit packed tower 10d) is connected to the inlet of the frontmost unit packed tower (e.g., unit packed tower 10a) via pipe 1, so that all unit packed towers are connected in an endless (annular) fashion. This configuration enables a fluid to circulate within the circulation system 100. The unit packed towers 10a to 10d may be the same or different in terms of internal shape, size, and adsorbent packing amount, and it is preferable to use unit packed towers 10a to 10d that are equivalent (preferably the same) in terms of internal shape, size, and adsorbent packing amount.

[0019] A circulation pump P1 for circulating the fluid in the direction of the arrow can be disposed within the circulation system 100. The circulation pump P1 is preferably a metering pump. In addition, in the circulation system 100, the piping 1 between two adjacent unit packed towers is provided with shutoff valves R1, R2, R3, and R4 that can shut off the flow of fluid to the downstream unit packed tower.

[0020] Between each of the shutoff valves R1 to R4 and the outlet of each of the unit packed towers 10a to 10d located upstream thereof, weakly adsorbent fraction withdrawal lines 2a, 2b, 2c, and 2d are branched off, respectively, for withdrawing fractions rich in components weakly adsorbable to the adsorbent Ab (referred to herein as "weakly adsorbent fractions to the adsorbent Ab" or simply "weakly adsorbent fractions"). Each of the weakly adsorbent fraction withdrawal lines 2a, 2b, 2c, and 2d is provided with a weakly adsorbent fraction withdrawal valve A1, A2, A3, and A4, respectively, that can open and close each weakly adsorbent fraction withdrawal line. Each of the weakly adsorbent fraction withdrawal lines 2a, 2b, 2c, and 2d is joined together into a single weakly adsorbent fraction joining pipe 2J. When any of the weakly adsorbent fraction withdrawal valves A1, A2, A3, and A4 is in an open state, the connection point between the weakly adsorbent fraction withdrawal line in which the opened weakly adsorbent fraction withdrawal valve is installed and piping 1 becomes the withdrawal port A for the weakly adsorbent fraction.

[0021] Similarly, between each of the shutoff valves R1 to R4 and the outlet of each of the unit packed towers 10a to 10d located upstream thereof, medium adsorbent fraction withdrawal lines 3a, 3b, 3c, and 3d are branched and disposed, for withdrawing fractions containing a large amount of medium adsorbent components for the adsorbent Ab (referred to herein as "medium adsorbent fractions for the adsorbent Ab" or simply "medium adsorbent fractions"). Each of the medium adsorbent fraction withdrawal lines 3a, 3b, 3c, and 3d is provided with a medium adsorbent fraction withdrawal valve B1, B2, B3, and B4, respectively, that can open and close each medium adsorbent fraction withdrawal line. Each of the medium adsorbent fraction withdrawal lines 3a, 3b, 3c, and 3d is joined together and combined into a single medium adsorbent fraction joining pipe 3J. When any of the medium adsorbent fraction withdrawal valves B1, B2, B3, and B4 is in an open state, the connection point between the medium adsorbent fraction withdrawal line in which the opened medium adsorbent fraction withdrawal valve is installed and piping 1 becomes the medium adsorbent fraction withdrawal port B.

[0022] Similarly, between each of the shutoff valves R1 to R4 and the outlet of each of the unit packed towers 10a to 10d located upstream thereof, there are branched strongly adsorbent fraction withdrawal lines 4a, 4b, 4c, and 4d for withdrawing fractions rich in strongly adsorbent components for the adsorbent Ab (referred to herein as "strongly adsorbent fractions for the adsorbent Ab" or simply "strongly adsorbent fractions"). The strongly adsorbent fraction withdrawal lines 4a, 4b, 4c, and 4d are respectively provided with strongly adsorbent fraction withdrawal valves C1, C2, C3, and C4 capable of opening and closing the respective strongly adsorbent fraction withdrawal lines. The strongly adsorbent fraction withdrawal lines 4a, 4b, 4c, and 4d are joined together into a single strongly adsorbent fraction joining pipe 4J. When any one of the strongly adsorbent fraction withdrawal valves C1, C2, C3, and C4 is in an open state, the connection point between the strongly adsorbent fraction withdrawal line in which the opened strongly adsorbent fraction withdrawal valve is installed and piping 1 becomes the strongly adsorbent fraction withdrawal port C.

[0023] Safety valves (or relief valves) (not shown) may be provided at appropriate locations in the circulation system 100 to prevent excessive pressure buildup in the circulation system 100. In addition, check valves T1, T2, T3, and T4 for preventing backflow are preferably provided between adjacent two unit packed columns.

[0024] As shown in FIG. 3 , the circulation system 100 is configured to be able to supply a stock solution 7 contained in a stock solution tank 6. The circulation system 100 is also configured to be able to supply one or more eluents. FIG. 3 shows an example in which four eluents are supplied. The stock solution 7 is supplied via a stock solution supply line 11 by a stock solution supply pump P2 capable of controlling the supply flow rate. The stock solution supply pump P2 is preferably a metering pump. The stock solution supply pump P2 is preferably one that adjusts the supply flow rate by feedback control, thereby controlling the amount of stock solution supplied to the circulation system. As shown in FIG. 3 , the stock solution supply line 11 branches into four stock solution supply branch lines 11a, 11b, 11c, and 11d. The stock solution can be supplied to the inlets of the respective unit packed towers 10a, 10b, 10c, and 10d via the stock solution supply branch lines 11a, 11b, 11c, and 11d. Each of the branch feed lines 11a, 11b, 11c, and 11d is provided with an openable / closable feed valve F1, F2, F3, or F4, and the feed is supplied to a unit packed tower connected downstream through the branch feed line having the open feed valve. When any of the feed valves F1, F2, F3, or F4 is opened, the connection between the branch feed line having the opened feed valve and the piping 1 becomes a feed port F.

[0025] Within the circulation system 100, a measuring device (not shown) is provided in at least one of the weakly adsorptive fraction junction pipe 2J, the moderately adsorptive fraction junction pipe 3J, and the strongly adsorptive fraction junction pipe 4J to measure the concentration of the component to be purified in the fraction containing the component to be purified and the volume of the recovered fraction during one feed cycle. The concentration of the component to be purified in the fraction containing the component to be purified can be measured, for example, using a UV detector or HPLC analysis after sampling. The volume of the recovered fraction can be measured, for example, using a flow meter or the like provided in the junction pipe. The circulation system 100 shown in FIG. 3 also includes a feedback mechanism (means) that calculates the amount of the component to be purified recovered (recovered volume) x based on the measured concentration of the component to be purified and the volume of the recovered fraction, and controls the amount of the component to be purified in the feed solution to be supplied in the next feed cycle so that it is equal to the recovered amount x of the component to be purified.

[0026] The circulation system 100 shown in FIG. 3 supplies four types of eluents with different desorption powers. The "desorption power" of an eluent refers to the strength of its ability to desorb components adsorbed on an adsorbent from the adsorbent. The eluent 9a contained in the eluent tank 8a is supplied to the eluent supply line 12 by the eluent supply pump P3, whose supply flow rate is controllable. The eluent 9b contained in the eluent tank 8b is supplied to the eluent supply line 13 by the eluent supply pump P4, whose supply flow rate is controllable. The eluent 9c contained in the eluent tank 8c is supplied to the eluent supply line 14 by the eluent supply pump P5, whose supply flow rate is controllable. Furthermore, the eluent 9d contained in the eluent tank 8d is supplied to the eluent supply line 15 by the eluent supply pump P6, whose supply flow rate is controllable. The eluent supply pumps P3 to P6 are preferably metering pumps. In addition, it is preferable that the supply flow rate of each eluent is adjusted in conjunction with feedback control of the stock solution supply pump P2, thereby controlling the amount of eluent supplied to the circulation system. That is, it is preferable to provide a mechanism (means) for controlling the supply flow rate of each eluent according to the controlled amount of stock solution. As shown in FIG. 3 , the eluent supply line 12 is branched into four eluent supply branch lines 12a, 12b, 12c, and 12d, and the eluent can be supplied to the inlets of each of the unit packed towers 10a, 10b, 10c, and 10d via each of the eluent supply branch lines 12a, 12b, 12c, and 12d. Each of the eluent supply branch lines 12a, 12b, 12c, and 12d is provided with an openable eluent supply valve E1a, E2a, E3a, or E4a, and the eluent is supplied to the unit packed tower connected downstream through the eluent supply branch line having the open eluent supply valve. Similarly, the eluent supply line 13 is branched into four eluent supply branch lines 13a, 13b, 13c, and 13d, the eluent supply line 14 is branched into four eluent supply branch lines 14a, 14b, 14c, and 14d, and the eluent supply line 15 is branched into four eluent supply branch lines 15a, 15b, 15c, and 15d, so that each eluent can be supplied to the inlet of each of the unit packed towers 10a, 10b, 10c, and 10d.The branch eluent supply lines 13a, 13b, 13c, and 13d are provided with openable and closable eluent supply valves E1b, E2b, E3b, and E4b, respectively. The branch eluent supply lines 14a, 14b, 14c, and 14d are provided with openable and closable eluent supply valves E1c, E2c, E3c, and E4c, respectively. The branch eluent supply lines 15a, 15b, 15c, and 15d are provided with openable and closable eluent supply valves E1d, E2d, E3d, and E4d, respectively. The connection point between the branch eluent supply line in which an open eluent supply valve is installed and the pipe 1 serves as an eluent supply port D. The number of eluent supply ports D will depend on the type of eluent used.

[0027] <Operation of the Circulation System> Next, the operation of the circulation system when carrying out the method of the present invention using the above-mentioned circulation system will be described, but the present invention is not limited to these embodiments except as specified in the present invention. In the simulated moving bed chromatographic separation method, the stock solution supply position, eluent supply position, and each adsorbent fraction extraction position are intermittently moved in the fluid circulation direction of the circulation system while maintaining a constant positional relationship. By repeating this operation, a moving bed processing operation that can continuously supply stock solution is realized in a simulated manner. In this specification, the entire process from the intermittent movement of each of the above positions (or from the initial setting of each position) to moving to the next position is referred to as "one set."

[0028] In the method of the present invention, the target liquid can be supplied to and withdrawn from the target location by appropriately adjusting the operation of pumps and the opening and closing of valves at various locations in the circulation system. That is, the methods for supplying target fluids and withdrawing target fractions in circulation systems are well known. The supply and withdrawal flow rates of each liquid can also be appropriately set depending on the objective, such as processing efficiency. For example, the flow rate of the circulation pump can be changed between the first raw solution supply cycle and the second or subsequent raw solution supply cycles. While the simulated moving bed method is not limited to this method, a gradient simulated moving bed method is preferred, and a step gradient simulated moving bed method is more preferred. Gradient simulated moving bed chromatography is simulated moving bed chromatography that allows the desorption strength of the eluent to be changed. In particular, a step gradient method is defined as a method in which the desorption strength of the eluent changes stepwise. Stepwise elution refers to a method in which the desorption strength of the eluent changes stepwise. Such a gradient simulated moving bed method is, for example, the simulated moving bed method described in JP 2020-85881 A. In particular, in a step gradient method, it is necessary to maintain each component adsorbed in the column in each substep, which makes it easy for the sample to overload the column and cause the extrusion tailing phenomenon. Therefore, the method of the present invention is particularly suitable for use in step gradient simulated moving bed chromatography.

[0029] In the method of the present invention, a stock solution supply cycle is comprised of a step (i) of supplying the stock solution from the stock solution supply port and a step (ii) of stopping the supply of the stock solution following the step (i), in which the amount x of the component to be purified recovered from the outlet X through the steps (i) and (ii) is measured, and a stock solution containing the component to be purified in an amount equal to the amount x of the component to be purified is supplied to the stock solution supply port in the next stock solution supply cycle. That is, in the method of the present invention, the amount of stock solution supplied in one stock solution supply cycle is determined by the amount x of the component to be purified recovered in the immediately preceding stock solution supply cycle or the first stock solution supply cycle, and a stock solution containing the component to be purified in an amount equal to the amount x of the component to be purified is supplied to the circulation system in the next stock solution supply cycle. By controlling the amount of stock solution (the amount of the component to be purified in the stock solution) in this manner, the amount of the component to be purified in the circulation system can be controlled to be constant in each stock solution supply cycle, thereby enabling stable separation and purification of each component. Typically, by controlling the amount of the component to be purified in the raw solution as described above, the amounts of components other than the component to be purified in the raw solution also become constant in each cycle. The amount of the component to be purified can be measured by a conventional method, for example, by the method described in the Examples. When there are two or more components to be purified, the "amount of the component to be purified" can be the total amount of each component to be purified.

[0030] The amount of the component to be purified extracted from the circulation system may be measured only in the first feed cycle of the raw solution, and this amount x of the component to be purified may be treated as the amount of the component to be purified in the second or subsequent feed cycles of the raw solution. In other words, the amount of the component to be purified in the second or subsequent feed cycles of the raw solution may not actually be measured, but the amount of the component to be purified in the second or subsequent feed cycles of the raw solution may be controlled by assuming that the amount of the component to be purified in the second or subsequent feed cycles of the raw solution is the same as the amount x of the component to be purified in the first feed cycle of the raw solution. In this case, the amount of the raw solution supplied in the second or subsequent feed cycles of the raw solution is constant. Furthermore, the amount x of the component to be purified extracted from the circulation system may be monitored in each feed cycle of the raw solution. In this case, the amount of the raw solution supplied in the second or subsequent feed cycles of the raw solution may be controlled based on the amount x of the component to be purified extracted from the circulation system in the immediately preceding feed cycle of the raw solution. This allows the effects of the present invention to be achieved more reliably and efficiently. In short, in the method of the present invention, the amount of raw liquid supplied in the current raw liquid supply cycle may be controlled based on the amount of the component to be purified extracted in the first raw liquid supply cycle, or based on the amount of the component to be purified extracted in the raw liquid supply cycle immediately preceding the current raw liquid supply cycle, or based on the average amount of each component to be purified extracted in multiple raw liquid supply cycles prior to the current raw liquid supply cycle (for example, the immediately preceding 1 to 5 raw liquid supply cycles).

[0031] The amount of feed of the raw solution in the first feed cycle can be appropriately set so that each component contained in the raw solution can be separated, taking into consideration the concentration of each component contained in the raw solution, the amount of adsorbent packed in each column, the number of packed towers, etc.

[0032] The method of the present invention can use at least one eluent, preferably two or more eluents with different desorption strengths, more preferably three or more eluents with different desorption strengths, even more preferably four or more eluents with different desorption strengths, even more preferably four to six eluents with different desorption strengths, and particularly preferably four or five eluents with different desorption strengths. The type of eluent is not particularly limited and is appropriately selected depending on the type of adsorbent and the type of components in the raw solution. For example, when an ion exchange resin is used as the adsorbent, multiple eluents with different desorption strengths can be prepared by changing the salt concentration of the eluent. For example, when a cation exchange resin is used, multiple eluents with different NaCl concentrations can be used as two or more eluents.

[0033] In the method of the present invention, the adsorbent packed in the unit packed column is appropriately selected depending on the multiple components contained in the raw solution and the component to be purified, and various adsorbents can be used. For example, strongly acidic cation exchange resins, weakly acidic cation exchange resins, strongly basic anion exchange resins, weakly basic anion exchange resins, synthetic adsorbents, zeolites, silica gel, functionally modified silica gel (preferably octadecylsilyl-modified silica gel), and other gel filtration chromatography materials can be used as the adsorbent.

[0034] [Simulated Moving Bed Chromatographic Separation System] The simulated moving bed chromatographic separation system of the present invention is a system for carrying out the method of the present invention. That is, the SMB separation system of the present invention is a SMB separation system that separates components in a stock solution based on differences in their adsorptivity to the adsorbent, using a circulation system in which a plurality of unit packed towers packed with an adsorbent are connected in series and endlessly via piping. The piping of the circulation system is provided with a stock solution supply port, an eluent supply port, an outlet X for a fraction containing the component to be purified, and an outlet Y for a fraction containing components other than the component to be purified. The system has a stock solution supply cycle consisting of step (i) in which the stock solution is supplied from the stock solution supply port and step (ii) in which the supply of the stock solution is stopped following step (i), a means for measuring (capable of measuring) the amount x of the component to be purified recovered from the outlet X through steps (i) and (ii), and a means for supplying (capable of supplying) a stock solution containing the component to be purified in an amount equal to the amount x of the component to be purified to the stock solution supply port in the next stock solution supply cycle. In the simulated moving bed chromatographic separation system of the present invention, the configuration of the circulation system, the raw solution, the components to be purified, each process, etc., can be referenced from the description of the method of the present invention described above. Note that the control of each process and each means described above is performed by a control unit (a computer or the like, not shown) and the measuring device, etc., built into the system shown in Figure 3. For example, each valve, pump, and measuring device shown in Figure 3 are connected to the control unit so as to be able to communicate, and their operation is controlled by the control unit.

[0035] The present invention will be described in more detail below based on examples, but the present invention is not limited to the following examples.

[0036] <Preparation of Stock Solution> A 20-mer oligonucleotide (desalted, lyophilized) synthesized by TriLink BioTechnology was obtained and dissolved in adsorption buffer (20 mmol / L Tris-HCl buffer) to prepare stock solution (F). The oligonucleotide concentration in the stock solution was 15 mg / mL. The NaCl concentration in the stock solution was 5 mM.

[0037] <Eluent> As the eluent, the solutions shown in Table 1 below were prepared.

[0038]

[0039] <Apparatus> A continuous chromatography apparatus (trade name: Octave 10, manufactured by Tosoh Corporation) was used.

[0040] <Adsorbent used in unit packed tower (column)> A strong anion exchanger (trade name: TSKgel SuperQ-5PW, manufactured by Tosoh Corporation) was used as the adsorbent.

[0041] <Column> The columns were 5 mm in diameter and 50 mm in length, and five columns were connected in series and endlessly via piping.

[0042] <Recovered fractions> The relationship between the contents of the fractions adsorbed to the adsorbent and the outlets for these fractions was as follows: Weakly adsorbed fraction: N-1 deletion product of the component to be purified, collected from outlet A. Moderately adsorbed fraction: 20-mer oligonucleotide (component to be purified), collected from outlet B. Strongly adsorbed fraction: N+1 adduct of the component to be purified, collected from outlet C.

[0043] <Operating Steps> The operating steps of the simulated moving bed chromatographic separation method of this example are shown in Figures 4 and 5. Figure 4 shows the steps in the first cycle, and Figure 5 shows the steps in the second cycle. In Figures 4 and 5, a square box represents one unit packed tower, and the numbers inside the box represent the unit packed tower number. The stock solution supply port is designated F, the eluent supply ports are designated Q, E, D1, D2, and S, the weakly adsorbent fraction outlet is designated A, the moderately adsorbent fraction outlet is designated B, and the strongly adsorbent fraction outlet is designated C. The stock solution is supplied from the stock solution supply port, and the eluent corresponding to the supply port is supplied from each eluent supply port. The separation method in this example is comprised of step A, which is a step of supplying a stock solution and an eluent and extracting each fraction, and step B, which is a step of moving each supply port and extraction port downstream while maintaining their relative positional relationship after step A is completed, and step A and step B are combined to form one set. Step A is comprised of substeps A1 to A6, and A1 to A6 are performed in this order.

[0044] Comparative Example 1 Under the conditions listed in Table 2 below, step A, consisting of substeps A1 to A6 below, and the subsequent step B were carried out in order, and operation was continued up to the third cycle (the raw solution supply cycle was repeated three times) to separate and recover the oligonucleotide, the component to be purified, from the raw solution. The amount of raw solution supplied was the same in each raw material supply cycle, and the amount of the component to be purified was 20 mg per raw material supply cycle. The amount of the component to be purified supplied in each raw material supply cycle exceeded the allowable load capacity of the column. The operating conditions listed below were set so that the purity of the component to be purified in the first cycle was 99% or higher. The same applies to the following examples.

[0045]

[0046] Example 1 Under the conditions listed in Table 3 below, Step A, consisting of substeps A1 to A6 below, and the subsequent Step B were carried out in order, and operation was continued up to cycle 5 (repeated five times with the stock solution supply cycle), to separate and recover oligonucleotides, the components to be purified, from the stock solution. The supply amount of stock solution was 20 mg of the component to be purified per stock solution supply cycle in the first cycle, and from cycle 2 onwards, the supply amount was set to an amount containing the component to be purified that was approximately the same as the amount of the component to be purified recovered in cycle 1 (16 mg of the component to be purified per stock solution supply cycle). Furthermore, the process times from cycle 2 onwards were also changed to the times listed in Table 3 below depending on the supply amount of stock solution.

[0047]

[0048] <Results> The purity and recovery rate of the purified components recovered in each cycle in the Comparative Examples and Examples are shown in the table below. Purity was measured by high-performance liquid chromatography. The column used for analysis was TSKgel-DNA NPR (manufactured by Tosoh Corporation). Recovery rate was calculated by 100 x [mass in fraction] / [mass in original solution].

[0049]

[0050] As shown in Table 4 above, in the comparative examples in which the target components were separated using a conventional simulated moving bed chromatographic separation method, the purities were all high, at over 99%, but the recovery rates were around 80%, resulting in poor yields. This indicates that only 80% of the target substance amount in the feed solution was always recovered, indicating that 20% of the target components supplied in each feed solution cycle accumulated in the column during operation. As the target components accumulated in the circulation system, the extrusion tailing phenomenon became more pronounced, and repeated operation of this type is thought to eventually render the separation system unable to maintain its performance. In contrast, in the examples in which the target components were separated using the method of the present invention, the purities were all over 99%, and while the recovery rate in the first cycle was 78.1%, recovery rates of approximately 100% were achieved from the second cycle onwards. In the examples, the amount of raw liquid supplied from the second cycle onwards was set to a supply amount of raw liquid containing approximately the same amount of the component to be purified as the amount of component to be purified recovered in the first cycle, so it was possible to recover almost all of the component to be purified supplied in each raw liquid supply cycle, demonstrating that stable operation was possible without excessive accumulation.

[0051] As described above, it has been demonstrated that the method of the present invention enables the separation and purification of target components with high purity and high recovery rate.

[0052] While the present invention has been described in connection with embodiments thereof, we do not intend to limit our invention to any of the details of the description unless otherwise specified, and believe that the claims should be construed broadly without departing from the spirit and scope of the invention as set forth in the appended claims.

[0053] This application claims priority based on Japanese Patent Application No. 2024-135426, filed on August 14, 2024, the contents of which are incorporated herein by reference as part of the present specification.

[0054] 100 Circulation system 10a, 10b, 10c, 10d Unit packed tower (column) Ab Adsorbent R1, R2, R3, R4 Shutoff valve 2a, 2b, 2c, 2d Weakly adsorbent fraction withdrawal line A1, A2, A3, A4 Weakly adsorbent fraction withdrawal valve 3a, 3b, 3c, 3d Medium adsorbent fraction withdrawal line B1, B2, B3, B4 Medium adsorbent fraction withdrawal valve 4a, 4b, 4c, 4d Strongly adsorbent fraction withdrawal line C1, C2, C3, C4 Strongly adsorbent fraction withdrawal valve T1, T2, T3, T4 Check valve 1 Piping 2J Weakly adsorbent fraction junction pipe 3J Medium adsorbent fraction junction pipe 4J Strongly adsorbent fraction junction pipe 6 Stock solution tank 7 Stock solution 8a, 8b, 8c, 8d Eluent tank 9a, 9b, 9c, 9d Eluent 11 Stock solution supply line 11a, 11b, 11c, 11d Stock solution supply branch line F1, F2, F3, F4 Stock solution supply valve 12, 13, 14, 15 Eluent supply lines 12a, 12b, 12c, 12d Eluent supply branch line 13a, 13b, 13c, 13d Eluent supply branch line 14a, 14b, 14c, 14d Eluent supply branch line 15a, 15b, 15c, 15d Eluent supply branch line E1a, E2a, E3a, E4a Eluent supply valve E1b, E2b, E3b, E4b Eluent supply valve E1c, E2c, E3c, E4c Eluent supply valve E1d, E2d, E3d, E4d Eluent supply valve P1 Circulation pump P2 Stock solution supply pump P3, P4, P5, P6 Eluent supply pump

Claims

1. A simulated moving bed chromatographic separation method using a circulation system in which a plurality of unit packed towers filled with adsorbent are connected in series and endlessly via piping to separate components in a stock solution based on differences in their adsorption strength to the adsorbent, wherein the piping of the circulation system is equipped with a stock solution supply port, an eluent supply port, an outlet X for a fraction containing a component to be purified, and an outlet Y for a fraction containing components other than the component to be purified, wherein in a stock solution supply cycle consisting of step (i) of supplying the stock solution from the stock solution supply port and step (ii) of stopping the supply of the stock solution following step (i), the amount x of the component to be purified recovered from outlet X through steps (i) and (ii) is measured, and a stock solution containing the component to be purified in an amount equal to the amount x of the component to be purified is supplied to the stock solution supply port in the next stock solution supply cycle.

2. The simulated moving bed chromatographic separation method according to claim 1, wherein the simulated moving bed is a gradient simulated moving bed.

3. A simulated moving bed chromatographic separation method according to claim 2, wherein the component to be purified is a component that is strongly or moderately adsorbent to the adsorbent.

4. A simulated moving bed chromatographic separation method according to any one of claims 1 to 3, wherein the amount of raw solution supplied in the second or subsequent raw solution supply cycles is controlled based on the amount x of the component to be purified recovered in the first raw solution supply cycle.

5. A simulated moving bed chromatographic separation method according to any one of claims 1 to 3, wherein the amount x of the component to be purified recovered in each feed cycle is monitored, and the amount of feed solution to be supplied in the next feed cycle is controlled based on the results of this monitoring.

6. A simulated moving bed chromatographic separation system that separates components in a stock solution based on differences in their adsorption strength to the adsorbent, using a circulation system in which a plurality of unit packed towers filled with adsorbent are connected in series and endlessly via piping, wherein the piping of the circulation system is provided with a stock solution supply port, an eluent supply port, an outlet X for fractions containing the component to be purified, and an outlet Y for fractions containing components other than the component to be purified, and wherein in a stock solution supply cycle consisting of step (i) in which the stock solution is supplied from the stock solution supply port and step (ii) in which the supply of the stock solution is stopped following step (i), the simulated moving bed chromatographic separation system has means for measuring the amount x of the component to be purified recovered from outlet X through steps (i) and (ii), and means for supplying a stock solution containing the component to be purified in an amount equal to the amount x of the component to be purified to the stock solution supply port in the next stock solution supply cycle.

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