Purification method and its use

Abstract

A cyclic chromatographic purification process for isolating a product P from a feed mixture F consisting of the product P and at least two further components corresponding to weakly adsorbable impurities W and strongly adsorbable impurities S, which uses only two chromatographic adsorbent sections as chromatographic stationary phases and comprises at least one basic sequence having at least one recycle step followed by only one purification step, preferably repeating the basic sequence cyclically.

Description

[Technical Field]

[0001] The present invention relates to a cyclic chromatographic method for producing high-purity therapeutic drugs originating from the chemical synthesis of peptides, oligonucleotides, and the like. [Background technology]

[0002] The purification process for active substances such as therapeutic peptides, oligonucleotides, and proteins typically involves a series of chromatographic steps.

[0003] In many cases of peptide and oligonucleotide production, the target compound is obtained by chemical synthesis. In this process, product-related impurities are generated in addition to the target compound, and these need to be removed in downstream processes.

[0004] Chromatography, due to its high selectivity, is an essential unit operation for removing product-related impurities. The goal of chromatography is to produce a product pool that meets purity specifications while maintaining high product yield and throughput.

[0005] Linear gradient chromatography is frequently used to achieve this objective. In linear gradient chromatography, after the product is bound to the stationary phase in the chromatographic adsorbent, the composition of the mobile phase, which is pumped through the adsorbent, is gradually changed over time at a constant gradient.

[0006] This results in the sequential desorption of impurities that elute early (weakly adsorbed), the product, and then impurities that elute later (strongly adsorbed). In the chromatogram, compounds that elute in this order appear as a series of peaks. The separation ability of the compounds can be improved by performing a gentler linear gradient, but this comes at the cost of increased processing time and decreased throughput. Similarly, the separation ability of compounds can be improved by reducing the mass of the starting material (feed) loaded into the column, but this also reduces throughput.

[0007] In preparative chromatography, throughput is crucial, limiting the duration of the gradient and thus causing overlap between impurity and product peaks in the preparative chromatogram. The highest product purity is typically obtained at the center of the product peak, and this portion of the chromatogram is collected as a pool at the adsorbent outlet during elution. Including side fractions containing products where impurity and product peaks overlap leads to a decrease in product pool purity. To avoid violating purity constraints, it is typically necessary to exclude some of the product in the side fractions from the product pool. Fractions of product not included in the product pool may even constitute the majority of the product contained in the starting material loaded onto the adsorbent. To avoid product loss, there is considerable interest in recovering the product contained in impure side fractions. Side fractions are often subjected to rechromatography; that is, the side fractions are used as a load material to perform the same or similar chromatographic unit operation. Through this operation, the product fraction can be recovered in purity, but separation is more difficult because this load material has a higher impurity content than the usual feed material. Rechromatography also has a range of operational disadvantages, including regulatory limitations, storage and handling of secondary fractions, and stability and quality control of secondary fractions.

[0008] Several processes have been proposed to automate the recycling of impure by-fractions in chromatography. These processes can be classified into single-adsorbent recycling processes and multiple-adsorbent recycling processes.

[0009] Single-adsorbent systems include recirculation of the chromatography profile through the same column. In a steady-state recirculation (SSR) process, fractions are collected from the beginning and end of the circulating chromatography profile, and new samples are injected into the profile.

[0010] In a multi-adsorbent recirculation process, it is possible to combine the internal recirculation of impure by-fractions from one adsorbent to another with the countercurrent principle, i.e., the use of the relatively opposite movement of the stationary and mobile phases, thereby improving the separation of products and impurities. Automated recirculation avoids the generation and collection of by-fractions, as well as their external storage, handling, and analysis, and only pure products are recovered from the process in high yield.

[0011] Multiple adsorbent processes that combine internal recirculation and countercurrent principles are known as pseudo-moving bed (SMB) processes. Early SMB processes were limited to the separation of two compounds (two-component separation) and could not be operated under linear gradient conditions; therefore, their applications were limited to separations that did not require a center cut in the chromatogram.

[0012] The concept of SMB was further developed to yield a very efficient process for center-cut (three-component system) separation with linear solvent gradient performance known as the "MCSGP" process (Multicolumn Countercurrent Solvent Gradient Purification) (see Patent Document 1). This process is well established in the industry. A number of adsorber and other chromatographic multi-adsorber technologies using internal recycle, such as the "gradient with steady state recycle" (GSSR) process, have been proposed.

[0013] Although MCSGP is described for column configurations of two to eight, in practice, a configuration of mainly two adsorbers is used because equipment with more columns has lower equipment complexity and higher operational flexibility.

[0014] The MCSGP process is designed based on the chromatogram of single-column batch chromatography. Generally, it is possible to obtain a product with a purity corresponding to the highest purity fraction from the batch chromatogram using MCSGP. In some cases, the possible maximum purity obtained by MCSGP is limited because overlapping impurities may extend far below the product peak.

Prior Art Documents

Patent Documents

[0015]

Patent Document 1

Summary of the Invention

[0016] An object of the present invention is to provide an improved MCSGP-like process that can achieve a higher product purity than the normal MCSGP process.

[0017] Surprisingly, by introducing an additional recirculation step to a process similar to the MCSGP process and combining it with a change in the loading scheme, it has been found that not only higher purity but also higher or equivalent productivity (throughput) at equivalent yields can be achieved.

[0018] Due to the proposed additional recirculation step, the chromatography profile is transferred from one chromatography adsorbent to another, and thus in-line dilution is applied, and this chromatography profile is re-adsorbed on the downstream adsorbent. Typically, this in-line dilution is carried out by essentially continuously adding an eluent without a modifier or with a low modifier content between two columns to reduce the elution strength of the downstream liquid and slow down the elution in the downstream column. While a typical modifier in the case of reverse-phase chromatography is an organic solvent, in the case of ion exchange, typical modifiers are salts (adjusting the ionic strength) or acids / bases (adjusting the pH). The in-line dilution is set at a level where complete adsorption of the compounds eluting from the upstream column is expected, and is not intended to promote further separation in the downstream column during the recirculation step. Therefore, it is expected that the partial separation of the compounds in the chromatography profile obtained by elution from the upstream will be nullified by the in-line dilution. In other words, due to the expected loss of separation by in-line dilution, the same situation as after the feed injection occurs.

[0019] Therefore, one skilled in the art should expect that the introduction of a recirculation step with normal or "excessive" in-line dilution will not lead to an improvement in product purity.

[0020] Furthermore, one skilled in the art should expect that the introduction of a recirculation step will take extra time, during which no additional feed is introduced and no product is produced, and thus the productivity (throughput) of the entire process will decrease due to the introduction of the recirculation step.

[0021] Surprisingly, it was found that the time loss caused by the introduction of the recirculation stage could be overcompensated by the increase in load. In addition, recirculation can achieve significantly higher purity.

[0022] In this method, two chromatography adsorbent sections are used as the chromatography stationary phase. Each adsorbent section may consist of a single column or several columns, in which case the columns of one section are always interconnected and never interrupted during the process.

[0023] The first adsorbent section has a first adsorbent section inlet and a first adsorbent section outlet, and the second adsorbent section has a second adsorbent section inlet and a second adsorbent section outlet.

[0024] This method includes an optional but preferred starting step, a recirculation step with one or more recirculation sequences (n≧1), and a purification step performed only once after the recirculation step. The recirculation and purification steps form a so-called basic sequence, which is repeated at least once (m≧1). An optional but preferred stopping step follows the desired number of basic sequences.

[0025] This basic principle is schematically shown in Figure 1.

[0026] Figure 2 illustrates the method for various cycle counts n=1, 2, 3, and 4 in the recirculation stage. All examples begin in the startup stage and end in the shutdown stage. In the startup stage, a feed mixture F is loaded onto a first adsorbent 1, which is usually pre-equilibriumized. The feed mixture contains not only the desired product P but also impurities, namely, less adsorbent impurities W and more adsorbent impurities S. Subsequently, the less adsorbent impurities W, i.e., impurities that adsorb less weakly to the stationary phase than product P, are eluted from this adsorbent with or without the continuation of the feed flow (as preferred in the example). The process then enters the recirculation stage.

[0027] When n=1, the recirculation step itself consists of one cycle, i.e., one interconnection (IC-R) step and one disconnection (BR) step. In the interconnection step, preferably with a gradient, the outlet of the first adsorbent 1 is connected to the inlet of the second adsorbent 2 while supplying the solvent (see below, in particular Figure 5). In-line dilution is performed between the adsorbents. In-line dilution is performed using a base solvent and therefore without a gradient.

[0028] Next, in the subsequent fragmentation step BR, the adsorbent is fragmented, and highly adsorbent impurities S, i.e., impurities that adsorb more strongly to the stationary phase than the product, are eluted from the first adsorbent 1, thereby re-equilibriumizing this adsorbent. Meanwhile, less adsorbent impurities W are eluted from the second adsorbent 2. This latter fragmentation step BR in the recirculation stage is optional.

[0029] When n=2, the recirculation step consists of the recirculation step for n=1 plus one more cycle, namely one more interconnection step and one more (optional) separation step. In the further interconnection step, the outlet of the former second adsorbent 2 is connected to the inlet of the former first adsorbent 1. Then, in the next further separation step, the adsorbent is separated, the strongly adsorbent impurities S are eluted from the second adsorbent 2, and the adsorbent is re-equilibriumized, while the weakly adsorbent impurities W are eluted from the first adsorbent 1. This latter further separation step is again optional.

[0030] When n=3, the recirculation step consists of the recirculation step for n=2 plus one more interconnection step and one more (optional) separation step. In the interconnection step, the outlet of the first adsorbent 1 is connected to the inlet of the second adsorbent 2. Next, in the separation step, the adsorbent is separated, the strongly adsorbent impurities S are eluted from the first adsorbent 1, and the adsorbent is re-equilibriumized, while the weakly adsorbent impurities W are eluted from the second adsorbent 2. This latter separation step is again optional.

[0031] When n=4, the recirculation step consists of the recirculation step for n=3 plus one more interconnection step and one more separation step. In the interconnection step, the outlet of the second adsorbent 2 is connected to the inlet of the first adsorbent 1. Next, in the separation step, the adsorbent is separated, and the strongly adsorbent impurities S are eluted from the second adsorbent 2, re-equilibriumizing the adsorbent, while the weakly adsorbent impurities W are eluted from the first adsorbent 1. This latter separation step is optional.

[0032] Generally, for any number of cycles (n>1), the recirculation stage consists of n-1 processes and the recirculation stage, plus one interconnection stage and one (optional) disconnection stage.

[0033] Generally, in the case of even n=2, 4, 6, ... recirculation stages, the outlet of the second adsorbent 2 is connected to the inlet of the first adsorbent 1 in the final interconnection step of the recirculation stage. Then, if implemented, in the following separation step, the adsorbent is separated, the strongly adsorbent impurities S are eluted from the second adsorbent 2, and this adsorbent is re-equilibriumized, while the weakly adsorbent impurities W are eluted from the first adsorbent 1. This latter separation step is optional.

[0034] Generally, in the case of an odd number n=1, 3, 5, ... recirculation stage, the outlet of the first adsorbent 1 is connected to the inlet of the second adsorbent 2 in the final interconnection step of the recirculation stage. Then, in the following splitting step, the adsorbent is split, and the strongly adsorbent impurities S are eluted from the first adsorbent 1, re-equilibriumizing this adsorbent, while the less adsorbent impurities W are eluted from the second adsorbent 2. This latter splitting step is optional.

[0035] The recirculation stage is followed by the purification stage. Each purification stage includes a first interconnection step IC1, a first batch (dispersion) step B1, a second interconnection step IC2, and a second batch (dispersion) step B2.

[0036] For n=1 and n=3, and for any odd natural number n, in the interconnection step IC1 of the purification stage, the outlet of the second adsorbent 2 is connected to the inlet of the first adsorbent 1. That is, the second adsorbent 2 is upstream of the first adsorbent 1, and in-line dilution is performed between the two adsorbents. In the subsequent batch step B1, the adsorbents are separated, the purified product P is eluted from the second adsorbent 2 and collected, while a new feed mixture F is loaded onto the first adsorbent 1. In the next interconnection step IC2, the adsorbents are connected, with the second adsorbent 2 upstream of the first adsorbent 1, and in-line dilution is performed between the two adsorbents.

[0037] In the next fragmentation step B2, the adsorbent is fragmented, the strongly adsorbent impurity S is eluted from the second adsorbent 2, and this adsorbent is re-equilibriumized, while the weakly adsorbent impurity W is eluted from the first adsorbent 1.

[0038] For n=2 and n=4, and for any even natural number n, in the interconnection step IC1 of the purification stage, the outlet of the first adsorbent 1 is connected to the inlet of the second adsorbent 2. That is, the first adsorbent 1 is upstream of the second adsorbent 2, and in-line dilution is performed between the two adsorbents. In the subsequent batch step B1, the adsorbents are separated, the purified product P is eluted from the first adsorbent 1 and collected, while a new feed mixture F is loaded into the second adsorbent 2. In the next interconnection step IC2, the adsorbents are connected, with the first adsorbent 1 upstream of the second adsorbent 2, and in-line dilution is performed between the two adsorbents.

[0039] In the next fragmentation step, the adsorbent is fragmented, the highly adsorbent impurity S is eluted from the first adsorbent 1, and this adsorbent is re-equilibriumized, while the less adsorbent impurity W is eluted from the second adsorbent 2.

[0040] For any number of recycle steps n times, after the desired number of basic sequences m times, the final purification step is followed by a stopping step as described in Figure 3.

[0041] Figure 3 shows the stopping stages for various recycle steps n=1, 2, 3, 4, .... A stopping stage follows the final purification step of the desired number of basic sequences, and this stopping stage itself consists of two parts: stopping stage I and stopping stage II.

[0042] The first stage, stop stage I, consists of the same steps as the final IC-R step and (optional) BR step of the preceding recirculation stage of the process.

[0043] The second stopping stage, stopping stage II, consists of the same steps as the preceding purification stage of the process, but with some modifications, and is extended by additional steps to elute product P and impurities S, however, this is carried out without further loading of a new feed mixture F.

[0044] For n=1, n=3, and any odd natural number n, the stopping step I consists of an interconnection step, where the outlet of the first adsorbent 1 is connected to the inlet of the second adsorbent 2. In-line dilution is performed between the adsorbents. In the next separation step, the adsorbents are separated, strongly adsorbent impurities S are eluted from the first adsorbent 1, and this adsorbent is re-equilibriumized, while weakly adsorbent impurities W are eluted from the second adsorbent 2. This latter separation step is optional.

[0045] For n=1 and n=3, and any odd natural number n, stopping stage II consists of interconnection step IC1, in which the outlet of the second adsorbent 2 is connected to the inlet of the first adsorbent 1. That is, the second adsorbent 2 is upstream of the first adsorbent 1, and in-line dilution is performed between the two adsorbents. In the subsequent batch step B1, the adsorbents are separated, and the purified product P is eluted from the second adsorbent 2 and collected. In this step, no new feed mixture F is loaded onto the first adsorbent 1. In the next interconnection step IC2, the adsorbents are connected, with the second adsorbent 2 upstream of the first adsorbent 1, and in-line dilution is performed between the two adsorbents. Subsequently, the purified product P is eluted from the first adsorbent 1 and collected, and highly adsorbent impurities S are eluted from the first adsorbent 1.

[0046] For n=2, n=4, and any even natural number n, stopping stage I consists of the same steps as stopping stage I when n is odd, but adsorbent 1 and adsorbent 2 are swapped. And for n=2, n=4, and any even natural number n, stopping stage II consists of the same steps as stopping stage II when n is odd, but adsorbent 1 and adsorbent 2 are swapped.

[0047] Figure 4 shows the tasks of two adsorbents in the startup phase for any number of times n. In the startup phase, the feed mixture F is loaded onto a pre-equilibriumized first adsorbent 1 (step B-SU-F). Subsequently, weakly adsorbent impurities W are eluted from this adsorbent (step B-SU-W). The linear gradient segment, flow rate, and switching time operated within the first adsorbent 1 in step B-SU-W preferably correspond to those selected for a single-column chromatogram.

[0048] Generally, as shown in the lower part of Figure 4, the operating parameters, namely the flow rate, gradient concentration, supply rate, and switching time of this method, can be derived from the single-adsorbent chromatogram. Therefore, this method can be designed and initialized based on the single-adsorbent chromatogram, allowing for more precise control during operation, for example, based on detector feedback.

[0049] Figure 5 shows the recirculation steps of this method when n=1.

[0050] The recirculation step itself consists of at least one sequence of interconnection (IC-R) and disconnection (BR) steps. In the first interconnection step (IC-R), the outlet of the first adsorbent 1 is connected to the inlet of the second adsorbent 2. The adsorbed compounds W, P, and S are eluted from the first adsorbent 1 to the second adsorbent 2 using a solvent gradient. Here, in-line dilution is performed between the adsorbents using a base solvent that does not contain modifiers or a solvent with a low modifier content, so that W, P, and S are strongly adsorbed onto the second adsorbent 2, and preferably, impurities W that are not even weakly adsorbed exit from the downstream adsorbent 2. Next, in the following first fragmentation step (BR), the adsorbent is fragmented, and the remaining impurities S are eluted from the first adsorbent 1 using a high, preferably constant, modifier concentration, after which the adsorbent 1 is re-equilibrated. Meanwhile, the less adsorbent impurities W are eluted from the second adsorbent 2 using a gradient with a low starting modifier concentration (see the dashed lines at the bottom of each step showing the modifier concentrations depending on the process). This latter fragmentation step BR is optional, and its importance depends on the purity and purity specifications of the product P in the feed mixture F. Lower purities correspond to higher content of W and S, so it will be considered to include a fragmentation step (BR) in the recirculation step to remove W and S. Again, as shown at the bottom of Figure 5, the operating parameters, namely the flow rate, gradient concentration, feed rate, and switching time of the method, can be derived from the single adsorbent chromatogram, where more precise control during operation is possible, for example, based on detector feedback.

[0051] When n=1, the stopping stage I is the same as the recirculation stage. Each recirculation stage is followed by a purification stage (Figures 2 and 6).

[0052] Figure 6 shows the purification steps of this method for n=1, 3, 5, ...

[0053] Each purification step includes a first interconnection step IC1, a first batch (dispersion) step B1, a second interconnection step IC2, and a second batch (dispersion) step B2.

[0054] In interconnection step IC1, the outlet of the second adsorbent 2 is connected to the inlet of the first adsorbent 1. That is, the second adsorbent 2 is upstream of the first adsorbent 1. In interconnection step IC1, a partially pure subfraction containing W and P (the overlapping region in the chromatogram (see schematic chromatogram at the bottom of Figure 6) ("zone 5")) is eluted from the second adsorbent 2 to the first adsorbent 1. During operation IC1, the flow containing W and P is diluted in-line before entering the first adsorbent 1, so that W and P are completely adsorbed when they enter the first adsorbent 1. In the subsequent batch step B1, the adsorbents are fragmented, the purified product P is eluted and collected from the second adsorbent 2, while a new feed mixture F is loaded onto the first adsorbent 1. In the next interconnection step IC2, the adsorbents are connected, with the second adsorbent 2 located upstream of the first adsorbent 1, eluting a partially pure subfraction containing P and S (the overlapping region in the chromatogram (see bottom of Figure 6) ("zone 7")) from the second adsorbent 2 to the first adsorbent 1. The flow containing P and S is diluted in-line before entering the first adsorbent 1, so that compounds P and S are completely adsorbed upon entering the first adsorbent 1. Again, as shown at the bottom of Figure 6, the operating parameters, namely the flow rate, gradient concentration, feed rate, and switching time of this method, can be derived from the single adsorbent chromatogram, allowing for more precise control during operation, for example, based on detector feedback.

[0055] Figure 7: To avoid the loss of product P at the end of the process operation, a stopping stage follows the purification stage, and this stopping stage itself consists of two parts: stopping stage I and stopping stage II.

[0056] For n=1, 3, 5…, the halting stage I is functionally identical to the recycle stage for n=1 shown in Figure 5. The halting stage I includes processes IC'-R and B'-R, which use the same conditions as the final sequence of the process recycle stage.

[0057] Figure 7 shows the termination stage II of this method for n=1, 3, 5, ...

[0058] Stopping step II includes essentially the same steps as the preceding purification step of the method, but without a feed (see Figure 6) (IC1-SD corresponds to IC1, B1-SD corresponds to B1, but without a feed, IC2-SD corresponds to IC2, and B2-SD corresponds to B2), except that it is extended by an additional step (B-SD-P) to elute product P from a first adsorbent previously located downstream, and an additional step (B-SD-S) to elute impurities S and clean and regenerate the same adsorbent. The conditions for collecting P in B-SD-P and B1-SD are preferably the same in terms of flow rate and concentration gradient. Similarly, the conditions for removing S in B-SD-S and B2-SD are preferably the same in terms of flow rate and concentration gradient.

[0059] If the preceding recirculation stage includes an even number of sequences (n=2, 4, 6…) of interconnection step IC-R and disconnection step BR, the positions of the adsorbents are swapped in steps IC1-SD, B1-SD, IC2-SD, and B2-SD. Specifically, in interconnection step IC1-SD, the outlet of the first adsorbent 1 is connected to the inlet of the second adsorbent 2, so the first adsorbent 1 is upstream of the second adsorbent 2. Similarly, the positions of the adsorbents are swapped in steps B1-SD, IC2-SD, and B2-SD.

[0060] In this process, gradients, particularly linear gradients, can and preferably be used in any step to enhance the separation of compounds and / or change the elution rate by changing the gradient slope.

[0061] By selecting a steeper gradient in the recirculation stage (IC-R and / or BR) than in the purification stage (IC1, B1, IC2, and / or B2), the recirculation stage can be completed more quickly, thus improving overall process productivity.

[0062] More generally, the present invention relates to a cyclic chromatographic purification method for isolating product P from a feed mixture F comprising product P and at least two further components corresponding to weakly adsorbed impurities W and strongly adsorbed impurities S.

[0063] In the proposed method, only two chromatographic adsorbent sections are used as the chromatographic stationary phase. The first adsorbent section has a first adsorbent section inlet and a first adsorbent section outlet, and the second adsorbent section has a second adsorbent section inlet and a second adsorbent section outlet.

[0064] The proposed method comprises at least one basic sequence having at least one recirculation step followed by only one purification step, where preferably the basic sequence is repeated cyclically at least twice.

[0065] According to the present invention, the above recirculation step consists of the following steps: a. Interconnection recirculation process IC-R, In this process, the adsorbent sections are interconnected, and the recirculation interconnection period t IC-R Inside, the outlet of the upstream adsorbent section is connected to the inlet of the downstream adsorbent section, and here, In the upstream adsorbent section, the eluent is loaded through the inlet of the upstream adsorbent section, and a fraction containing a weakly adsorbent impurity W in a range that overlaps with at least the product P, the product P, and a strongly adsorbent impurity S in a range that overlaps with at least the product P is eluted from the upstream adsorbent section to the downstream adsorbent section, and The flow exiting the upstream adsorbent outlet is diluted in-line before it enters the downstream adsorbent inlet; b. Any batch recirculation process BR, Here, the recycle batch period t B-R The above adsorbent section is divided inside, and The preceding upstream adsorbent section of step a is cleaned to remove and regenerate highly adsorbent impurities S, and the eluent is loaded into the inlet of the preceding downstream adsorbent section of step a to elute less adsorbent impurities W that do not overlap with the product P; The recirculation sequence comprises at least one recirculation sequence, preferably just one sequence, or at least two, or at least three, or at least four recirculation sequences, After each recirculation sequence in process a and process b, the adsorbent sections are switched sequentially, with each recirculation sequence occurring at least once.

[0066] According to the present invention, the recirculation stage is followed by only one purification stage. This purification stage consists of the following steps: c. First interconnection purification process IC1, In this process, the adsorbent sections are interconnected, and here, the first interconnection purification period t IC1 Inside, the outlet of the upstream adsorbent section is connected to the inlet of the downstream adsorbent section. The eluent is loaded onto the upstream adsorbent via the upstream adsorbent inlet, and the product fraction containing the overlapping weakly adsorbent impurities W and product P is eluted from the upstream adsorbent to the downstream adsorbent section, and The flow exiting the upstream adsorbent section outlet is diluted in-line before entering the downstream adsorbent section inlet; d. First batch purification process B1, Here, the first batch purification period t B1 The adsorbent section is divided, the product P is eluted from the previous upstream adsorbent section, and the feed mixture F is supplied to the inlet of the previous downstream adsorbent section; e. Second interconnection purification process IC2, Here, the second interconnection purification period t IC2 Inside, the outlet of the upstream adsorbent section is connected to the inlet of the downstream adsorbent section. The eluent is loaded into the upstream adsorbent section via the inlet of the upstream adsorbent section, and the product fraction containing the overlapping product P and the highly adsorbent impurities S is eluted from the upstream adsorbent to the downstream adsorbent, and The flow exiting the upstream adsorbent section outlet is diluted in-line before entering the downstream adsorbent section inlet; f. Second batch purification process B2, Here, the second purification batch period t B2 The adsorbent is broken up inside, the previous upstream adsorbent is cleaned and regenerated, and the eluent is loaded into the previous downstream adsorbent inlet; This includes, in order, the upstream adsorbent section of the final interconnection recirculation step in the preceding recirculation stage performs the function of the downstream adsorbent section, and the downstream adsorbent section of the final interconnection recirculation step in the preceding recirculation stage performs the function of the upstream adsorbent section.

[0067] As noted above, preferably, an eluent gradient is used at least once or in all stages.

[0068] Preferably, in the interconnection step of the method, an eluent having a gradient in the form of a time-varying modifier concentration is loaded into the upstream section inlet via the upstream adsorbent inlet, and the flow exiting the upstream adsorbent section outlet is inline diluted with an eluent without a modifier, or with an eluent having a different modifier concentration than that of the upstream adsorbent section inlet, preferably a lower modifier concentration than that of the upstream adsorbent section inlet, before it enters the downstream adsorbent section inlet.

[0069] More preferably, in a batch process that does not involve elution of the purified product, the previous upstream adsorbent is cleaned and regenerated with an eluent having a higher modifier concentration than at the end of the preceding interconnection recirculation process, or with an eluent containing a different modifier, or with a purification solution, and an eluent having a gradient in the form of a time-varying modifier concentration is loaded into the inlet of the previous downstream adsorbent.

[0070] In a batch step of a method involving the elution of the purified product, preferably, an eluent having a gradient in the form of a time-varying modifier concentration is loaded onto a previous upstream adsorbent via the upstream adsorbent inlet.

[0071] In the recirculation step IC-R described above, according to a preferred embodiment, an eluent having a gradient in the form of a time-varying modifier concentration is loaded into the upstream adsorbent section via the upstream adsorbent section inlet, and the flow exiting the upstream adsorbent outlet is inline diluted with an eluent that does not contain a modifier, or with an eluent having a different modifier concentration than that of the upstream adsorbent section inlet, before it enters the downstream adsorbent inlet.

[0072] In yet another preferred embodiment, in any batch recirculation step BR, the previous upstream adsorbent section of the preceding step a is cleaned and regenerated with an eluent having a higher modifier concentration than at the end of the preceding interconnection recirculation step, or with an eluent containing different modifiers, or with a cleansing solution, and an eluent having a gradient in the form of a time-varying modifier concentration is loaded into the inlet of the previous downstream adsorbent section of step a to elute weakly adsorbent impurities W in a range that does not overlap with the product P.

[0073] A further preferred embodiment is characterized in that, in the purification step of the first interconnection purification step IC1, an eluent having a gradient in the form of a time-varying modifier concentration is loaded into the upstream adsorbent section via the upstream adsorbent inlet, and the flow exiting the upstream adsorbent section outlet is inline diluted with an eluent that does not contain a modifier, or with an eluent having a different modifier concentration than that of the upstream adsorbent section inlet, before it enters the downstream adsorbent section inlet.

[0074] Furthermore, in the first batch purification step B1 described above, it is preferable to elute the product P from the preceding upstream adsorbent section by loading an eluent having a gradient in the form of a time-varying modifier concentration into the upstream adsorbent section through its inlet.

[0075] In the second interconnection purification step IC2 described above, preferably, an eluent having a gradient in the form of a time-varying modifier concentration is loaded into the upstream adsorbent section inlet via the upstream adsorbent inlet, and the flow exiting the upstream adsorbent section outlet is inline diluted with an eluent without a modifier (basic solvent only) or with an eluent having a different modifier concentration than that of the upstream adsorbent section inlet before it enters the downstream adsorbent section inlet.

[0076] Furthermore, in the second batch purification step B2 described above, it is preferable to purify and regenerate the previous upstream adsorbent with an eluent having a higher modifier concentration than at the end of the preceding interconnection recirculation step, or with an eluent containing a different modifier, or with a purification solution, and load an eluent having a gradient in the form of a time-varying modifier concentration into the inlet of the previous downstream adsorbent.

[0077] The modifier is preferably selected from the group consisting of an organic solvent or inorganic solvent (or a mixture thereof) different from the base solvent (or a mixture thereof) of the eluent, or an electrolyte in such an organic solvent or inorganic solvent (or a mixture thereof), and is preferably selected from a dissolved salt, pH, or a combination thereof.

[0078] Preferably, the base solvent is an organic solvent or an inorganic solvent (or a mixture thereof), particularly water, or a mixture of water and at least one organic solvent, optionally containing a buffer salt, acid, or base, or a combination thereof. For example, the base solvent may be a mixture of water (99.9%) and trifluoroacetic acid (TFA, 0.1%), and the modifier may be a mixture of 9.9% water, 0.1% TFA, and 90.0% acetonitrile. Alternatively, the modifier may be 100% acetonitrile. In another example, the base solvent may be a 25 mM aqueous phosphate buffer solution (pH 7.0), and the modifier may be a 25 mM phosphate buffer solution and 500 mM NaCl (pH 7.0).

[0079] The base solvent is typically water, or a mixture of water and one or more salts and / or organic solvents in a smaller proportion than water (e.g., acetonitrile and trifluoroacetic acid), especially when biomolecules obtained from biochemical processes are to be separated. This base solvent will be referred to as Solvent A below. To establish a gradient, this is mixed with a further solvent or solvent mixture different from the base solvent (mixture). This further solvent may be, for example, a mixture of the same solvents as the base solvent, but in different proportions (e.g., in the above example, water with a higher proportion of acetonitrile). Especially in the case of biomolecules, this further solvent is typically again water-based, but with a higher proportion of organic solvents. To establish a gradient, a modifier mixture containing components at a considerably lower concentration than the base solvent (e.g., if the base solvent is, for example, water, then organic solvents (or multiple), salts, pH, or a combination thereof) is typically provided as an eluent at the start of the gradient and gradually mixed with the further solvent by a gradient pump to produce a corresponding controlled gradient. Feed mixtures may be supplied in various solvents, or in a base solvent, or they may be supplied as a mixture of the original solutions (typically aqueous solutions) in a mixture of a base solvent of appropriate concentration.

[0080] In at least one or all of the above stages, preferably a linear eluent gradient is used in the form of a gradient of modifier concentration changing over time.

[0081] As described above, preferably, a starting stage is carried out before carrying out the first interconnected recirculation step of the first recirculation stage, in which during the first batch start-up period t B-SU-F the adsorbent is segmented, and the feed mixture F is supplied to the inlet of the adsorbent section that becomes the upstream adsorbent section of the first interconnected recirculation step of the first recirculation stage, while the adsorbent section that becomes the downstream adsorbent section of the first interconnected recirculation step of the first recirculation stage is equilibrated or already equilibrated and in an inactive state, and preferably subsequently, during the second batch start-up period t B-SU-W the adsorbent is segmented, and weakly adsorbing impurities W are eluted from the adsorbent section to which the feed mixture F was supplied in the preceding first batch start-up step, while the other adsorbents are equilibrated or already equilibrated and in an inactive state.

[0082] After the above first batch start-up period t B-SU-F and before the above second batch start-up period t B-SU-W the eluent can be supplied to the inlet of the adsorbent section that becomes the upstream adsorbent section of the first interconnected recirculation step of the first recirculation stage, and the eluent either does not contain a modifier (only the base solvent) or can have a modifier concentration essentially corresponding to the starting modifier concentration applied during the first batch start-up period or in the absence of the second batch start-up step of the first interconnected recirculation step.

[0083] Furthermore, during the above second batch start-up period, an eluent having a gradient in the form of a modifier concentration changing over time can be loaded at the inlet of the adsorbent section to which the feed mixture F was supplied.

[0084] A stopping step can be performed after the completion of at least one basic sequence, preferably two or more basic sequences, where the stopping step consists of the following steps: a'. Interconnect stop recirculation process IC'-R, In this process, the upstream adsorbent section of the preceding final second interconnection purification process IC2 is positioned downstream, and the other adsorbent sections are positioned upstream, the adsorbent sections are interconnected, and there is a stop-recirculation interconnection period t. IC’-R-SD Inside, the outlet of the upstream adsorbent section is connected to the inlet of the downstream adsorbent section. Here, the eluent is loaded into the upstream adsorbent section via the inlet of the upstream adsorbent section, and a fraction containing a weakly adsorbent impurity W in a range that overlaps with at least the product P, and a strongly adsorbent impurity S in a range that overlaps with at least the product P, is eluted from the upstream adsorbent section to the downstream adsorbent section, and The flow exiting the upstream adsorbent outlet is diluted in-line before entering the downstream adsorbent inlet; b'. Any batch stop recirculation process B'-R, In this process, the adsorbent section is separated, and here, a stop-recirculation batch period t B’-R The above adsorbent section is divided inside, and The preceding upstream adsorbent section of step a' is cleaned and regenerated, and the eluent is loaded into the inlet of the preceding downstream adsorbent section of step a to elute weakly adsorbent impurities W within a range that does not overlap with the product P; It includes them in order.

[0085] Preferably, the following steps are followed: c'. First interconnection termination purification process IC1-SD, In this process, the adsorbent sections are interconnected, and here, the first interconnection termination purification period t IC1’ Inside, the outlet of the upstream adsorbent section is connected to the inlet of the downstream adsorbent section. The eluent is loaded onto the upstream adsorbent via the upstream adsorbent inlet, and the product fraction containing the overlapping weakly adsorbent impurities W and product P is eluted from the upstream adsorbent to the downstream adsorbent section, and The flow exiting the upstream adsorbent section outlet is diluted in-line before entering the downstream adsorbent section inlet; d'. First batch stop purification process B1-SD, Here, the first batch purification period t B1’ The adsorbent section is divided within it, and product P is eluted from the previous upstream adsorbent section, and the previous downstream adsorbent section is either idling or the eluent is supplied to the inlet of the previous downstream adsorbent section; e'. Second interconnection termination purification process IC2-SD, Here, the second interconnection termination and purification period t IC2’ Inside, the outlet of the upstream adsorbent section is connected to the inlet of the downstream adsorbent section. The eluent is loaded into the upstream adsorbent section via the inlet of the upstream adsorbent section, and the product fraction containing the overlapping product P and the highly adsorbent impurities S is eluted from the upstream adsorbent to the downstream adsorbent, and The flow exiting the upstream adsorbent section outlet is diluted in-line before entering the downstream adsorbent section inlet; f'. Second batch stop purification process B2-SD, Here, the second purification batch period t B2’ The adsorbent is broken up inside, the previous upstream adsorbent is cleaned and regenerated, and the eluent is loaded into the previous downstream adsorbent inlet; This process continues in sequence, where the upstream adsorbent section of the interconnection stop recirculation process performs the function of the downstream adsorbent section, and the downstream adsorbent section of the interconnection stop recirculation process performs the function of the upstream adsorbent section.

[0086] Typically, g'. The first final stop batch process B-SD-P, Here, the first final stop batch period t B-SD-PWithin this, the first adsorbent and the second adsorbent are separated, the product P is eluted from the previous downstream adsorbent, and the other adsorbent is either idling or regenerating; h'. Any second final stop batch process B-SD-S, Here, the second final stop batch period t B-SD-S Within the first adsorbent and the second adsorbent are separated, and the highly adsorbent impurity S is eluted from the previous downstream adsorbent, while the other adsorbent is in an idling state or is regenerated; And so it continues.

[0087] In the above interconnection stop recirculation step IC'-R, the upstream adsorbent section of the preceding final second interconnection purification step IC2 can be loaded with an eluent having a constant composition or a gradient in the form of a time-varying modifier concentration, where the flow exiting the upstream adsorbent outlet is inline diluted with an eluent without modifiers or with an eluent having a different modifier concentration than that of the upstream adsorbent section inlet before it enters the downstream adsorbent inlet.

[0088] In the batch stop recirculation process B'-R described above, the upstream adsorbent section from the preceding process a can be cleaned and regenerated with an eluent having a higher modifier concentration than at the end of the preceding interconnection recirculation process, or with an eluent containing a different modifier, or with a cleansing solution. Here, an eluent having a gradient in the form of a time-varying modifier concentration is loaded into the inlet of the downstream adsorbent section from the preceding process a' to elute weakly adsorbent impurities W within a range that does not overlap with the product P.

[0089] In the first interconnection termination purification step IC1-SD described above, an eluent having a gradient in the form of a time-varying modifier concentration can be loaded onto the upstream adsorbent via the upstream adsorbent inlet, and the flow exiting the upstream adsorbent section outlet is inline diluted with an eluent that does not contain a modifier, or with an eluent having a different modifier concentration than that at the upstream adsorbent section inlet, before it enters the downstream adsorbent section inlet.

[0090] In the first batch stop purification step B1-SD described above, product P can be eluted from the previous upstream adsorbent section by loading an eluent having a gradient in the form of a time-varying modifier concentration through the upstream adsorbent inlet, while the previous downstream adsorbent section is either idling or an eluent, preferably a base solvent, is supplied to the inlet of the previous downstream adsorbent section.

[0091] In the second interconnection termination purification step IC2-SD described above, the upstream adsorbent section can be loaded with an eluent of a constant composition or an eluent having a gradient in the form of a time-varying modifier concentration, via the upstream adsorbent section inlet. The flow exiting the upstream adsorbent section outlet is inline diluted with an eluent without modifiers or with an eluent having a different modifier concentration than that at the upstream adsorbent section inlet before it enters the downstream adsorbent section inlet.

[0092] Furthermore, in the second batch stop purification step B2-SD described above, the previous upstream adsorbent can be cleaned and regenerated with an eluent having a higher modifier concentration than at the end of the preceding interconnection recirculation step, where the eluent is loaded into the previous downstream adsorbent inlet.

[0093] Finally, in the first final stop batch step B-SD-P described above, product P can be eluted from the previous downstream adsorbent with an eluent having a gradient in the form of a time-varying modifier concentration, where the other adsorbent is idling, regenerating, and / or In the first and final second final stop batch process B-SD-S described above, highly adsorbent impurities S can be eluted from the previous downstream adsorbents using an eluent having a higher modifier concentration than at the end of the preceding interconnection recirculation process, at which point the other adsorbents are either idling or being regenerated.

[0094] A linear gradient having various gradients and / or flow rates is preferably used in the recirculation and / or purification stages, where preferably a higher gradient and / or flow rate is used in the recirculation stage than in the purification stage.

[0095] In yet another preferred embodiment, in most of the method (in particular all except steps IC2 and IC2-SD) or all interconnection steps, an eluent having a gradient in the form of a time-varying modifier concentration is loaded into the upstream section inlet via the upstream adsorbent inlet, and the flow exiting the upstream adsorbent section outlet is inline diluted with an eluent without a modifier, or with an eluent having a different modifier concentration than that of the upstream adsorbent section inlet, before it enters the downstream adsorbent section inlet, and In batch processes that do not involve elution of the purified product, the previous upstream adsorbent is cleaned and regenerated with an eluent having a higher modifier concentration than at the end of the preceding interconnection recirculation process, and, where applicable, an eluent having a gradient in the form of a time-varying modifier concentration is loaded into the previous downstream adsorbent inlet, or In a batch step of a method involving the elution of the purified product, an eluent having a gradient in the form of a time-varying modifier concentration is loaded onto the upstream adsorbent via the upstream adsorbent inlet, and The modifier is selected from the group consisting of a solvent or mixture thereof different from the base solvent or mixture thereof of the eluent, an electrolyte in such a solvent or mixture thereof, preferably selected from a dissolved salt or pH, or a combination thereof, where preferably the base solvent is water, or a mixture of water and at least one organic solvent, or a mixture of water and one or more salts and / or organic solvents in a smaller proportion than water, and more preferably the modifier is an organic solvent, or a mixture of water and at least one organic solvent, wherein the concentration of the at least one organic solvent is higher than in the base solvent, water, or a salt concentration and / or H different from that in the base solvent. + A mixture of water having a concentration, and The above modifier concentration is applied during the process in the following steps: The recirculation processes (IC-R, IC'-R, IC1, IC1-SD), except in the case of the next final stop batch process (B-SD-P), and If applicable, the process includes product elution (B1, B1-SD, B-SD-P) and, if applicable, further recirculation (IC2, IC2-SD) (wherein the above recirculation (IC2, IC2-SD), the modifier concentration may fluctuate or may be kept constant only in the recirculation process), From the lowest concentration at the start of elution of weakly adsorbent impurities W (B-SU-W, BR, B'-R, B2, B2-SD) in a range that does not overlap with product P, After increasing the concentration to the maximum level, preferably linearly, the elution of highly adsorbent impurities S (BR, B'-R, B2, B2-SD, B-SD-S) begins.

[0096] In this process, it is possible to use adsorbents with different dimensions or different stationary phases, or adsorbents with both different dimensions and different stationary phases. Elution of product P is always performed from the same adsorbent, as is the loading of new feed material. For example, in the recirculation step, which involves only a single sequence of IC-R and BR (n=1), the main purification task is performed by adsorbent 2 (recirculation of W / P, elution of product P, recirculation of P / S). Therefore, it is possible to maximize separation efficiency by using small particle chromatographic resin in adsorbent 2 for this task, while it is possible to use large particle resin to accommodate the recirculation flow in adsorbent 1. Due to the in-line dilution of W / P and P / S in steps IC1 and IC2, the flow of eluent entering adsorbent 1 is greater than the flow exiting adsorbent 2, so adsorbent 1 may benefit from larger particles, which have lower back pressure than small particle resin under flow.

[0097] Furthermore, the present invention relates to the uses described above for the purification of biomolecules selected from the group consisting of nucleic acid molecules, preferably DNA and RNA molecules, proteins, peptides, carbohydrates, lipids, and combinations and modifications thereof, and fragments, of natural or synthetic origin.

[0098] Further embodiments of the present invention are provided in the dependent claims.

[0099] Preferred embodiments of the present invention are described below with reference to the drawings, which are intended to illustrate preferred embodiments of the present invention and are not intended to limit the present invention. [Brief explanation of the drawing]

[0100] [Figure 1] Figure 1 shows a general scheme of this process, including the startup phase, the recirculation phase, the purification phase, and the shutdown phase. [Figure 2]Figure 2 shows, in more detail, the logic of this process (startup phase, recirculation phase, purification phase), including the adsorbent connections for n=1 to 4 sequences in the recirculation phase, and the flow entering and leaving the adsorbent. Feed / elute flows that do not contain W, P, S, or F are not specifically marked. [Figure 3] Figure 3, more specifically, shows the logic of this process (stopping phase), including the adsorbent connections and the flow entering and leaving the adsorbent for n=1 to 4 sequences in the recirculation phase. Feed / elute flows that do not contain W, P, S, or F are not specifically marked. [Figure 4] Figure 4 is a diagram illustrating the tasks of two adsorbents in the initial stage for a single-column chromatogram (shown at the bottom), schematically showing the linear gradient segments (thick dashed lines) used in the process in the case of a linear gradient operation. Thin vertical dashed lines schematically indicate section boundaries, and in Figures 4 to 7, vertical sections designated as zones and separated by thin vertical dashed lines indicate various functions of the column in each zone. The lower right side shows a chromatogram containing product P, a less adsorbent impurity W that elutes faster than product P to the left of product P, and a more adsorbent impurity S that elutes slower than product P to the right of product P. Where indicated as a dashed line at the bottom of zone 2, it indicates the applicable feed concentration at the column inlet in zone 2. In the chromatogram zones (zones 4-8 in Figures 4, 6, and 7, and zones 4-6 in Figure 5), the lower dashed lines indicate the modifier concentrations used for gradients applied at the column inlet in each zone during the elution of less adsorbent impurities W and product P (zones 4-7 in Figures 4, 6, and 7, and zones 4 and 5 in Figure 5), and the higher, but usually constant, modifier concentrations applied during the elution of S, which does not overlap with P (zone 8 in Figures 4, 6, and 7, and zone 6 in Figure 5). [Figure 5]Figure 5 shows the tasks of two adsorbents in the recirculation step for a single-column chromatogram (shown at the bottom), schematically illustrating the linear gradient segment used in the process in the case of a linear gradient operation. [Figure 6] Figure 6 shows the tasks of two adsorbents in the purification step for a single-column chromatogram (shown at the bottom), schematically illustrating the linear gradient segment used in the process in the case of a linear gradient operation. [Figure 7] Figure 7 shows the task of two adsorbents in stopping stage II for a single-column chromatogram (shown at the bottom), schematically illustrating the linear gradient segment used in the process in the case of a linear gradient operation. [Figure 8-1] Figure 8a) shows the superposition of UV profiles for the process and the single-column reference run at the termination stage, and Figure 8b) shows the superposition of product concentration values ​​determined by offline HPLC analysis for the process and the single-column reference run at the termination stage. [Figure 8-2] Figure 8c) shows the superposition of product purity values ​​determined by offline HPLC analysis at the termination stage of the process and single-column reference run, and Figure 8d) shows the superposition of impurity content values ​​determined by offline HPLC analysis at the termination stage of the process and single-column reference run. The main peak of the highly adsorbent impurity S is indicated by the arrow in Figure 8d). [Figure 9] Figure 9 shows a superposition of the purity / yield curves for this process and a single-column reference run. [Figure 10]Figure 10a) shows the internal chromatogram of a normal MCSGP process recorded at the column outlet of the upstream column through steps IC1, B1, IC2, and B2; b) shows the internal chromatogram of the purification stage of this process for n=1, recorded at the column outlet of the upstream column through steps IC1, B1, IC2, and B2; and c) shows the internal chromatogram of the purification stage of this process for n=2, recorded at the column outlet of the upstream column through steps IC1, B1, IC2, and B2. The rectangular area indicates the product collection interval, the arrow labeled "W" points to the location of weakly adsorbed impurities, and the arrow labeled "S" points to the location of strongly adsorbed impurities. [Figure 11] Figure 11a) shows the chromatogram of the batch reference process at a floor height of 10 cm, and b) shows the chromatogram of the batch reference process at a floor height of 20 cm. The rectangular areas indicate the product collection interval, the arrows labeled "W" point to the location of weakly adsorbed impurities, and the arrows labeled "S" point to the location of strongly adsorbed impurities. [Figure 12] Figure 12 shows product pool purity versus productivity for 1. the process with n=1 sequences in the recirculation stage, 2. the process with n=2 sequences in the recirculation stage, the MCSGP process, a single-column batch reference process at a floor height of 10 cm, and a single-column batch reference process at a floor height of 20 cm. For batch processes, the figure also shows purity / productivity curves for points corresponding to product pools of various sizes. [Modes for carrying out the invention]

[0101] Example 1: Purification of Angiotensin II A feed solution containing 2.1 g / L angiotensin II from chemical synthesis was prepared in solvent A (5% acetonitrile (ACN) in water + 0.1% trifluoroacetic acid (TFA)). Solvents A and B (50% ACN in water + 0.1% TFA) were used for chromatography. The feed purity was %W=4.76, %P=91.67, and %S=3.57 (in all cases, area %) based on analytical HPLC.

[0102] The process was performed using two columns with a column volume of 2.5 mL each (0.46 cm inner diameter × 15 cm floor height). For reference, a single-column run was performed using the same material.

[0103] This process was executed on the Contichrom CUBE system (ChromaCon) using the parameters shown in Table 1.

[0104] To investigate the effect of the recirculation step, a shutdown step was performed immediately after the recirculation step, and the product peak was fractionated in a non-cyclic, discontinuous process. A comparison of fractionated shutdown with fractionation using a single-column reference run revealed that the product peak was broader in this process, as seen in the UV detector signal recorded at the column outlet (Figure 8a) and confirmed by offline analysis using analytical HPLC (Figure 8b). Furthermore, the purity achieved as determined by analytical HPLC was higher over a considerably wider range of fractions in this process (Figure 8c). Offline HPLC analysis confirmed that the highly adsorbent impurity S was significantly delayed in this process, allowing for the inclusion of a larger fraction in the product pool (Figure 8d).

[0105] Plotting the purity-yield curves of the batch reference process and the present process (Figure 9) reveals that, with the same initial load of 16 g / L, the present process yields a higher yield for a given purity. The individual points on the yield-purity curve represent pools of varying sizes obtained by grouping the fractions.

[0106] [Table 1]

[0107] Example 2: Numerical simulation of oligonucleotide purification This process was simulated for the purification of oligonucleotides by anion exchange chromatography. The feed mixture for the simulation consisted of 20 mers of dsDNA oligonucleotides, impurities W and S: P = 2.865 g / L (87.2% purity), W1 = 0.13 g / L, W2 = 0.23 g / L, S1 = 0.08 g / L, S2 = 0.13 g / L. The gradient is shown as G in Figures 10 and 11.

[0108] Two columns with an inner diameter of 0.5 cm and a floor height of 10 cm, packed with YMC SmartSep Q30, were used. For the simulation, a mechanism model based on the Vilang-Muir adsorption isotherm was used and calibrated using a series of single-column gradient experiments with various gradient slopes and loads.

[0109] Figure 10a shows an internal chromatogram of a typical MCSGP process according to Patent Document 1, recorded at the column outlet of the upstream column through steps IC1, B1, IC2, and B2.

[0110] Similarly, Figures 10b and 10c show internal chromatograms of the purification steps for n=1 (Figure 10b) and n=2 (Figure 10c), i.e., for one or two recirculation steps, recorded at the column outlet of the upstream column through steps IC1, B1, IC2, and B2, respectively. The shaded rectangular areas in Figures 10a-c indicate the product collection interval. Within the product collection window, it can be seen that the product collected in the product elution window becomes much purer as the number of recirculation steps increases, from n=0 (normal MCSGP process, Figure 10a) through n=1 (one recirculation step, Figure 10b) to n=2 (two recirculation steps, Figure 10c). This is a result of better removal of impurities "W" and "S", indicated by the arrows in Figures 10a-c.

[0111] The operating parameters of the simulated system to be examined are shown in the table.

[0112] [Table 2] [Table 3] [Table 4]

[0113] For comparison, Figures 11a and 11b show chromatograms of the batch reference process at floor heights of 10 cm and 20 cm, respectively. Similar to Figures 10a-c, the rectangular areas indicate the product collection intervals, the arrows labeled "W" point to the locations of relevant overlapping, weakly adsorbed impurities, and the arrows labeled "S" point to the locations of relevant overlapping, strongly adsorbed impurities. It can be seen that the more recirculation steps used, the lower the amount of impurities W and S in the product pool.

[0114] These results are compared in terms of product pool purity versus productivity and are shown in Figure 12. In addition to the dual-column process, a single-column reference process was simulated. In the single-column reference process, product pools of various sizes corresponding to pools with varying product and impurity content, and therefore varying productivity and purity, were extracted from the simulated chromatograms.

[0115] This process demonstrates that it is possible to achieve a much higher purity (over 97.5% purity for both n=1 and n=2) than the reference batch process (less than 97.0% purity) with comparable productivity. Compared to the "conventional" MCSGP process described in Patent Document 1, this process achieves higher purity (MCSGP purity of less than 96.0%), but with slightly lower productivity. Furthermore, Figure 12 confirms that the product purity can be further increased by increasing the number of recirculation step sequences n (purity = 97.8% for n=1 compared to 98.4% for n=2), but this comes at the expense of productivity (productivity = 3.4 g / L / h for n=1 compared to 2.3 g / L / h for n=2). [Explanation of symbols]

[0116] IC1 purification stage, first interconnection step IC2 purification stage, second interconnection step Interconnection process for IC-R recirculation stage or stop stage I IC1-SD Stop Phase II First Interconnection Process IC2-SD Second interconnection process in shutdown phase II B1 The first fragmentation step (batch process) in the purification stage. B2 The second separation process (batch process) in the purification stage. BR Recirculation stage or stopping stage I interruption process (batch process) B1-SD Stopping Phase II, First Disruption Process (Batch Process) B2-SD Second division process (batch process) of shutdown stage II B-SU-F Startup phase interruption process (supply process) B-SU-W Starting Phase Separation Process (W Dissolution Process) B-SD-P stopping stage fragmentation process (product elution process) B-SD-S stopping stage separation process (S elution process) t IC1 Duration of stage IC1 t IC2 Duration of stage IC2 t IC-R Duration of stage IC-R t B1 Duration of process B1 t B2 Duration of process B2 t B-R Duration of process BR t B-SU-F Duration of process B-SU-F t B-SU-W Duration of process B-SU-W t B-SD-P Duration of process B-SD-P t B-SD-S Duration of process B-SD-S P product Feed mixture containing FW, P, and S W is an impurity with weak adsorption properties. S: Strongly adsorbent impurities

Claims

1. A cyclic chromatography purification method for isolating the product (P) from a feed mixture (F) consisting of the product (P) and at least two further components corresponding to weakly adsorbed impurities (W) and strongly adsorbed impurities (S), wherein The above method uses only two chromatography adsorbent sections as the chromatography stationary phase, wherein the first adsorbent section (1) has a first adsorbent section inlet and a first adsorbent section outlet, and the second adsorbent section (2) has a second adsorbent section inlet and a second adsorbent section outlet. The method comprises at least one basic sequencing having at least one recirculation step followed by only one purification step, The basic sequence may be repeated cyclically at least twice. The aforementioned recirculation step consists of at least one recirculation sequence, The aforementioned recirculation sequence is a. Interconnect Recirculation Process (IC-R), In this process, the adsorbent sections are interconnected, and the recirculation interconnection period (t ＩＣ－Ｒ ) The outlet of the upstream adsorbent section is connected to the inlet of the downstream adsorbent section, Here, the eluent is loaded into the upstream adsorbent section via the inlet of the upstream adsorbent section, and a fraction containing the weakly adsorbent impurity (W) in a range that overlaps with at least the product (P), the product (P), and the strongly adsorbent impurity (S) in a range that overlaps with at least the product (P) is eluted from the upstream adsorbent section to the downstream adsorbent section, and The flow exiting the upstream adsorbent outlet is diluted in-line before entering the downstream adsorbent inlet; Includes, The aforementioned recirculation sequence is b. Batch recirculation process (B-R), Here, the recycle batch period (t Ｂ－Ｒ ) The adsorbent section is divided in the middle, The preceding upstream adsorbent section of step a is cleaned to remove and regenerate highly adsorbent impurities (S), and the eluent is loaded into the inlet of the preceding downstream adsorbent section of step a to elute less adsorbent impurities (W) that do not overlap with the product (P); It may include, After each recirculation sequence in steps a and b, the adsorbent sections are switched in sequence, where the number of recirculation sequences is one or more, and Each recycling stage is followed by a single purification stage. The aforementioned purification step consists of the following steps: c. First interconnection purification step (IC1), In this process, the adsorbent sections are interconnected, and here, a first interconnection purification period (t ＩＣ１ ) The outlet of the upstream adsorbent section is connected to the inlet of the downstream adsorbent section, The eluent is loaded onto the upstream adsorbent via the upstream adsorbent inlet, and the product fraction containing overlapping weakly adsorbent impurities (W) and products (P) is eluted from the upstream adsorbent to the downstream adsorbent section, The flow exiting the upstream adsorbent section outlet is diluted in-line before entering the downstream adsorbent section inlet; d. First batch purification process (B1), Here, the first batch purification period (t Ｂ１ The adsorbent section is divided in the process, the product (P) is eluted from the previous upstream adsorbent section, and the feed mixture (F) is supplied to the inlet of the previous downstream adsorbent section; e. Second interconnection purification process (IC2), Here, the second interconnection purification period (t ＩＣ２ ) The outlet of the upstream adsorbent section is connected to the inlet of the downstream adsorbent section, The eluent is loaded into the upstream adsorbent section via the inlet of the upstream adsorbent section, and the product fraction containing the overlapping product (P) and highly adsorbent impurities (S) is eluted from the upstream adsorbent to the downstream adsorbent, The flow exiting the upstream adsorbent section outlet is diluted in-line before entering the downstream adsorbent section inlet; f. Second batch purification process (B2), Here, the second purification batch period (t Ｂ２ The adsorbent is broken up in the process, the previous upstream adsorbent is cleaned and regenerated, and the eluent is loaded into the previous downstream adsorbent inlet; The method comprising the steps in order, wherein the upstream adsorbent section of the final interconnection recirculation step of the preceding recirculation stage performs the function of the downstream adsorbent section, and the downstream adsorbent section of the final interconnection recirculation step of the preceding recirculation stage performs the function of the upstream adsorbent section.

2. The method according to claim 1, wherein an eluent gradient is used in at least one or all of the steps, the eluent gradient may be a linear eluent gradient, wherein a gradient slope higher than that of the purification steps (IC1, B1, IC2, and / or B2) may be selected in the recirculation step (IC-R and / or B-R).

3. The method according to claim 1 or 2, wherein, in the interconnection step of the method, an eluent having a gradient in the form of a time-varying modifier concentration is loaded into the upstream section inlet via the upstream adsorbent inlet, and the flow exiting the upstream adsorbent section outlet is inline diluted with an eluent without a modifier or with an eluent having a different modifier concentration than that of the upstream adsorbent section inlet before it enters the downstream adsorbent section inlet.

4. In a batch process of a method that does not involve elution of the purified product, the previous upstream adsorbent is cleaned and regenerated, and an eluent having a gradient in the form of a time-varying modifier concentration is loaded into the inlet of the previous downstream adsorbent, and / or The method according to claim 1, wherein, in a batch step of a method involving the elution of a purified product, an eluent having a gradient in the form of a time-varying modifier concentration is loaded onto a prior upstream adsorbent via the upstream adsorbent inlet.

5. The method according to claim 1, wherein in a batch step of a method without elution of the purified product, the previous upstream adsorbent is cleaned and regenerated with an eluent having a higher modifier concentration than at the end of the preceding interconnection recirculation step, or with an eluent containing a different modifier, or with a cleansing solution, and an eluent having a gradient in the form of a time-varying modifier concentration is loaded into the inlet of the previous downstream adsorbent.

6. In the recirculation step of the interconnection-recirculation process (IC-R), an eluent having a gradient in the form of a time-varying modifier concentration is loaded into the upstream adsorbent section via the inlet of the upstream adsorbent section, and the flow exiting the upstream adsorbent outlet is inline diluted with an eluent without a modifier, or with an eluent having a different modifier concentration than that of the inlet of the upstream adsorbent section, before it enters the downstream adsorbent inlet, and / or The method according to claim 1, wherein in the batch recirculation step (B-R), the upstream adsorbent section prior to step a is cleaned and regenerated, and an eluent having a gradient in the form of a time-varying modifier concentration is loaded into the inlet of the downstream adsorbent section prior to step a to elute weakly adsorbent impurities (W) in a range that does not overlap with the product (P).

7. The method according to claim 1, wherein in the batch recirculation step (B-R), the upstream adsorbent section prior to step a is cleaned and regenerated with an eluent having a higher modifier concentration than at the end of the preceding interconnection recirculation step, or with an eluent containing a different modifier, or with a cleansing solution, and an eluent having a gradient in the form of a time-varying modifier concentration is loaded into the inlet of the downstream adsorbent section prior to step a to elute weakly adsorbent impurities (W) in a range that does not overlap with the product (P).

8. In the purification step of the first interconnection purification step (IC1), an eluent having a gradient in the form of a time-varying modifier concentration is loaded into the upstream adsorbent section via the upstream adsorbent inlet, and the flow exiting the upstream adsorbent section outlet is inline diluted with an eluent that does not contain a modifier, or with an eluent having a different modifier concentration than that of the upstream adsorbent section inlet, and / or In the first batch purification step (B1), the product (P) is eluted from the section by loading an eluent having a gradient in the form of a time-varying modifier concentration into the previous upstream adsorbent section through its inlet, and / or In the second interconnection purification step (IC2), an eluent having a gradient in the form of a time-varying modifier concentration is loaded into the upstream adsorbent section inlet via the upstream adsorbent section inlet, and the flow exiting the upstream adsorbent section outlet is inline diluted with an eluent that does not contain a modifier, or with an eluent having a different modifier concentration than that of the upstream adsorbent section inlet, and / or The method according to claim 1, wherein in the second batch purification step (B2), the previous upstream adsorbent is cleaned, regenerated, and an eluent having a gradient in the form of a time-varying modifier concentration is loaded into the inlet of the previous downstream adsorbent.

9. The method according to claim 1, wherein in the second batch purification step (B2), the previous upstream adsorbent is purified and regenerated with an eluent having a higher modifier concentration than at the end of the preceding interconnection recirculation step, or with an eluent containing a different modifier, or with a purification solution, and an eluent having a gradient in the form of a time-varying modifier concentration is loaded into the inlet of the previous downstream adsorbent.

10. The method according to claim 2, wherein the modifier is selected from the group consisting of an organic solvent or inorganic solvent or a mixture thereof different from the base solvent or mixture thereof of the eluent, or an electrolyte in such an organic solvent or inorganic solvent (or a mixture thereof), and the electrolyte may be selected from a dissolved salt, pH, or a combination thereof.

11. The base solvent is water, or a mixture of water and at least one organic solvent, or a mixture of water and one or both of the salts and / or organic solvents in a smaller proportion than water, The method according to claim 10, wherein the modifier is an organic solvent, or a mixture of water and at least one organic solvent, wherein the concentration of the at least one organic solvent is higher than that in the base solvent, water, or a mixture of water and at least one organic solvent, wherein the salt concentration or H+ concentration is different from that of the base solvent.

12. The method according to claim 1, wherein in at least one or all of the steps described above, the eluent gradient is used in the form of a gradient of increasing modifier concentration over time, and the eluent gradient may be linear.

13. Before carrying out the first interconnection recirculation step of the first recirculation step, a starting step is carried out, in which step, First batch start period (t Ｂ－ＳＵ－Ｆ ) divides the adsorbent (1, 2) within it, and, The feed mixture (F) is supplied to the inlet of the adsorbent section (1), which is the upstream adsorbent section of the first interconnection recirculation step of the first recirculation step. On the other hand, the adsorbent section (2), which is the downstream adsorbent section of the first interconnection recirculation step of the first recirculation stage, is either equilibrated or has already been equilibrated and is in an inactive state, Second batch start period (t Ｂ－ＳＵ－Ｗ The method according to claim 1, wherein the adsorbents (1, 2) are divided in the adsorbent section (1) to which the feed mixture (F) was supplied in a preceding first batch starting step, and the other adsorbent (2) is equilibrated or has already been equilibrated and is inactive.

14. Before performing the first interconnection recirculation step of the first recirculation step, a starting step is performed, in which step, During the first batch start period (t B-SU-F), the adsorbents (1, 2) are divided, and The feed mixture (F) is supplied to the inlet of the adsorbent section (1), which is the upstream adsorbent section of the first interconnection recirculation step of the first recirculation step. On the other hand, the adsorbent section (2), which is the downstream adsorbent section of the first interconnection recirculation step of the first recirculation stage, is either equilibrated or has already been equilibrated and is in an inactive state, The method according to claim 1, wherein, during the second batch initiation period (t B-SU-W), the adsorbents (1, 2) are further divided, and the poorly adsorbent impurities (W) are eluted from the adsorbent section (1) to which the feed mixture (F) was supplied in the preceding first batch initiation step, while the other adsorbent (2) is equilibrated or has already been equilibrated and is inactive.

15. After said first batch start-up period (t Ｂ－ＳＵ－Ｆ ), and before said second batch start-up period (t Ｂ－ＳＵ－Ｗ ), an eluent is supplied to an inlet of said adsorbent section which becomes an upstream adsorbent section of a first interconnected recirculation step of said first recirculation stage, and said eluent contains no modifier or has a modifier concentration essentially corresponding to an initial modifier concentration applied during said first batch start-up period or in the absence of a second batch start-up period of said first interconnected recirculation step, and / or The method according to claim 13, wherein during the second batch start-up period, an eluent having a gradient in the form of a time-varying modifier concentration is loaded into the inlet of the adsorbent section (1) supplied with the feed mixture (F).

16. After at least one basic sequence, or after two or more basic sequences have been completed, a halt phase is performed. The aforementioned stopping stage is a'. interconnection outage recirculation process (IC'-R); In this process, the upstream adsorbent section of the preceding final second interconnection purification process (IC2) is positioned downstream, and the other adsorbent sections are positioned upstream, and the adsorbent sections are interconnected, and there is a stop-recirculation interconnection period (t ＩＣ’－Ｒ－ＳＤ ) The outlet of the upstream adsorbent section is connected to the inlet of the downstream adsorbent section, Here, the eluent is loaded into the upstream adsorbent section via the inlet of the upstream adsorbent section, and a fraction containing the weakly adsorbent impurity (W) in a range that overlaps with at least the product (P), the product (P), and the strongly adsorbent impurity (S) in a range that overlaps with at least the product (P) is eluted from the upstream adsorbent section to the downstream adsorbent section, and The flow exiting the upstream adsorbent outlet is diluted in-line before entering the downstream adsorbent inlet; Includes, The aforementioned stopping step occurs after the process interconnection stopping and recirculation process (IC'-R), b'. Batch stop recirculation process (B'-R) In this process, the adsorbent section is separated, and here, a stop-recirculation batch period (t Ｂ’－Ｒ ) The adsorbent section is divided in the middle, The preceding upstream adsorbent section of step a' is cleaned and regenerated, and the eluent is loaded into the inlet of the preceding downstream adsorbent section of step a to elute weakly adsorbent impurities (W) within a range that does not overlap with the product (P); It may include, The following steps follow: c'. First interconnection termination and purification process (IC1-SD), In this process, the adsorbent sections are interconnected, and here, a first interconnection termination purification period (t ＩＣ１’ ) The outlet of the upstream adsorbent section is connected to the inlet of the downstream adsorbent section, The eluent is loaded onto the upstream adsorbent via the upstream adsorbent inlet, and the product fraction containing overlapping weakly adsorbent impurities (W) and products (P) is eluted from the upstream adsorbent to the downstream adsorbent section, The flow exiting the upstream adsorbent section outlet is diluted in-line before entering the downstream adsorbent section inlet; d'. First batch stop purification process (B1-SD), Here, the first batch purification period (t Ｂ１’ The adsorbent section is divided during this process, and the product (P) is eluted from the previous upstream adsorbent section, and the previous downstream adsorbent section is either idling or the eluent is supplied to the inlet of the previous downstream adsorbent section; e'. Second interconnection termination and purification process (IC2-SD), Here, the second interconnection termination and purification period (t ＩＣ２’ ) The outlet of the upstream adsorbent section is connected to the inlet of the downstream adsorbent section, The eluent is loaded into the upstream adsorbent section via the inlet of the upstream adsorbent section, and the product fraction containing the overlapping product (P) and highly adsorbent impurities (S) is eluted from the upstream adsorbent to the downstream adsorbent, The flow exiting the upstream adsorbent section outlet is diluted in-line before entering the downstream adsorbent section inlet; f'. Second batch stop purification process (B2-SD), Here, the second purification batch period (t Ｂ２’ The adsorbent is broken up in the process, the previous upstream adsorbent is cleaned and regenerated, and the eluent is loaded into the previous downstream adsorbent inlet; Next, g'. First final stop batch process (B-SD-P), Here, the first final stop batch period (t Ｂ－ＳＤ－Ｐ ) in which the first adsorbent and the second adsorbent are separated, and the product (P) is eluted from the previous downstream adsorbent, and the other adsorbent is in an idling state or is regenerated; This continued in order, The aforementioned stopping step occurs after the first final stopping batch process (B-SD-P), h'. Second final stop batch process (B-SD-S), Here, the second final stop batch period (t Ｂ－ＳＤ－Ｓ ) In the process, the first adsorbent and the second adsorbent are separated, and highly adsorbent impurities (S) are eluted from the previous downstream adsorbent, while the other adsorbent is in an idling state or is regenerated; It may include, The method according to claim 1, wherein the upstream adsorbent section of the interconnection stop recirculation process performs the function of the downstream adsorbent section, and the downstream adsorbent section of the interconnection stop recirculation process performs the function of the upstream adsorbent section.

17. In the aforementioned interconnection stop recirculation step (IC'-R), the preceding final second interconnection purification step (IC2) loads the upstream adsorbent section with an eluent having a constant composition or a gradient in the form of a time-varying modifier concentration, and here, the flow exiting the upstream adsorbent outlet is inline diluted with an eluent without modifiers or with an eluent having a different modifier concentration than that of the upstream adsorbent section inlet, and / or In the batch stop recirculation step (B'-R), the upstream adsorbent section prior to step a is cleaned and regenerated, and an eluent having a gradient in the form of a time-varying modifier concentration is loaded into the inlet of the downstream adsorbent section prior to step a' to elute weakly adsorbent impurities (W) within a range that does not overlap with the product (P), and / or In the first interconnection termination purification step (IC1-SD), an eluent having a gradient in the form of a time-varying modifier concentration is loaded onto the upstream adsorbent via the upstream adsorbent inlet, and the flow exiting the upstream adsorbent section outlet is inline diluted with an eluent that does not contain a modifier, or with an eluent having a different modifier concentration than that of the upstream adsorbent section inlet, and / or In the first batch stop purification step (B1-SD), the product (P) is eluted from the previous upstream adsorbent section by loading an eluent having a gradient in the form of a time-varying modifier concentration through the upstream adsorbent inlet, and the previous downstream adsorbent section is either idling or the eluent is supplied to the previous downstream adsorbent section inlet, the eluent may be in the form of a base solvent, and / or In the second interconnection termination purification step (IC2-SD), the upstream adsorbent section is loaded with an eluent having a constant composition or a gradient in the form of a time-varying modifier concentration, via the inlet of the upstream adsorbent section, and the flow exiting the outlet of the upstream adsorbent section is inline diluted with an eluent that does not contain a modifier, or with an eluent having a different modifier concentration than that of the inlet of the upstream adsorbent section, before it enters the inlet of the downstream adsorbent section, and / or In the second batch stop purification step (B2-SD), the previous upstream adsorbent is cleaned and regenerated, and the eluent is loaded into the previous downstream adsorbent inlet, and / or In the first final stop batch step (B-SD-P), the product (P) is eluted from the previous downstream adsorbent with an eluent having a gradient in the form of a time-varying modifier concentration, and the other adsorbents are idling, regenerated, and / or The method according to claim 16, wherein in the first final second final stop batch step (B-SD-S), highly adsorbent impurities (S) are eluted from the previous downstream adsorbent, and other adsorbents are in an idling state or are regenerated.

18. The method according to claim 1, wherein a linear gradient having various slopes and / or flow rates is used in the recirculation step and / or the purification step.

19. In most or all interconnection steps of the above method, an eluent having a gradient in the form of a time-varying modifier concentration is loaded into the upstream section inlet via the upstream adsorbent inlet, and the flow exiting the upstream adsorbent section outlet is inline diluted with an eluent without a modifier, or with an eluent having a different modifier concentration than that of the upstream adsorbent section inlet, before it enters the downstream adsorbent section inlet, In batch processes that do not involve elution of the purified product, the previous upstream adsorbent is cleaned and regenerated with the eluent, and, where applicable, the eluent having a gradient in the form of a time-varying modifier concentration is loaded into the previous downstream adsorbent inlet, or In a batch step of a method involving the elution of the purified product, an eluent having a gradient in the form of a time-varying modifier concentration is loaded onto the upstream adsorbent via the upstream adsorbent inlet, and The modifier is selected from the group consisting of a solvent or mixture thereof different from the base solvent or mixture thereof of the eluent, and an electrolyte in such a solvent or mixture thereof, the electrolyte may be selected from a dissolved salt, pH, or a combination thereof, the base solvent may be water, or a mixture of water and at least one organic solvent, or a mixture of water and one or more salts and / or organic solvents in a smaller proportion than water, the modifier is an organic solvent, or a mixture of water and at least one organic solvent, wherein the concentration of the at least one organic solvent is higher than that of the base solvent, water, or a salt concentration or H different from that of the base solvent ＋ It may be a mixture of water having a concentration, and, The modifier concentration is adjusted during the process by the following steps: The recirculation process (IC-R, IC'-R, IC1, IC1-SD), except in the case of the next final stop batch process (B-SD-P), and If applicable, the process of elution of the product (B1, B1-SD, B-SD-P), and if applicable, the process of further recirculation (IC2, IC2-SD) (wherein the modifier concentration may fluctuate during the recirculation (IC2, IC2-SD), or may be kept constant only in the case of the recirculation (IC2, IC2-SD)). The method according to claim 2, wherein the elution of the weakly adsorbent impurities (W) (B-SU-W, B-R, B'-R, B2, B2-SD) in a range that does not overlap with the product (P) is increased from the lowest concentration at the start of the elution to the highest concentration, or increased linearly, before the elution of the strongly adsorbent impurities (S) (B-R, B'-R, B2, B2-SD, B-SD-S) begins.

20. Use of the method of Claim 1 for the purification of biomolecules of natural or synthetic origin, which may be selected from the group consisting of nucleic acid molecules including DNA molecules and RNA molecules, proteins including antibodies, peptides, carbohydrates, lipids, and combinations and modifications thereof, and fragments.

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