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 agents of chemically synthesized origin, such as peptides and oligonucleotides. [Background technology]

[0002] The purification process of 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, a process that generates product-related impurities in addition to the target compound, which need to be removed in downstream processes.

[0004] Due to its high selectivity, chromatography 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] To this end, linear gradient chromatography is frequently used, in which, after binding of the product to the stationary phase in the chromatographic adsorbent, the composition of the mobile phase pumped through the adsorbent is gradually changed over time at a constant rate.

[0006] This causes sequential desorption of the early eluting (weakly adsorbed) impurity, the product, and the late eluting (strongly adsorbed) impurity. In a chromatogram, compounds eluting in this order appear as a series of peaks. By implementing a gentler linear gradient, the resolution of the separated compounds can be improved, but this comes at the expense of increased processing time and reduced throughput. Similarly, the resolution of the compounds can be increased by reducing the mass of starting material (feed) loaded onto the column, but this also reduces throughput.

[0007] In preparative chromatography, throughput is very important, so the duration of the gradient is limited, which causes overlap of the impurity and product peaks in the preparative chromatogram. The highest product purity is typically obtained in the center of the product peak, and this part of the chromatogram is collected as a pool at the adsorbent outlet during elution. Including side fractions containing products with overlapping impurity and product peaks causes a decrease in product pool purity. To avoid violation of the purity constraint, it is typically necessary to exclude some of the products in the side fractions from the product pool. The product fractions not included in the product pool may even constitute a large part of the product contained in the starting material loaded on the adsorbent. To avoid product loss, there is a high interest in recovering the products contained in the impure side fractions. The side fractions are often subjected to rechromatography, i.e., the same or similar chromatographic unit operations are performed using the side fractions as load materials. Through this operation, a pure product fraction can be recovered, but the separation is more difficult because this load material has a higher impurity content than the normal feed material. Rechromatography also has a series of operational disadvantages including regulatory restrictions, storage and handling of the subfractions, stability and quality control of the subfractions.

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

[0009] Single adsorbent installations involve recirculation through the same column of a chromatographic profile. In a steady-state recirculation (SSR) process, fractions are collected from the beginning and end of a recirculating chromatographic profile and a new sample is injected within the profile.

[0010] The multiple adsorbent recycle process allows for the combination of internal recycle of impure subfractions from one adsorbent to another, and the use of the countercurrent principle, i.e., the relative opposite movement of the stationary and mobile phases, thereby improving the separation of products and impurities. Automatic recycle avoids the generation and collection of subfractions, their external storage, handling, and analysis, and only pure products are recovered from the process in high yield.

[0011] Multiple adsorbent processes combining internal recycle and countercurrent principles are known as simulated moving bed (SMB) processes. Early SMB processes were limited to the separation of two compounds (binary separations) and could not be operated under linear gradient conditions, limiting their application to separations that did not require a center cut in the chromatogram.

[0012] Further development of the SMB concept has led to a highly efficient process for center-cut (ternary) separations with linear solvent gradient capabilities known as the "MCSGP" process (Multicolumn Countercurrent Solvent Gradient Purification) (see US Pat. No. 5,399,433). This process is well established in the industry. Other chromatographic multi-adsorbent techniques using multiple adsorbents and internal recycle have been proposed, such as the "gradient with steady state recycle" (GSSR) process.

[0013] Although MCSGPs have been described for two to eight column configurations, in practice, primarily two adsorbent configurations are used due to lower instrumental complexity and greater operational flexibility for installations that use more columns.

[0014] The MCSGP process is designed based on the chromatogram of a single-column batch chromatography. Generally, it is possible to obtain a product with a purity corresponding to the purest fraction from the batch chromatogram using MCSGP. In some cases, overlapping impurities may extend far below the product peak, limiting the maximum possible purity obtainable with MCSGP. [Prior art documents] [Patent documents]

[0015] [Patent Document 1] European Patent Application Publication No. 1877769 Summary of the Invention

[0016] The present invention aims to provide a further improved MCSGP-like process, which can reach higher product purity than the conventional MCSGP process.

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

[0018] The proposed additional recirculation step transfers the chromatographic profile from one chromatographic adsorbent to another, whereby an in-line dilution is applied so that the chromatographic profile is re-adsorbed in the downstream adsorbent. Typically, this in-line dilution is performed by essentially constantly adding modifier-free or low modifier content eluent between the two columns to reduce the elution strength of the downstream liquid and slow down the elution in the downstream column. In the case of reversed-phase chromatography, typical modifiers are organic solvents, whereas in ion exchange, typical modifiers are salts (adjusting ionic strength) or acids / bases (adjusting 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. Thus, it is expected that the partial separation of the compounds in the chromatographic profile obtained by elution from the upstream will be nullified by the in-line dilution. In other words, the in-line dilution creates the same situation as after feed injection, with the expected loss of separation.

[0019] Therefore, one skilled in the art would expect that the introduction of a recycle step with normal or "excessive" in-line dilution would not lead to improved product purity.

[0020] Furthermore, one skilled in the art would expect that the introduction of a recycle step would reduce the overall productivity (throughput) of the process since the recycle step takes up extra time during which no additional feed is introduced and no product is produced.

[0021] Surprisingly, it has been found that the time loss caused by the introduction of a recycle stage can be overcompensated by increasing the load. In addition, significantly higher purities can be achieved by recycle.

[0022] The method uses two chromatographic adsorbent sections as chromatographic stationary phases, where one adsorbent section may consist of one single column but also of several columns, in which case the columns of one section are always interconnected and never disconnected during the process.

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

[0024] The method comprises an optional but preferred start-up phase, a recirculation phase with one or several recirculation sequences (n≧1) and a purification phase carried out only once after the recirculation phase. The recirculation and purification phases form a so-called basic sequence, which is repeated at least once (m≧1). After the desired number of basic sequences, an optional but preferred stop-down phase follows.

[0025] This basic principle is illustrated diagrammatically in FIG.

[0026] Figure 2 shows the method for different cycle times n = 1, 2, 3, 4 in the recycle phase. All examples begin with a start-up phase and end with a stop-down phase. In the start-up phase, a feed mixture F is loaded onto a first adsorbent body 1, usually pre-equilibrated. The feed mixture contains not only the desired product P, but also impurities, namely a less adsorbable impurity W and a more adsorbable impurity S. Subsequently, the less adsorbable impurity W, i.e. an impurity that adsorbs less strongly to the stationary phase than the product P, is eluted from this adsorbent body, with or without the (exemplary preferred) continuation of the feed flow. The process then enters the recycle phase.

[0027] In the case of 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, the outlet of the first adsorber 1 is connected to the inlet of the second adsorber 2, preferably with a solvent supply with a gradient (see further below, in particular FIG. 5). An in-line dilution is carried out between the adsorber. An in-line dilution is carried out with a base solvent, thus without a gradient.

[0028] Then, in a next splitting step BR, the adsorbents are split and the strongly adsorbed impurities S, i.e. impurities which adsorb more strongly to the stationary phase than the product, are eluted from the first adsorbent 1 and this adsorbent is re-equilibrated, while the weakly adsorbed impurities W are eluted from the second adsorbent 2. This latter splitting step BR in the recycle phase is optional.

[0029] In the case of n=2, the recycle step consists of the recycle step of n=1 plus one more cycle, i.e. one more interconnection step and one more (optional) splitting step. In the further interconnection step, the outlet of the former second adsorber 2 is connected to the inlet of the former first adsorber 1. Then, in a following further splitting step, the adsorber is split and the strongly adsorber impurity S is eluted from the second adsorber 2 and this adsorber is re-equilibrated, while the weakly adsorber impurity W is eluted from the first adsorber 1. This latter further splitting step is again optional.

[0030] In the case of n=3, the recycle step consists of the recycle step of n=2 plus one further interconnection step and one further (optional) splitting step. In the interconnection step, the outlet of the first adsorber 1 is connected to the inlet of the second adsorber 2. Then, in a subsequent splitting step, the adsorber is split and the strongly adsorber impurities S are eluted from the first adsorber 1 and this adsorber is re-equilibrated, while the weakly adsorber impurities W are eluted from the second adsorber 2. This latter splitting step is again optional.

[0031] In the case of n=4, the recycle step consists of the recycle step of n=3 plus one more interconnection step and one more splitting step. In the interconnection step, the outlet of the second adsorber 2 is connected to the inlet of the first adsorber 1. Then, in a subsequent splitting step, the adsorber is split and the strongly adsorber impurity S is eluted from the second adsorber 2 and this adsorber is re-equilibrated, while the weakly adsorber impurity W is eluted from the first adsorber 1. This latter splitting step is optional.

[0032] In general, for any number of cycles (n>1), the recycle step consists of n-1 process and recycle steps plus one further interconnect step and one further (optional) disconnect step.

[0033] Typically, for recycle stages with even numbers n=2, 4, 6, ..., in the last interconnection step of the recycle stage, the outlet of the second adsorber 2 is connected to the inlet of the first adsorber 1. Then, in a next splitting step, if performed, the adsorber is split and the strongly adsorber impurities S are eluted from the second adsorber 2 and this adsorber is re-equilibrated, while the weakly adsorber impurities W are eluted from the first adsorber 1. This latter splitting step is optional.

[0034] Typically, for odd number n=1, 3, 5, ... recycle steps, in the last interconnection step of the recycle step, the outlet of the first adsorber 1 is connected to the inlet of the second adsorber 2. Then, in a subsequent splitting step, the adsorber is split and the strongly adsorber impurities S are eluted from the first adsorber 1 and this adsorber is re-equilibrated, while the weakly adsorber impurities W are eluted from the second adsorber 2. This latter splitting step is optional.

[0035] The recirculation steps are followed by purification steps, each of which includes a first interconnection step IC1, a first batch (disconnection) step B1, a second interconnection step IC2 and a second batch (disconnection) step B2.

[0036] For n=1 and n=3 and all odd natural numbers n, in the interconnection step IC1 of the purification stage, the outlet of the second adsorber 2 is connected to the inlet of the first adsorber 1, i.e. the second adsorber 2 is upstream of the first adsorber 1 and an in-line dilution is performed between the two adsorber. In the subsequent batch step B1, the adsorber is disconnected and the purified product P is eluted from the second adsorber 2 and collected, while a fresh feed mixture F is loaded into the first adsorber 1. In the next interconnection step IC2, the adsorber is connected and the second adsorber 2 is upstream of the first adsorber 1 and an in-line dilution is performed between the two adsorber.

[0037] In the next disruption step B2, the adsorber is disrupted and the strongly adsorptive impurities S are eluted from the second adsorber 2 and this adsorber is re-equilibrated, while the weakly adsorptive impurities W are eluted from the first adsorber 1.

[0038] For n=2 and n=4 and all even natural numbers n, in the interconnection step IC1 of the purification stage, the outlet of the first adsorber 1 is connected to the inlet of the second adsorber 2, i.e. the first adsorber 1 is upstream of the second adsorber 2 and an in-line dilution is performed between the two adsorber. In the subsequent batch step B1, the adsorber is disconnected and the purified product P is eluted from the first adsorber 1 and collected, while a fresh feed mixture F is loaded into the second adsorber 2. In the next interconnection step IC2, the adsorber is connected and the first adsorber 1 is upstream of the second adsorber 2 and an in-line dilution is performed between the two adsorber.

[0039] In the next splitting step, the adsorber is split and the strongly adsorber impurity S is eluted from the first adsorber 1 and this adsorber is re-equilibrated, while the weakly adsorber impurity W is eluted from the second adsorber 2.

[0040] For any number n of recycle steps, after any desired number m of basic sequences, the final purification step is followed by a stop step as illustrated in FIG.

[0041] Figure 3 shows the stopping phase for various numbers of recirculation phases n = 1, 2, 3, 4, .... The last purification phase of the basic sequence of the desired number of times is followed by a stopping phase, which itself consists of two phases: stopping phase I and stopping phase II.

[0042] The first stage, shutdown stage I, consists of the same IC-R and (optional) BR steps as at the end of the preceding recycle stage of the process.

[0043] The second stoppage stage, stoppage stage II, consists of the same steps as the preceding purification stage of the process with some modifications, but extended by an additional step of eluting product P and an additional step of eluting impurity S, except that it is operated without an additional load of fresh feed mixture F.

[0044] For n=1 and n=3 and all odd whole numbers n, the stop stage I consists of an interconnection step, in which the outlet of the first adsorber 1 is connected to the inlet of the second adsorber 2. An in-line dilution is performed between the adsorber. In a subsequent splitting step, the adsorber is split and the strongly adsorber impurity S is eluted from the first adsorber 1 and this adsorber is re-equilibrated, while the weakly adsorber impurity W is eluted from the second adsorber 2. This latter splitting step is optional.

[0045] For n=1 and n=3 and all odd natural numbers n, the stop stage II consists of an interconnection step IC1, in which the outlet of the second adsorber 2 is connected to the inlet of the first adsorber 1, i.e. the second adsorber 2 is upstream of the first adsorber 1 and an in-line dilution is carried out between the two adsorber. In a subsequent batch step B1, the adsorber is disconnected and the purified product P is eluted from the second adsorber 2 and collected. In this step, no new feed mixture F is loaded into the first adsorber 1. In a next interconnection step IC2, the adsorber is connected, the second adsorber 2 is upstream of the first adsorber 1 and an in-line dilution is carried out between the two adsorber. Subsequently, the purified product P is eluted from the first adsorber 1 and collected and the highly adsorbable impurities S are eluted from the first adsorber 1.

[0046] For n=2 and n=4 and all even natural numbers n, stop-down phase I consists of the same steps as stop-down phase I when n is odd, but with adsorbents 1 and 2 interchanged, and for n=2 and n=4 and all even natural numbers n, stop-down phase II consists of the same steps as stop-down phase II when n is odd, but with adsorbents 1 and 2 interchanged.

[0047] 4 shows the task of two adsorbents in the start-up phase for any number n. In the start-up phase, the pre-equilibrated first adsorbent 1 is loaded with the feed mixture F (step B-SU-F). Subsequently, weakly adsorbed impurities W are eluted from this adsorbent (step B-SU-W). The linear gradient segments, flow rates and switching times operated in the first adsorbent 1 in step B-SU-W preferably correspond to those selected for the single-column chromatogram.

[0048] In general, the operating parameters, i.e., flow rates, gradient concentrations, feed rates, and switching times of the method, can be derived from the single adsorbent chromatogram, as shown in the lower part of Figure 4. Thus, the method can be designed and initialized based on the single adsorbent chromatogram, where much more precise control during operation is possible, for example, based on detector feedback.

[0049] FIG. 5 shows the recycle stage of the method for n=1.

[0050] The recycle stage itself consists at least of one sequence of interconnection (IC-R) and disconnection (BR) steps. In the first interconnection step (IC-R), the outlet of the first adsorber 1 is connected to the inlet of the second adsorber 2. The adsorbed compounds W, P and S are eluted from the first adsorber 1 into the second adsorber 2 using a solvent gradient, where W, P and S are strongly adsorbed on the second adsorber 2, while the impurity W, which is preferably not even weakly adsorbed, leaves the downstream adsorber 2, due to the in-line dilution performed between the adsorber with a base solvent without modifier or a solvent with a low modifier content. Then, in the next first splitting step (BR), the adsorbent is split and the remaining impurities S are eluted from the first adsorbent 1 using a high, preferably constant, modifier concentration before re-equilibrating the adsorbent 1, while the weakly adsorbed impurities W are eluted from the second adsorbent 2 using a gradient with a low starting modifier concentration (see the lower dashed lines under each step showing the modifier concentrations according to the process). This latter splitting step BR is optional and its importance depends on the purity and purity specifications of the product P in the feed mixture F. Since a lower purity corresponds to a higher content of W and S, it will be considered to include a splitting step (BR) in the recycle stage to remove W and S. Again, as shown in the lower part of FIG. 5, the operating parameters, i.e. flow rates, gradient concentrations, feed rates and switching times of the method, can be derived from the single adsorbent chromatogram, where even more precise control during operation is possible, for example based on detector feedback.

[0051] When n=1, the stopping step I is identical to the recycling step. Each recycling step is followed by a purification step (FIG. 2, FIG. 6).

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

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

[0054] In the interconnection step IC1, the outlet of the second adsorber 2 is connected to the inlet of the first adsorber 1, i.e. the second adsorber 2 is upstream of the first adsorber 1. In the interconnection step IC1, a partially pure subfraction containing W and P (the overlapping region ("zone 5") in the chromatogram (see schematic chromatogram at the bottom of FIG. 6)) is eluted from the second adsorber 2 into the first adsorber 1. During the operation of IC1, the stream containing W and P is diluted in-line before entering the first adsorber 1, so that W and P are completely adsorbed when they enter the first adsorber 1. In the subsequent batch step B1, the adsorber is disconnected and the purified product P is eluted and collected from the second adsorber 2, while a fresh feed mixture F is loaded into the first adsorber 1. In the next interconnection step IC2, the adsorbents are connected, the second adsorbent 2 being upstream of the first adsorbent 1, and the partially pure subfraction containing P and S (the overlapping region ("zone 7") in the chromatogram (see the bottom part of FIG. 6)) is eluted from the second adsorbent 2 into the first adsorbent 1. Due to the in-line dilution of the stream containing P and S before entering the first adsorbent 1, the compounds P and S are completely adsorbed when they enter the first adsorbent 1. Again, as shown in the bottom part of FIG. 6, the operating parameters, i.e. flow rates, gradient concentrations, feed rates and switching times of the method, can be derived from the single adsorbent chromatograms, where even more precise control during operation is possible, for example based on detector feedback.

[0055] FIG. 7: To avoid losses of product P at the end of the process run, the purification step is followed by a stoppage step, which itself consists of two stages: stoppage step I and stoppage step II.

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

[0057] FIG. 7 shows the stopping phase II of the method for n=1, 3, 5, . . .

[0058] The stop stage II comprises essentially the same steps as the previous purification stage of the method, but without a feed (see FIG. 6) (IC1-SD corresponds to IC1, B1-SD corresponds to B1 but without a feed, IC2-SD corresponds to IC2, B2-SD corresponds to B2), but is extended by an additional step (B-SD-P) of eluting the product P from the first adsorber, previously in a downstream position, and an additional step (B-SD-S) of eluting the impurity S, cleaning and regenerating the same adsorber. The conditions for the collection of P in B-SD-P and B1-SD are preferably identical in terms of flow rate and concentration gradient. Similarly, the conditions for the removal of S in B-SD-S and B2-SD are preferably identical in terms of flow rate and concentration gradient.

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

[0060] In the process, gradients, particularly linear gradients, can be and preferably will be used at any step to enhance separation of the compounds and / or modify elution rates by altering the slope of the gradient.

[0061] By choosing a higher gradient slope in the recycle step steps (IC-R and / or BR) than in the purification step steps (IC1, B1, IC2, and / or B2), the recycle step will be completed sooner, thus increasing the overall process productivity.

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

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

[0064] The proposed method comprises at least one basic sequence with at least one recycle step followed by exactly one purification step, whereby preferably the basic sequence is cyclically repeated at least twice.

[0065] According to the invention, the recycling step comprises the following steps: a. Interconnection recirculation process IC-R, In this process, the adsorbent sections are interconnected and have a recirculation interconnection period t IC-R an upstream adsorber section outlet connected to a downstream adsorber section inlet, wherein The upstream adsorber section is loaded with an eluent via an inlet of the upstream adsorber section, and a fraction including weakly adsorbable impurities W at least to an extent overlapping with the product P, the product P, and strongly adsorbable impurities S at least to an extent overlapping with the product P is eluted from the upstream adsorber section to the downstream adsorber section; and The flow exiting the upstream adsorber outlet is diluted in-line before entering the downstream adsorber inlet; b. An optional batch recycle step BR; where the recirculation batch period t B-R the adsorber section is severed during The upstream adsorber section prior to the preceding step a is cleaned to remove strongly adsorptive impurities S and regenerated, and an eluent is loaded into the inlet of the downstream adsorber section prior to the preceding step a to elute weakly adsorptive impurities W in the range not overlapping with the product P; at least one recirculation sequence, preferably only one, or at least two, or at least three, or at least four recirculation sequences, After each recycle sequence of steps a and b, the adsorber sections are switched in sequence, with the recycle sequence being performed one or more times.

[0066] According to the invention, the recycling step is followed by only one purification step, which comprises the following steps: c. First interconnect purification step IC1; In this process, the adsorbent sections are interconnected, where a first interconnected purification period t IC1 an outlet of the upstream adsorber section is connected to an inlet of the downstream adsorber section; the upstream adsorber is loaded with an eluent via the upstream adsorber inlet, and a product fraction comprising overlapping weakly adsorbable impurities W and product P is eluted from the upstream adsorber into the downstream adsorber section; and The flow exiting the upstream adsorber section outlet is diluted in-line before entering the downstream adsorber section inlet; d. First batch purification step B1; Here, the first batch purification period t B1 the adsorber section is disconnected during the process, and product P is eluted from the former upstream adsorber section, and feed mixture F is supplied to the inlet of the former downstream adsorber section; e. A second interconnect purification step IC2; Here, the second interconnection purification period t IC2 an outlet of the upstream adsorber section is connected to an inlet of the downstream adsorber section; The upstream adsorber section is loaded with an eluent via an upstream adsorber section inlet, and a product fraction containing overlapping product P and highly adsorbable impurity S is eluted from the upstream adsorber to the downstream adsorber; and The flow exiting the upstream adsorber section outlet is diluted in-line before entering the downstream adsorber section inlet; f. A second batch purification step B2; Here, the second purification batch period t B2 during which the adsorbent is disconnected, and the former upstream adsorbent is cleaned and regenerated, and eluent is loaded into the former downstream adsorbent inlet; in order, where the upstream adsorber section of the last interconnected recycle step of the preceding recycle stage assumes the function of the downstream adsorber section, and the downstream adsorber section of the last interconnected recycle step of the preceding recycle stage assumes the function of the upstream adsorber section.

[0067] As pointed out above, preferably an eluent gradient is used in at least one or all of the steps.

[0068] Preferably, in the interconnection step of the method, the upstream section inlet is loaded with an eluent having a gradient in the form of a time-varying modifier concentration via the upstream adsorber inlet, and the flow leaving the upstream adsorber section outlet is diluted in-line with a modifier-free eluent or with an eluent having a different modifier concentration than the inlet of the upstream adsorber section, preferably a lower modifier concentration than the inlet of the upstream adsorber section, before entering the downstream adsorber section inlet.

[0069] More preferably, in a batch step of the process without elution of a purified product, the previous upstream adsorber is cleaned and regenerated, for example with an eluent having a higher modifier concentration than at the end of the preceding interconnected recycle step, or with an eluent containing a different modifier, or with a cleaning solution, and an eluent having a gradient in the form of a time-varying modifier concentration is loaded into the previous downstream adsorber inlet.

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

[0071] In the recirculation stage in the interconnected recirculation process IC-R, according to a preferred embodiment, the upstream adsorber section is loaded with an eluent having a gradient in the form of a time-varying modifier concentration via the upstream adsorber section inlet, and the flow leaving the upstream adsorber outlet is diluted in-line with a modifier-free eluent or with an eluent having a different modifier concentration than the inlet of the upstream adsorber section before entering the downstream adsorber inlet.

[0072] According to yet another preferred embodiment, in any batch recycle step BR, the upstream adsorber section prior to the preceding step a is cleaned and regenerated, for example with an eluent having a higher modifier concentration than at the end of the preceding interconnected recycle step, or with an eluent containing a different modifier, or with a cleaning solution, and an eluent having a gradient in the form of a time-varying modifier concentration is loaded at the inlet of the downstream adsorber section prior to step a to elute weakly adsorbable impurities W in a range that does not overlap with the product P.

[0073] Yet another preferred embodiment is characterized in that, in said purification stage in said first interconnected purification step IC1, the upstream adsorbent section is loaded via the upstream adsorbent inlet with an eluent having a gradient in the form of a time-varying modifier concentration, and the flow leaving the upstream adsorbent section outlet is diluted in-line with a modifier-free eluent or with an eluent having a modifier concentration different from that at the inlet of the upstream adsorbent section before entering the downstream adsorbent section inlet.

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

[0075] In the second interconnected purification step IC2, the upstream adsorber section inlet is preferably loaded with an eluent having a gradient in the form of a time-varying modifier concentration via the upstream adsorber section inlet, and the flow leaving the upstream adsorber section outlet is diluted in-line with a modifier-free eluent (base solvent only) or with an eluent having a different modifier concentration than the inlet of the upstream adsorber section before entering the downstream adsorber section inlet.

[0076] Also, in the above-mentioned second batch purification step B2, it is preferred to clean and regenerate the previous upstream adsorbent with an eluent having a higher modifier concentration than at the end of the preceding interconnected recirculation step, or with an eluent containing a different modifier, or with a cleaning solution, and to load an eluent having a gradient in the form of a time-varying modifier concentration at the inlet of the previous downstream adsorbent.

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

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

[0079] The base solvent is usually water or a mixture of water with a small proportion of one or more salts and / or organic solvents (e.g. with acetonitrile and trifluoroacetic acid) compared to water, particularly when biological molecules, for example obtained from biochemical processes, are to be separated, and this base solvent will be referred to below as solvent A. To establish the gradient, it is mixed with a further solvent or solvent mixture different from the base solvent (mixture). This further solvent can be, for example, a mixture of the same solvents as the base solvent, but with different proportions (e.g. in the above example, water with a higher proportion of acetonitrile). In particular in the case of biological molecules, this further solvent is typically again based on water, but with an increased proportion of organic solvent. To establish the gradient, a modifier mixture, which typically contains a significantly lower concentration of components different from the base solvent (e.g. organic solvent(s), salts or pH, or a combination thereof, when the base solvent is, for example, water), is provided as eluent at the beginning of the gradient and is mixed incrementally with the further solvent by a gradient pump to provide a corresponding controlled gradient. The feed mixture may be provided in various solvents, or may be provided in a base solvent, or the feed mixture may be provided as a mixture of an original solution (typically an aqueous solution) in a mixture with an appropriate concentration of a base solvent.

[0080] In at least one or all of the above steps, a preferably linear eluent gradient is used with a time-varying gradient of increasing modifier concentration.

[0081] As mentioned above, preferably, before carrying out the first interconnect recirculation step of the first recirculation stage, a start-up step is carried out, in which First batch start-up period t B-SU-F The adsorbent is divided into two parts, and feeding the feed mixture F to an inlet of an adsorber section which is the upstream adsorber section of the first interconnected recycle step of the first recycle stage; On the other hand, the adsorber section that is to be the downstream adsorber section of the first interconnected recycle step of the first recycle stage is equilibrated or has already been equilibrated and is inactive, and Preferably, a second batch start-up period t B-SU-W During this step, the adsorbent is split and weakly adsorbable impurities W are eluted from the adsorbent section to which feed mixture F was supplied in the preceding first batch start-up step, while the other adsorbent is being equilibrated or has already been equilibrated and is in an inactive state.

[0082] The first batch start-up period t B-SU-F After the second batch start-up period t B-SU-W Prior to this, an eluent may be fed to the inlet of the adsorber section which will be the upstream adsorber section of the first interconnected recirculation step of the first recirculation stage, and said eluent may be modifier-free (base solvent only) or have a modifier concentration which essentially corresponds to the starting modifier concentration applied during said first batch start-up period or in the absence of the second batch start-up step of the first interconnected recirculation step.

[0083] Additionally, during said second batch start-up period, the inlet of the adsorber section fed with feed mixture F can be loaded with an eluent having a gradient in the form of a time-varying modifier concentration.

[0084] After completion of at least one base sequence, preferably two or more base sequences, a stopping step can be carried out, which comprises the following steps: a'. Interconnect stop recirculation process IC'-R, In this step, the upstream adsorber section of the last preceding second interconnected purification step IC2 takes the downstream position and another adsorber section takes the upstream position, the adsorber sections are interconnected, and there is a stop recirculation interconnection period t IC’-R-SD an outlet of the upstream adsorber section is connected to an inlet of the downstream adsorber section; Here, an eluent is loaded into the upstream adsorber section via an inlet of the upstream adsorber section, and a fraction containing weakly adsorbable impurities W at least to an extent overlapping with the product P, the product P, and strongly adsorbable impurities S at least to an extent overlapping with the product P is eluted from the upstream adsorber section to the downstream adsorber section; and The flow exiting the upstream adsorber outlet is diluted in-line before entering the downstream adsorber inlet; b'. Optional batch stop recycle step B'-R; In this process, the adsorber section is disconnected, where the stop recycle batch period t B’-R the adsorber section is severed during The previous upstream adsorber section of the preceding step a' is cleaned and regenerated, and an eluent is loaded into the inlet of the previous downstream adsorber section of step a to elute weakly adsorptive impurities W in the range not overlapping with the product P; in order.

[0085] Preferably, this is followed by the steps of: c'. First interconnection stop purification step IC1-SD; In this process, the adsorbent sections are interconnected, where a first interconnection stop purification period t IC1’ an outlet of the upstream adsorber section is connected to an inlet of the downstream adsorber section; The upstream adsorber is loaded with an eluent via the upstream adsorber inlet, and a product fraction containing overlapping weakly adsorbable impurities W and product P is eluted from the upstream adsorber into the downstream adsorber section; and The flow exiting the upstream adsorber section outlet is diluted in-line before entering the downstream adsorber section inlet; d'. First batch stop purification step B1-SD; Here, the first batch purification period t B1’ during which the adsorber section is disconnected and product P is eluted from the previous upstream adsorber section and the previous downstream adsorber section is either idled or eluent is supplied to the inlet of the previous downstream adsorber section; e'. second interconnection stop purification step IC2-SD, Here, the second interconnection stop purification period t IC2’ an outlet of the upstream adsorber section is connected to an inlet of the downstream adsorber section; The upstream adsorber section is loaded with an eluent via an upstream adsorber section inlet, and a product fraction containing overlapping product P and highly adsorbable impurity S is eluted from the upstream adsorber to the downstream adsorber; and The flow exiting the upstream adsorber section outlet is diluted in-line before entering the downstream adsorber section inlet; f'. Second batch stop purification step B2-SD; Here, the second purification batch period t B2’ during which the adsorbent is disconnected, and the former upstream adsorbent is cleaned and regenerated, and eluent is loaded into the former downstream adsorbent inlet; follows in sequence, where the upstream adsorber section of the interconnected stop recycle step assumes the function of the downstream adsorber section, and the downstream adsorber section of the interconnected stop recycle step assumes the function of the upstream adsorber section.

[0086] Typically, g'. the first final stop batch process B-SD-P; Here, the first final stop batch period t B-SD-Pduring which the first and second adsorbents are decoupled and product P is eluted from the previous downstream adsorbent and the other adsorbent is idled or regenerated; h'. optional second final stopping batch step B-SD-S, Here, the second final stop batch period t B-SD-S during which the first and second adsorber are decoupled, and the highly adsorptive impurities S are eluted from the previous downstream adsorber, and the other adsorber is idled or regenerated; continues.

[0087] In the above interconnected stop recycle step IC'-R, the upstream adsorber section of the preceding and last second interconnected purification step IC2 can be loaded via the upstream adsorber section inlet with an eluent of constant composition or with an eluent having a gradient in the form of a time-varying modifier concentration, where the flow leaving the upstream adsorber outlet is diluted in-line with a modifier-free eluent or with an eluent having a modifier concentration different from that at the inlet of the upstream adsorber section before entering the downstream adsorber inlet.

[0088] In the above batch stop recycle step B'-R, the previous upstream adsorber section of the previous step a can be cleaned and regenerated with an eluent having a higher modifier concentration than at the end of the previous interconnected recycle step, or with an eluent containing a different modifier, or with a cleaning solution, where an eluent having a gradient in the form of a time-varying modifier concentration is loaded at the inlet of the previous downstream adsorber section of step a' to elute weakly adsorbable impurities W in a range that does not overlap with the product P.

[0089] In the above first interconnected stop purification step IC1-SD, the upstream adsorber can be loaded with an eluent having a gradient in the form of a time-varying modifier concentration via the upstream adsorber inlet, and the flow leaving the upstream adsorber section outlet is diluted in-line with a modifier-free eluent or with an eluent having a different modifier concentration than the inlet of the upstream adsorber section before entering the downstream adsorber section inlet.

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

[0091] In the above-mentioned second interconnected stop purification step IC2-SD, the upstream adsorber section can be loaded via the upstream adsorber section inlet with an eluent of constant composition or with an eluent having a gradient in the form of a time-varying modifier concentration, and the flow leaving the upstream adsorber section outlet is diluted in-line with a modifier-free eluent or with an eluent having a different modifier concentration than the inlet of the upstream adsorber section before entering the downstream adsorber section inlet.

[0092] Further, in said second batch stop purification step B2-SD, the former upstream adsorber can be cleaned and regenerated with an eluent having a higher modifier concentration than at the end of the preceding interconnected recycle step, where the eluent is loaded into the former downstream adsorber inlet.

[0093] Finally, in said first final stop batch step B-SD-P, the 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, while the other adsorbent is idle or regenerated, and / or In said first final second final stop batch step B-SD-S, the strongly adsorbed impurities S can be eluted from the previous downstream adsorber with an eluent having a higher modifier concentration than at the end of the preceding interconnected recycle step, while the other adsorber is idled or regenerated.

[0094] A linear gradient with different slopes and / or flow rates is preferably used in the recirculation stage and / or purification stage, where preferably a higher slope and / or higher flow rate is used in the recirculation stage than in the purification stage.

[0095] According to yet another preferred embodiment, in most (in particular all except step IC2 and step IC2-SD) or all interconnected steps of the method, the upstream section inlet is loaded with an eluent having a gradient in the form of a time-varying modifier concentration via the upstream adsorber inlet, the flow leaving the upstream adsorber section outlet is diluted in-line with a modifier-free eluent or with an eluent having a modifier concentration different from that at the inlet of the upstream adsorber section before entering the downstream adsorber section inlet, and In a batch step of the process without elution of a purified product, the previous upstream adsorber is cleaned and regenerated with an eluent having a higher modifier concentration than at the end of the preceding interconnected recycle step, and, if applicable, an eluent having a gradient in the form of a time-varying modifier concentration is loaded into the previous downstream adsorber inlet, or In a batch step of the process involving elution of the purified product, the previous upstream adsorber is loaded via the upstream adsorber inlet with an eluent having a gradient in the form of a time-varying modifier concentration, 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, electrolytes in such a solvent or mixture thereof, preferably selected from dissolved salts or pH, or a combination thereof, wherein preferably said 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 minor proportion compared to water, more preferably said modifier is an organic solvent, or a mixture of water and at least one organic solvent, in which the concentration of said at least one organic solvent is higher than in the base solvent, salt concentrations and / or H concentrations different from those in water or the base solvent. + and The modifier concentration is adjusted during the process by the following steps: Recirculation processes (IC-R, IC'-R, IC1, IC1-SD) except for the final stop batch process (B-SD-P), and where applicable, steps of product elution (B1, B1-SD, B-SD-P) and, where applicable, further steps of recycling (IC2, IC2-SD), during which the modifier concentration may vary or may be kept constant only in the case of the recycling steps, From the lowest concentration at the start of elution of the weakly adsorbed impurity W in the range not overlapping with the product P (B-SU-W, BR, B'-R, B2, B2-SD) over the entire After a maximum, preferably linear, increase in concentration, the elution of the strongly adsorbed impurity S (BR, B'-R, B2, B2-SD, B-SD-S) begins.

[0096] In the process, it is possible to use adsorbents with different dimensions or with different stationary phases, or with both different dimensions and different stationary phases. Elution of product P is always done from the same adsorbent, as is the loading of new feed material. For example, in the case of a recycle stage involving only a single sequence (n=1) of IC-R and BR, the main purification task is performed by adsorbent 2 (recycle of W / P, elution of product P, recycle of P / S). It is therefore possible to use small particle chromatographic resin in adsorbent 2 for this task to maximize the separation capacity, whereas large particle resin can be used to receive the recycle stream in adsorbent 1. Because the eluent flow entering adsorbent 1 is higher than the flow leaving adsorbent 2 due to the in-line dilution of W / P and P / S in steps IC1 and IC2, adsorbent 1 may benefit from larger particles that have lower back pressure under flow than small particle resins.

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

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

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

[0100] [Figure 1] FIG. 1 shows a general scheme of the process, including start-up, recycle, purification and shutdown steps. [Diagram 2]Figure 2 shows in more detail the logic of the process (start-up, recycle, purification stages) including the adsorbent connections and the streams entering and leaving the adsorbent for n=1 to 4 sequences in the recycle stage. Feed / eluent streams that do not contain W, P, S, or F are not specifically marked. [Diagram 3] Figure 3 shows in more detail the logic of the process (stopping stage) including the adsorbent connections and the streams entering and leaving the adsorbent for n=1 to 4 sequences in the recycle stage. Feed / eluent streams that do not contain W, P, S or F are not specifically marked. [Figure 4] Figure 4 shows the task of the two adsorbents in the start-up phase for a single column chromatogram (shown at the bottom) and shows diagrammatically the linear gradient segment (thick dashed line) used in the process in the case of linear gradient operation. Thin vertical dashed lines show diagrammatically the boundaries of the sections, and in Figures 4 to 7 the vertical sections designated as zones and separated by thin vertical dashed lines show the various functions of the column in each zone, with the lower right hand side showing a chromatogram with product P, a weaker impurity W eluting earlier than product P to the left of product P, and a more strongly adsorbed impurity S eluting later than product P to the right of product P, and the applicable feed concentration in that zone at the inlet of the column in zone 2, shown as a dashed line at the bottom of zone 2. In the chromatographic zones (zones 4 to 8 in Figures 4, 6 and 7, and zones 4 to 6 in Figure 5), the lower dashed lines indicate the modifier concentration used for the gradient applied at the inlet of the column in each zone during elution of the less adsorbable impurity W and product P (zones 4 to 7 in Figures 4, 6 and 7, and zones 4 and 5 in Figure 5), and the higher, but usually constant, modifier concentration applied during the stage of elution of S, which does not overlap with P (zone 8 in Figures 4, 6 and 7, and zone 6 in Figure 5). [Diagram 5]FIG. 5 is a diagram showing the task of two adsorbents in the recycle stage for a single column chromatogram (shown at the bottom) and shows, in schematic form, the linear gradient segment used in the process in the case of linear gradient operation. [Figure 6] FIG. 6 is a diagram showing the task of two adsorbents in the purification step for a single column chromatogram (shown at the bottom) and shows, in schematic form, the linear gradient segment used in the process in the case of linear gradient operation. [Figure 7] FIG. 7 is a diagram showing the task of the two adsorbents in the stopping stage II for a single-column chromatogram (shown at the bottom) and shows, in schematic form, the linear gradient segment used in the process in the case of linear gradient operation. [Figure 8-1] FIG. 8 a) shows an overlay of the UV profile of the stop phase of the process and a single-column reference run, and b) shows an overlay of the product concentration values ​​determined by offline HPLC analysis of the stop phase of the process and a single-column reference run. [Figure 8-2] 8 c) shows an overlay of product purity values ​​determined by offline HPLC analysis of the stop phase of the process and a single-column reference run, and d) shows an overlay of impurity content values ​​determined by offline HPLC analysis of the stop phase of the process and a single-column reference run, with the main peak of the strongly adsorbing impurity S indicated by an arrow in d). [Figure 9] FIG. 9 shows the overlay of purity / yield curves of the process and a single column reference run. [Figure 10]FIG. 10 a) shows an internal chromatogram of a typical MCSGP process recorded at the column outlet of the upstream column through steps IC1, B1, IC2, and B2; b) shows an 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 an 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, where the rectangular areas indicate product collection intervals, the arrows marked "W" indicate the locations of weakly adsorbable impurities, and the arrows marked "S" indicate the locations of strongly adsorbable impurities. [Figure 11] Figure 11 a) shows a chromatogram of the batch reference process at a bed height of 10 cm and b) shows a chromatogram of the batch reference process at a bed height of 20 cm. The rectangular areas indicate the product collection intervals, the arrows marked "W" point to the location of the weakly adsorbing impurities and the arrows marked "S" point to the location of the strongly adsorbing impurities. [Figure 12] Figure 12 shows product pool purity versus productivity for 1. the present process with n=1 sequence in the recycle stage, 2. the present process with n=2 sequence in the recycle stage, the MCSGP process, a single-column batch reference process with a bed height of 10 cm, and a single-column batch reference process with a bed height of 20 cm. For the batch process, the figure also shows the purity / productivity curves for points corresponding to product pools of various sizes. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[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) + 0.1% trifluoroacetic acid (TFA) in water). Solvent A and solvent B (50% ACN + 0.1% TFA in water) were used for chromatographic runs. Feed purity was %W = 4.76, %P = 91.67, %S = 3.57 (area % based on analytical HPLC in all cases).

[0102] The process was carried out using two columns (0.46 cm internal diameter x 15 cm bed height) with a column volume of 2.5 mL. For reference, a single column run was carried out using the same materials.

[0103] The process was carried out in a Contichrom CUBE system (ChromaCon) using the parameters shown in Table 1.

[0104] To investigate the effect of the recycle step, a stop step was performed immediately after the recycle step and the product peak was fractionated in a non-cyclic, non-continuous process. Comparison of the fractionated shutdown with the fractionation of a single-column reference run shows 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 achieved purity as determined by analytical HPLC was higher over a much wider range of fractions in this process (Figure 8c). Offline HPLC analysis confirmed that the highly adsorbing impurity S was significantly retarded in this process, allowing the inclusion of more fractions in the product pool (Figure 8d).

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

[0106] [Table 1]

[0107] Example 2: Numerical simulation of oligonucleotide purification The process was simulated for the purification of oligonucleotides by anion exchange chromatography. The simulated feed mixture consisted of a 20-mer dsDNA oligonucleotide, 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 of 0.5 cm inner diameter and 10 cm bed height packed with YMC SmartSep Q30 were used. For the simulations, a mechanistic model based on the Bailangmuir adsorption isotherm was used and calibrated using a series of single-column gradient experiments with various gradient slopes and loads.

[0109] FIG. 10a shows the internal chromatogram of a typical MCSGP process according to US Pat. No. 6,399,433 recorded at the column outlet of the upstream column through steps IC1, B1, IC2, and B2.

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

[0111] The operating parameters of the simulated system investigated are given in the table.

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

[0113] For comparison, Figure 11a and b show chromatograms of the batch reference process at bed heights of 10 cm and 20 cm, respectively. As in Figure 10a-c, the rectangular areas indicate the product collection intervals, the arrows labeled "W" point to the locations of associated overlapping weakly adsorptive impurities, and the arrows labeled "S" point to the locations of associated overlapping strongly adsorptive impurities. It can be seen that as more recycle stages are used, the product pool contains much lower amounts of impurities W and S.

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

[0115] It can be seen that the present process allows obtaining much higher purity (>97.5% for both n=1 and n=2) than the reference batch process (<97.0% purity) at comparable productivity. Compared to the "normal" MCSGP process according to US Pat. No. 5,999,433, the present process achieves higher purity (<96.0% MCSGP purity), but with slightly lower productivity. Furthermore, Fig. 12 confirms that by increasing the number n of sequences of recycle steps, the product purity can be further increased (purity=97.8% for n=1 vs. 98.4% for n=2), but at the expense of productivity (productivity=3.4g / L / h for n=1 vs. 2.3g / L / h for n=2). [Explanation of symbols]

[0116] IC1 First interconnection step in the purification stage IC2 Second interconnection step of the purification stage IC-R Interconnection process of recirculation phase or shutdown phase I IC1-SD First interconnect step of Stop Phase II IC2-SD Second Interconnect Step of Stop Phase II B1 First division process in the purification stage (batch process) B2 Second division process in the purification stage (batch process) BR Recycle stage or stop stage I separation process (batch process) B1-SD First division step of the shutdown phase II (batch step) B2-SD Second decoupling step of shutdown phase II (batch step) B-SU-F Start-up stage separation process (supply process) B-SU-W Start-up phase separation process (W elution process) B-SD-P Stopping Phase Splitting Step (Product Elution Step) B-SD-S Stop stage separation step (S elution step) t IC1 Duration of phase IC1 t IC2 Duration of stage IC2 t IC-R Duration of stage IC-R t B1 Duration of step B1 t B2 Duration of step 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 Weakly adsorbable impurities S Highly adsorbable impurities

Claims

1. 1. A cyclic chromatographic purification process for isolating a product (P) from a feed mixture (F) which comprises said product (P) and at least two further components corresponding to weakly adsorbable impurities (W) and strongly adsorbable impurities (S), comprising: The method uses only two chromatographic adsorbent sections as the chromatographic stationary phase, where a first adsorbent section (1) has a first adsorbent section inlet and a first adsorbent section outlet, and a second adsorbent section (2) has a second adsorbent section inlet and a second adsorbent section outlet; The method comprises at least one basic sequence having at least one recycling step followed by only one purification step, wherein preferably the basic sequence is cyclically repeated at least twice, and The recycling step comprises the following steps: a. Interconnect Recirculation Process (IC-R), In this process, the adsorber sections are interconnected and have a recirculation interconnection period (t ＩＣ－Ｒ ) the upstream adsorber section outlet is connected to the downstream adsorber section inlet; wherein an eluent is loaded into the upstream adsorber section via an inlet of the upstream adsorber section, and a fraction containing the weakly adsorbable impurities (W) at least to an extent overlapping with the product (P), the product (P), and the strongly adsorbable impurities (S) at least to an extent overlapping with the product (P) is eluted from the upstream adsorber section to the downstream adsorber section; and the flow exiting the upstream adsorber outlet is diluted in-line prior to entering the downstream adsorber inlet; b. An optional batch recycle step (B-R); where the recirculation batch period (t Ｂ－Ｒ the adsorber section is interrupted during The upstream adsorber section prior to the preceding step a is cleaned to remove strongly adsorptive impurities (S) and regenerated, and an eluent is loaded into the inlet of the downstream adsorber section prior to step a to elute weakly adsorptive impurities (W) in the range not overlapping with the product (P); at least one recirculation sequence including After each recycle sequence of steps a and b, the adsorber sections are switched in sequence, where the number of recycle sequences is one or more, and Each recycling step is followed by exactly one purification step, The purification step comprises the following steps: c. a first interconnect purification step (IC1); In this process, the adsorber sections are interconnected, where a first interconnected purification period (t ＩＣ１ the upstream adsorber section outlet is connected to the downstream adsorber section inlet during the upstream adsorber is loaded with an eluent via the upstream adsorber inlet, and a product fraction comprising overlapping weakly adsorbable impurities (W) and product (P) is eluted from the upstream adsorber into the downstream adsorber section; and the flow exiting the upstream adsorber section outlet is diluted in-line prior to entering the downstream adsorber section inlet; d. a first batch purification step (B1); Here, the first batch purification period (t Ｂ１ ) the adsorber section is disconnected and product (P) is eluted from the former upstream adsorber section and feed mixture (F) is supplied to the inlet of the former downstream adsorber section; e. A second interconnect purification step (IC2); Here, the second interconnection purification period (t ＩＣ２ the upstream adsorber section outlet is connected to the downstream adsorber section inlet during the upstream adsorber section is loaded with an eluent via the upstream adsorber section inlet, and a product fraction comprising overlapping product (P) and highly adsorbable impurities (S) is eluted from the upstream adsorber into the downstream adsorber; and the flow exiting the upstream adsorber section outlet is diluted in-line prior to entering the downstream adsorber section inlet; f. a second batch purification step (B2); Here, the second purification batch period (t Ｂ２ ) the adsorbent is disconnected and the former upstream adsorbent is cleaned and regenerated, and eluent is loaded into the former downstream adsorbent inlet; in order, wherein the upstream adsorbent section of the last interconnected recirculation step of the preceding recirculation stage performs the function of the downstream adsorbent section, and the downstream adsorbent section of the last interconnected recirculation step of the preceding recirculation stage performs the function of the upstream adsorbent section.

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

3. 3. The method according to claim 1 or 2, wherein in the interconnection step of the method, the upstream section inlet is loaded with an eluent having a gradient in the form of a time-varying modifier concentration via the upstream adsorber inlet, and the flow leaving the upstream adsorber section outlet is diluted in-line with a modifier-free eluent or with an eluent having a modifier concentration different from that of the inlet of the upstream adsorber section before entering the downstream adsorber section inlet.

4. In a batch step of the process without elution of a purified product, the previous upstream adsorber is preferably cleaned and regenerated with an eluent having a higher modifier concentration than at the end of the preceding interconnected recycle step, or with an eluent containing a different modifier, or with a cleaning solution, and an eluent having a gradient in the form of a time-varying modifier concentration is loaded at the inlet of the previous downstream adsorber, and / or 2. The method of claim 1, wherein in a batch step of the method involving elution of the purified product, the former upstream adsorber is loaded via the upstream adsorber inlet with an eluent having a gradient in the form of a time-varying modifier concentration.

5. In the recycle step of the interconnected recycle process (IC-R), the upstream adsorber section is loaded with an eluent having a gradient in the form of a time-varying modifier concentration via the upstream adsorber section inlet, and the flow leaving the upstream adsorber outlet is diluted in-line with a modifier-free eluent or with an eluent having a different modifier concentration than the inlet of the upstream adsorber section before entering the downstream adsorber inlet; and / or 2. The method according to claim 1, wherein in an optional batch recycle step (B-R), the upstream adsorber section prior to the preceding step a is cleaned and regenerated, preferably with an eluent having a higher modifier concentration than at the end of the preceding interconnected recycle step, or with an eluent containing a different modifier, or with a cleaning solution, and an eluent having a gradient in the form of a time-varying modifier concentration is loaded at the inlet of the downstream adsorber section prior to step a to elute the weakly adsorbable impurities (W) in a range not overlapping with the product (P).

6. During the purification stage in the first interconnected purification step (IC1), the upstream adsorber section is loaded via the upstream adsorber inlet with an eluent having a gradient in the form of a time-varying modifier concentration, and the flow leaving the upstream adsorber section outlet is diluted in-line with a modifier-free eluent or with an eluent having a different modifier concentration than the inlet of the upstream adsorber section before entering the downstream adsorber section inlet; and / or In said first batch purification step (B1), eluting the product (P) from the former upstream adsorber section by loading said section via its inlet with an eluent having a gradient in the form of a time-varying modifier concentration, and / or In the second interconnected purification step (IC2), the upstream adsorber section inlet is loaded with an eluent having a gradient in the form of a time-varying modifier concentration via the upstream adsorber section inlet, and the flow leaving the upstream adsorber section outlet is diluted in-line with a modifier-free eluent or with an eluent having a different modifier concentration than the inlet of the upstream adsorber section before entering the downstream adsorber section inlet; and / or 2. The method according to claim 1, wherein in the second batch purification step (B2) the previous upstream adsorbent is cleaned and regenerated, preferably with an eluent having a higher modifier concentration than at the end of the preceding interconnected recirculation step, or with an eluent containing a different modifier, or with a cleaning solution, and an eluent having a gradient in the form of a time-varying modifier concentration is loaded at the inlet of the previous downstream adsorbent.

7. said modifier is selected from the group consisting of an organic or inorganic solvent or mixtures thereof different from the base solvent or mixtures thereof of said eluent, electrolytes in such organic or inorganic solvent (or mixtures thereof), preferably selected from dissolved salts or pH, or combinations thereof; wherein the base solvent is preferably 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, one or both of which are present in a small proportion compared to water, More preferably, the modifier is an organic solvent or a mixture of water and at least one organic solvent, the mixture having a higher concentration of the at least one organic solvent than in the base solvent, water or a mixture of water and at least one organic solvent, the mixture having a different salt concentration or H+ concentration than in the base solvent.

8. 2. The method according to claim 1, wherein in at least one or all of said steps a preferably linear eluent gradient is used with a time-varying gradient of increasing modifier concentration.

9. Prior to performing the first interconnect recirculation step of the first recirculation stage, a start-up step is performed, which step comprises: First batch start-up period (t Ｂ－ＳＵ－Ｆ The adsorbent (1, 2) is divided into two portions, feeding a feed mixture (F) to an inlet of said adsorber section (1), which is the upstream adsorber section of the first interconnected recycle step of said first recycle stage; On the other hand, the adsorber section (2), which is to be the downstream adsorber section of the first interconnected recycle step of the first recycle stage, is equilibrated or has already been equilibrated and is inactive; and Preferably, the second batch start-up period (t Ｂ－ＳＵ－Ｗ 2. The method according to claim 1, wherein during the first batch start-up step, the adsorbents (1, 2) are split and weakly adsorbable impurities (W) are eluted from the adsorbent section (1) to which the feed mixture (F) was supplied in the preceding first batch start-up step, while the other adsorbent (2) is equilibrated or has already been equilibrated and is inactive.

10. The first batch start-up period (t Ｂ－ＳＵ－Ｆ ) and after the second batch start-up period (t Ｂ－ＳＵ－Ｗ and / or, before the first interconnected recirculation step, an eluent is supplied to the inlet of the adsorber section which will be the upstream adsorber section of the first interconnected recirculation step of the first recirculation stage, and the eluent does not contain a modifier or has a modifier concentration which essentially corresponds to the starting modifier concentration applied during the first batch start-up period or in the absence of the second batch start-up period of the first interconnected recirculation step; 10. The method according to claim 9, wherein during the second batch start-up period, an eluent having a gradient in the form of a time-varying modifier concentration is loaded at the inlet of the adsorber section (1) fed with the feed mixture (F).

11. After completion of at least one base sequence, preferably two or more base sequences, a stopping step is carried out, said stopping step comprising the following steps: a'. interconnection outage recirculation process (IC'-R); In this step, the upstream adsorber section of the last preceding second interconnected purification step (IC2) takes the downstream position and another adsorber section takes the upstream position, the adsorber sections are interconnected, and a stop recycle interconnection period (t ＩＣ’－Ｒ－ＳＤ the upstream adsorber section outlet is connected to the downstream adsorber section inlet during wherein an eluent is loaded into the upstream adsorber section via an inlet of the upstream adsorber section, and a fraction containing the weakly adsorbable impurities (W) at least to an extent overlapping with the product (P), the product (P), and the strongly adsorbable impurities (S) at least to an extent overlapping with the product (P) is eluted from the upstream adsorber section to the downstream adsorber section; and the flow exiting the upstream adsorber outlet is diluted in-line prior to entering the downstream adsorber inlet; b'. An optional batch stop recycle step (B'-R); In this process, the adsorber section is disconnected, where a stop recycle batch period (t Ｂ’－Ｒ the adsorber section is interrupted during The upstream adsorber section prior to the preceding step a' is cleaned and regenerated, and an eluent is loaded into the inlet of the downstream adsorber section prior to step a to elute weakly adsorptive impurities (W) in the range not overlapping with the product (P); followed by the steps of: c'. First interconnect stop purification step (IC1-SD); In this process, the adsorber sections are interconnected, where a first interconnection stop purification period (t ＩＣ１’ the upstream adsorber section outlet is connected to the downstream adsorber section inlet during the upstream adsorber is loaded with an eluent via the upstream adsorber inlet, and a product fraction comprising overlapping weakly adsorbable impurities (W) and product (P) is eluted from the upstream adsorber into the downstream adsorber section; and the flow exiting the upstream adsorber section outlet is diluted in-line prior to entering the downstream adsorber section inlet; d'. First batch stop purification step (B1-SD); Here, the first batch purification period (t Ｂ１’ ) the adsorber section is disconnected and product (P) is eluted from the previous upstream adsorber section and the previous downstream adsorber section is either idled or eluent is fed to the inlet of the previous downstream adsorber section; e'. Second interconnect stop purification step (IC2-SD); Here, the second interconnection stop purification period (t ＩＣ２’ the upstream adsorber section outlet is connected to the downstream adsorber section inlet during the upstream adsorber section is loaded with an eluent via the upstream adsorber section inlet, and a product fraction comprising overlapping product (P) and highly adsorbable impurities (S) is eluted from the upstream adsorber into the downstream adsorber; and the flow exiting the upstream adsorber section outlet is diluted in-line prior to entering the downstream adsorber section inlet; f'. Second batch stop purification step (B2-SD); Here, the second purification batch period (t Ｂ２’ ) the adsorbent is disconnected and the former upstream adsorbent is cleaned and regenerated, and eluent is loaded into the former downstream adsorbent inlet; Next, g'. First final shutdown batch step (B-SD-P); Here, the first final stop batch period (t Ｂ－ＳＤ－Ｐ during which the first and second adsorber are decoupled and product (P) is eluted from the previous downstream adsorber and the other adsorber is idled or regenerated; h'. Optionally but preferably, a second final shutdown batch step (B-SD-S); Here, the second final stop batch period (t Ｂ－ＳＤ－Ｓ ) the first and second adsorber are decoupled and the highly adsorptive impurities (S) are eluted from the previous downstream adsorber and the other adsorber is idled or regenerated; 2. The method of claim 1 , wherein the upstream adsorber section of the interconnected cessation recycle step performs the function of the downstream adsorber section, and the downstream adsorber section of the interconnected cessation recycle step performs the function of the upstream adsorber section.

12. In the interconnected stop recycle step (IC'-R), the upstream adsorber section of the preceding last second interconnected purification step (IC2) is loaded via the upstream adsorber section inlet with an eluent of constant composition or with an eluent having a gradient in the form of a time-varying modifier concentration, wherein the flow leaving the upstream adsorber outlet is diluted in-line with a modifier-free eluent or with an eluent having a modifier concentration different from that at the inlet of the upstream adsorber section before entering the downstream adsorber inlet, and / or In said batch stop recycle step (B'-R), the upstream adsorber section before the preceding step a is cleaned and regenerated, and an eluent having a gradient in the form of a time-varying modifier concentration is loaded at the inlet of the downstream adsorber section before step a' to elute weakly adsorptive impurities (W) in a range not overlapping with the product (P), and / or In the first interconnection stop purification step (IC1-SD), the upstream adsorber is loaded via the upstream adsorber inlet with an eluent having a gradient in the form of a time-varying modifier concentration, and the flow leaving the upstream adsorber section outlet is diluted in-line with a modifier-free eluent or with an eluent having a different modifier concentration than the inlet of the upstream adsorber section before entering the downstream adsorber section inlet; and / or in said first batch stop purification step (B1-SD), the product (P) is eluted from the previous upstream adsorber section by loading an eluent having a gradient in the form of a time-varying modifier concentration via said upstream adsorber inlet, and the previous downstream adsorber section is either idled or an eluent, preferably in the form of said base solvent, is fed to the previous downstream adsorber section inlet; and / or In the second interconnection stop purification step (IC2-SD), the upstream adsorber section is loaded via the upstream adsorber section inlet with an eluent of constant composition or with an eluent having a gradient in the form of a time-varying modifier concentration, and the flow leaving the upstream adsorber section outlet is diluted in-line with a modifier-free eluent or with an eluent having a different modifier concentration than the inlet of the upstream adsorber section before entering the downstream adsorber section inlet; and / or In the second batch stop purification step (B2-SD), the former upstream adsorber is cleaned and regenerated, and the eluent is loaded into the former downstream adsorber inlet; and / or In said first final stop batch step (B-SD-P), the product (P) is eluted from the previous downstream adsorber with an eluent having a gradient in the form of a time-varying modifier concentration, and the other adsorber is idled or regenerated, and / or 12. The method according to claim 11, wherein in the first final second final shutdown batch step (B-SD-S) the strongly adsorbed impurities (S) are eluted from the previous downstream adsorber and the other adsorber is idled or regenerated.

13. 2. The method of claim 1, wherein a linear gradient with different slopes and / or flow rates is used in the recycle step and / or in the purification step.

14. In most or all of the interconnection steps of the method, the upstream section inlet is loaded with an eluent having a gradient in the form of a time-varying modifier concentration via the upstream adsorber inlet, and the flow leaving the upstream adsorber section outlet is diluted in-line with a modifier-free eluent or with an eluent having a different modifier concentration than the inlet of the upstream adsorber section before entering the downstream adsorber section inlet; and In batch steps of the process without elution of the purified product, the previous upstream adsorber is cleaned and regenerated with eluent, and, if applicable, the eluent having a gradient in the form of a time-varying modifier concentration is loaded into the previous downstream adsorber inlet, or In a batch step of the process involving elution of the purified product, the previous upstream adsorber is loaded via the upstream adsorber inlet with an eluent having a gradient in the form of a time-varying modifier concentration, and said modifier is selected from the group consisting of a solvent or mixture thereof different from the base solvent or mixture thereof of said eluent, electrolytes in such solvent or mixture thereof, preferably selected from dissolved salts or pH, or a combination thereof, wherein preferably said 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 minor proportion compared to water, more preferably said modifier is an organic solvent, or a mixture of water and at least one organic solvent, wherein the concentration of said at least one organic solvent is higher than that of said base solvent, water, or a mixture of water having a salt concentration or H+ concentration different from that of said base solvent, and The modifier concentration is controlled during the process by the following steps: Recycle steps (IC-R, IC'-R, IC1, IC1-SD) except for the final stop batch step (B-SD-P), and a step of product elution (B1, B1-SD, B-SD-P), if applicable, and a step of further recirculation (IC2, IC2-SD), if applicable, where during said recirculation (IC2, IC2-SD) said modifier concentration can vary or can be kept constant only in the case of said recirculation (IC2, IC2-SD), From the minimum concentration at the start of elution of the weakly adsorbed impurity (W) in the range not overlapping with the product (P) (B-SU-W, B-R, B'-R, B2, B2-SD) over the entire 3. The method according to claim 2, wherein after a maximum, preferably linear, increase in concentration, the elution of the strongly adsorbing impurity (S) begins (B-R, B'-R, B2, B2-SD, B-SD-S).

15. 2. Use of the method according to claim 1 for the purification of biological molecules, of natural or synthetic origin, preferably selected from the group consisting of nucleic acid molecules including DNA and RNA molecules, proteins including antibodies, peptides, carbohydrates, lipids, and combinations and modifications and fragments thereof.

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