RU2776950C2 - Method for control, assessment and regulation of cyclic chromatographic purification process - Google Patents

Abstract

FIELD: purification.

SUBSTANCE: present invention relates to methods for the control, assessment and regulation of cyclic chromatographic purification processes. A method for the control, assessment and regulation of a cyclic chromatographic purification process including at least two adsorbers is proposed. The method includes at least following stages: a) control of a chromatogram, including the measurement of at least one current signal in liquid, proportional to the concentration; b) assessment of the chromatogram, including the comparison of at least one of the specified current signals proportional to the concentration, measured at the stage (a), with its threshold value; c) regulation of chromatographic purification process by means of the adaptation of the end of the current phase based on the comparison during the stage (b) and the beginning of the following phase. In this case, the sequence of stages a)-c) is performed in the specified order at least twice. There are impurities in impurities, which are adsorbed less than product (W), and impurities, which are adsorbed more than product (S). In this case, the current signal value is the absolute signal value, its integral, its slope or its slope sign, or a combination thereof.

EFFECT: invention allows for provision of an improved method for the control, assessment and regulation of a cyclic chromatographic purification process.

15 cl, 10 dwg

Description

FIELD OF TECHNOLOGY

The present invention relates to methods for monitoring, evaluating and regulating cyclic chromatographic purification processes.

PRIOR ART

Chromatographic processes are used to isolate products from complex mixtures.

In the case of "difficult" chromatographic separations, the target product is accompanied by side compounds (impurities) with very similar adsorption properties. In chromatograms obtained by conventional single adsorber chromatography, this results in overlapping portions of the product peaks and peaks of various by-compounds at the front and back of the product peak, requiring isolation by cutting out the center of the peak. This separation is also called the triple separation problem, since the compounds to be separated can be grouped into three classes on the chromatogram - early eluting (poorly adsorbing) impurities/side compounds, centrally eluting "product" and late eluting (strongly adsorbing) impurities/side compounds. Accordingly, the overlapping fractions at the front and back of the product peak contain both the desired product and impurities/by-products, and usually these fractions have to be discarded because their purity is not up to the established specifications. This means that in such a ternary separation problem, a high purity product fraction can only be obtained by reducing the yield. The addition of fractions containing by-products increases the yield by including more of the target compound, but reduces the purity by including by-products/impurities. This situation is also known as the trade-off between yield and purity.

In order to isolate the product contained in the fractions contaminated with by-products, various chromatographic processes have been proposed using recirculation methods, the purpose of which was to alleviate the problem of trade-off between yield and purity. Methods using a single adsorber and collecting fractions contaminated with by-products in separate tanks for subsequent purification have been proposed in cases where re-treatment is compatible with regulatory requirements.

In some processes, the by-products contaminated fractions are recycled directly to the same adsorber without an intermediate storage step.

Chromatographic processes using more than one chromatographic adsorber make it possible to combine the use of internal recirculation of fractions contaminated with by-products and the application of the principles of countercurrent chromatography, that is, the relative movement in opposite directions of the stationary phase (adsorbent material) and mobile phase (fluid media). Through internal recycling of the co-contaminated fractions, the target compound contained in these fractions can first be transferred directly from one adsorber to another without a storage step in an external tank and then isolated using countercurrent chromatography principles and recovered.

The efficient combination of internal recirculation and the principles of countercurrent chromatography allows the target compound to be obtained simultaneously in high yield and high purity in multi-absorber processes. These processes are also known as pseudo-moving bed (SMB) processes. Previously, the use of SMB processes has been limited to binary separations, ie separations of two target compounds or separation of a target compound and one set of either early eluting or late eluting impurities/side compounds. A ternary separation of the product, whose peak overlaps with the side compound peaks at the front and back of the product peak, can only be achieved by combining the two SMB processes, potentially requiring a concentration step between the two SMB systems. The complexity of such systems has hindered their practical application and has led to the development of alternative ternary separation methods using the principles of countercurrent chromatography. In addition, linear solvent gradients, which are important for the separation of compounds with similar adsorption properties, cannot be used in SMB processes.

In this regard, a very efficient process for ternary separations with the possibility of using a linear solvent gradient is known as "multicolumn countercurrent solvent gradient purification" (MCSGP; from English: multicolumn countercurrent solvent gradient purification) and is widely used in industry (see the publication EP- A-1 877 769). Such a process has been described using 2 to 8 adsorbers.

Other chromatographic multi-adsorber techniques using multiple adsorbers and internal recirculation have also been proposed, such as the "gradient chromatography with stable recirculation" (GSSR; from English: gradient with steady state recycle) process. Most of the previously described applications of multi-adsorber countercurrent chromatographic processes have been in the dual-adsorber MCSGP process, which has the advantage of low equipment complexity and high operational flexibility in terms of flow rates used, switching times, and linear solvent gradient operation.

US-A-2017241992 proposes a method for regulating and/or controlling and/or optimizing a chromatographic process, which uses at least 2 columns operating alternately, and the workflow can be carried out so that at least 2 columns operate in a connected and disconnected states, with the columns changing positions after such a sequence of connected and disconnected states, and downstream of at least one or relative to each column is a detector capable of detecting the target product and/or impurities as they pass the detector.

Publication WO 2014/166799 relates to a chromatographic purification method for isolating a fraction of the target product from a mixture using 2 chromatographic columns (adsorbers); it refers to the ways in which such a process is carried out, and in this context it also refers to the regulation and/or control and/or optimization of processes. The method includes the following stages, which are carried out at least once during the cycle: the first stage (B1) of autonomous operation, during which the adsorbers are disconnected for a certain period of time, and the first adsorber is loaded with the initial mixture through the inlet using the first volumetric flow rate , and waste is drained through its outlet; at the same time, the target product is obtained from the second adsorber through its outlet, after which the second adsorber is regenerated; the first stage (IC1) of operation of the adsorbers in the connected state, during which the outlet of the first adsorber is connected to the inlet of the second adsorber for a certain period of time, while the first adsorber is loaded through the inlet with the initial mixture in a volume exceeding its dynamic capacity, with using a second space flow rate that is the same as or greater than the first space flow rate, and waste is drained through the outlet of the second adsorber; the second stage (B2) of autonomous operation, similar to the first stage (B1) of operation in autonomous mode, but with the change of adsorbers in places; the second stage (IC2) of operation of adsorbers in the connected state, similar to the first stage (IC1) of operation of adsorbers in the connected state of adsorbers, but with the change of adsorbers in places.

SUMMARY OF THE INVENTION

One of the objectives of the present invention is to provide an improved method for monitoring, evaluating and regulating a chromatographic cycle purification process.

In the proposed method, the chromatographic profile of the elution, that is, a signal proportional to the concentration, is controlled, and while the elution is still ongoing, an action is performed that has an immediate effect. In addition, the method does not require a chromatographic model describing the separation, nor a control algorithm in which the action taken is based on the final difference between the actual value and the set value (setpoint). In contrast, the method uses threshold values and initiates control actions when the threshold values are reached or exceeded. To determine setpoints (setpoints) in the method, certain knowledge about the chromatographic separation is required, which can be obtained by evaluating the model gradient chromatogram. In some cases, the setpoints can be determined automatically, for example relative to the maximum peak. Therefore, the position of the chromatogram relative to the elution time/volume (x-axis) is irrelevant for determining setpoints that are based only on the height of the chromatogram (y-axis).

The proposed method of controlling the process solves the problem of fluctuations in environmental and operating parameters and differences in the efficiency of the functioning of adsorbers, which can lead to suboptimal efficiency of countercurrent processes. When using the proposed method, the process is steadily carried out at the setpoint level.

The proposed method includes the elements of a) essentially continuous monitoring, b) evaluation of the chromatogram, preferably at the outlet of the adsorber performing elution tasks, and c) triggering a control action based on the evaluated information.

More specifically, the present invention relates to a method for monitoring, evaluating and regulating a cyclic chromatographic purification process, including at least two adsorbers (preferably no more than two adsorbers, no more than 3 adsorbers, or no more than 4 adsorbers), through which the liquid with the initial mixture is passed (Load) containing the target components of the product (or compound) (P) and impurities (W, S).

Typically, the adsorbers operate alternately, with at least two adsorbers undergoing a sequence of phases of operation in connected and disconnected states, and after such a sequence of phases of operation in connected and disconnected states, the adsorbers are reversed. In the case of two adsorbers, for example two columns, this sequence is strictly defined. In this context, it should be noted that any of these two adsorbers can also be a group of two, three, four or even more adsorbers (columns) that are connected in series with each other, and this state of serial connection is maintained during the process without being disconnected. .

In the case where there are more than two adsorbers, the sequences of the phases of operation in connected and disconnected states are such that always after phase 11 of operation of the adsorbers in connected state, during which two adsorbers (or two groups of adsorbers) are connected, there follows a phase B1 of independent operation of the adsorbers , during which the same two adsorbers are operating, and the product P is eluted from the adsorber (or group of adsorbers) that was previously located upstream. Therefore, as shown in FIG. 3-8 and described below, control is also feasible in processes that use 3 or more columns, since it only requires the presence of phases of the adsorbers in a connected state and offline, and a signal proportional to the concentration, as described in more detail below , preferably measured at the outlet of at least one of these identical adsorbers, preferably at the outlet of the upstream adsorber, or at the outlets of two adsorbers, or at the outlets of all adsorbers. The signal proportional to the concentration can also preferably be measured in at least one of the following positions: at the outlet of the adsorber in phase (B1) of the autonomous elution of the target product, at the outlet of the adsorber located upstream during phases (I1, I2) work in the connected state, and preferably only in these positions. In parallel with each such sequence I1, B1, which includes two adsorbers or two groups of adsorbers, additional adsorbers or groups of adsorbers can independently perform other tasks, such as purification, balancing or reaction tasks.

Parallel to each phase I1, B1, as well as parallel to phases I2, B2, which include two additional adsorbers or two groups of adsorbers, additional adsorbers or groups of adsorbers can independently perform other tasks, for example, purification, balancing or reaction tasks. An example of such a process using three adsorbers is shown in FIG. 10a. Accordingly, in a process using 3 adsorbers or 3 groups of adsorbers, the positions of the adsorbers during the processes only change after phase B2. Similarly, it is possible to carry out the process using 4 adsorbers or 4 groups of adsorbers as shown in FIG. 10b.

The processes disclosed in EP 1877769 B1 also do not contradict the present invention and can be carried out using the idea of the present invention, which consists in the need for a phase of operation of the adsorbers in a connected state and a subsequent phase of autonomous operation of the adsorbers, i.e. the present invention is also applicable in this case. (for example, to the processes presented in Fig. 20, Fig. 22 or Fig. 23 in the publication EP 1877769 B1). In EP 1 877 769 B1, the connected state adsorber phases corresponding to "I1" and "I2" are referred to as "CCL" and the offline adsorber phases "B1" and "B2" are referred to as "BL". With regard to impurities (W, S), only one impurity (W or S) may be present, i.e. a (binary) distribution is possible with impurities (W) adsorbing less than the product, or only impurities (S) adsorbing stronger than the product. It is also possible that there are impurities (W) adsorbed more weakly than the product and impurities (S) adsorbed more strongly than the product.

The cleaning process includes at least two different phases (I, B):

- at least one phase (I) of operation of the adsorbers in a connected state, during which the two adsorbers are connected to each other so that the outlet of the upstream adsorber is in fluid (hydraulic) connection with the inlet of the adsorber located downstream downstream, and

- at least one phase (B) of autonomous operation, during which at least one adsorber is not in liquid connection with other adsorbers, and during which the target components of the product (P) are isolated in a purified form, preferably from a detached adsorber.

According to the present invention, the method includes at least the following steps:

a. monitoring the chromatogram, including measuring in the liquid at least one current signal value proportional to the concentration;

b. evaluating the chromatogram comprising comparing at least one of the current signals proportional to the concentration measured in step a. with a threshold signal value;

c. adjusting the chromatographic purification process by adapting the end of the current phase based on the comparison made in step b. and the start of the next phase;

moreover, the sequence of stages a.-s. are performed in this order one after the other at least twice in one separation process.

Thus, the process is carried out cyclically by repeating the sequence of steps a.-s. at the desired or required frequency.

In a characteristic case, the process is carried out cyclically by repeating the sequence of steps a.-s. within a certain period of time, for example - within a period of time ranging from 10 minutes to 200 hours, or (often) - in the range from 12 hours to 52 hours, or from 24 hours to 48 hours. Alternatively, the process is carried out cyclically by repeating the sequence of steps a.-s. depending on the supplied volume, that is, the sequence of stages a.-s. repeat until the entire volume of the initial mixture has been processed.

For example, in the case of a method based on continuous fermentation, the sequence of stages and.-with. can be carried out cyclically until the fermentation process is completed, which can take place over several weeks or months, for example, from 1 week to 20 weeks or from 4 weeks to 8 weeks, or, if the process is carried out continuously, the sequence of stages a.-c. repeat until replacement or cleaning of adsorbers is required.

According to a first preferred embodiment of the present invention, in the case of a signal proportional to concentration, at least its absolute value, its integral, its slope and the sign of the slope are taken into account, and preferably a combination of these parameters is taken into account, most preferably a combination of the absolute value and the sign of the slope.

The concentration proportional signal measured in step a. can preferably be measured at the outlet of at least one adsorber, preferably at the outlets of two adsorbers or at the outlets of all adsorbers. The signal proportional to the concentration can also preferably be measured in at least one or only one of the following positions: at the outlet of the adsorber in phase (B1) of the autonomous elution of the target product, at the outlet of the adsorber located upstream during phases (I1, I2) of the operation of adsorbers in the connected state. That is, the signal proportional to the concentration is preferably measured only at the outlet of the upstream adsorber of the two active adsorbers, and in the case of a stand-alone phase, at the outlet of the absorber eluting the product.

According to a preferred embodiment of the present invention, the purification process comprises at least four different phases (I1, B1, I2, B2) in the following order:

- at least one first phase (I1) of operation of the adsorbers in a connected state, during which the two adsorbers are connected to each other so that the outlet of the upstream adsorber is in fluid communication with the inlet of the adsorber located downstream , the solvent enters through the inlet to the upstream adsorber, and the desired target components (P) and poorly adsorbed impurities (W) move from the upstream adsorber to the downstream adsorber, preferably until until essentially only the target components of the product (P) exit the outlet of the upstream adsorber, preferably an intermediate dilution is carried out between the upstream adsorber and the downstream adsorber;

- at least one first phase (B1) of autonomous operation, during which the adsorbers are not in liquid connection, and during which the solvent through the inlet enters the adsorber located upstream during the first phase (I1) of operation of the adsorbers in a connected condition, and through the outlet of this adsorber eluting the product, the target components of the product (P) are collected, while the liquid containing the initial mixture (Load) enters through the inlet into the adsorber located downstream during the first phase (I1) of operation adsorbers in the connected state, and through the outlet of this adsorber in the characteristic case, weakly adsorbed impurities are collected;

- at least one second phase (I2) of the adsorbers in the connected state, during which the two adsorbers are connected to each other so that the outlet of the adsorber, which was upstream during the first phase (I1) of the connected state, is connected to adsorber inlet downstream during the first phase (I1) of operation in the connected state, the solvent enters through the inlet into the upstream adsorber, and the desired target compounds (P) and strongly adsorbable impurities (S) move out of the adsorber located upstream into the adsorber located downstream, and preferably between the adsorber located upstream and the adsorber located downstream, carry out intermediate dilution;

- at least one second phase (B2) of autonomous operation, during which the adsorbers are not in liquid connection, and during which the solvent through the inlet enters the adsorber located upstream during the second phase (I2) of operation of the adsorbers in the connected condition, and through the outlet of this upstream adsorber, strongly adsorbed impurities (S) are collected, while the solvent enters through the inlet into the downstream adsorber during the second phase (I2) of the operation of the adsorbers in the connected state, and through the outlet of this adsorber, formerly located downstream, weakly adsorbed impurities are collected,

moreover, the functions of the phases (I1, B1, I2, B2) are performed synchronously or, preferably, sequentially, and they are performed cyclically at least twice, moreover, when the cycle is carried out after or at the moment of switching, the adsorber previously located upstream during the second phase ( B2) autonomous operation is moved so that it becomes the adsorber located downstream during the next first phase (I1) of operation of adsorbers in a connected state, and the adsorber previously located downstream during the second phase (B2) autonomous operation, is moved so that it becomes the upstream adsorber during the subsequent first phase (I1) of operation of the adsorbers in the connected state. This process is essentially the same as the process depicted in FIG. 1 and detailed below. In the context of this purification method, step a. preferably comprises measuring at least one current value of the signal proportional to the concentration in the liquid at the outlet of the upstream adsorber during the first phase (I1) of the operation of the adsorbers in the connected state, and preferably measuring at least one parameter selected from its absolute magnitude and sign of its slope, preferably a combination of these two parameters is measured.

It is possible to measure the absolute value and preferably also the sign of the slope of the signal and in case the threshold value of the absolute value is exceeded, the next first autonomy phase (B1) can be started, either in the form of a first autonomy phase with a fixed duration, or in the form of a first autonomy phase, having a duration that varies depending on further monitoring, evaluation and regulation.

After exceeding the threshold value of the absolute value, it is possible to provide a fixed delay until the start of the next first phase (B1) of autonomous operation, either in the form of a first phase of autonomous operation with a fixed duration, or in the form of a first phase of autonomous operation of adsorbers in autonomous mode, having a duration that varies depending on from further monitoring, evaluation and regulation.

After exceeding the first threshold value of the absolute value, a minimum fixed delay can be provided and after exceeding the second threshold value of the absolute value, preferably with checking the additional condition that the signal slope is positive, the next first phase (B1) of autonomous operation is started, or in the form of the first phase of autonomous operation with a fixed duration, or in the form of the first phase of autonomous operation, having a duration that varies depending on further control, evaluation and regulation.

If we are talking about the first phase B1 autonomous, having a duration that varies depending on further monitoring, evaluation and regulation, then this applies, for example, to the regulation of the duration of the stage of elution of the product, which is described in detail below.

These different ways of determining when to start the product elution step can also be combined.

Stage a. may include measuring at least one current value of the signal proportional to the concentration in the liquid at the outlet of the adsorber eluting the product during the first phase (B1) of autonomous operation, and preferably at least one parameter is measured, selected from the absolute value of the signal and the sign its slope is preferably measured by a combination of these two parameters. In the context of such a measurement, after the absolute value of the signal has fallen below a threshold value, preferably under (continuous) control of the additional condition that the slope of the signal is negative, it is possible to start the next second phase (I2) of the operation of the adsorbers in the connected state, or in the form of a second phases of adsorbers in the connected state with a fixed duration, or in the form of a second phase of the adsorbers in the connected state, having a duration that varies depending on further monitoring, evaluation and regulation.

Stage a. may also include measuring at least one current value of the signal proportional to the concentration in the liquid at the outlet of the upstream adsorber during the second phase (I2) of the operation of the adsorbers in the connected state, and preferably measure at least one parameter selected from the absolute value of the signal and the sign of its slope, and preferably a combination of these two parameters is measured, and after the decrease in the absolute value of the signal below the threshold value, preferably under control of the additional condition that the signal slope is negative, you can start the next second phase (B2) autonomous operation, either in the form of a second phase of autonomous operation with a fixed duration, or in the form of a second phase of autonomous operation of the adsorbers, having a duration that varies depending on further monitoring, evaluation and regulation.

A cycle chromatographic process may use at least two adsorbers, and each cycle may include at least two adsorber coupled phases during which the two adsorbers are in fluid bond to internally recirculate various partially purified fractions (W/P, P /S) containing side compounds. During stage a. preferably, at least one parameter, selected from the absolute value of the signal and its slope, is measured at the outlet of the respective upstream adsorber.

A change in the sign of the slope can also be used as a criterion for the control action.

The elution gradient of the process can have a constant slope with respect to the volume of liquid mobile phase used in the process during both phases of adsorber operation in the connected state and the first phase of autonomous operation (phases I1, B1, I2), or isocratic operation is possible (slope, equal to zero).

The threshold value for terminating one process phase and starting a new process phase can preferably be determined from a signal proportional to the concentration recorded during the same or a previous cycle of the chromatographic process.

In addition, a modulating action may be initiated based on failure to reach a certain threshold value within a predetermined elution volume, or time, or concentration gradient.

The concentration proportional signal is typically based on a measurement of visible light, UV, infrared, fluorescence, Raman scattering, ionic strength, electrical conductivity, or refractive index.

A preferred method of monitoring the chromatogram includes monitoring a concentration-proportional signal at the outlet of at least one adsorber, preferably at both outlets if two adsorbers are used, and preferably at the outlets of all adsorbers if more than two adsorbers are used.

Monitoring of a signal proportional to concentration can be done by detecting a UV signal, which can be the absolute value of the signal, its integral, its slope, or the sign of its slope.

A preferred method for evaluating a chromatogram is to compare the current value of at least one of said parameters with a predetermined value, and to take a control action based on this comparison.

The preferred control action is to stop the current ongoing phase, for example the phase IC1 of the internal recirculation of the product, and the beginning of the next phase, for example the phase B1 of the collection of the product.

It has been found that the control method can compensate for changes in process parameters such as mobile phase composition and temperature, adsorber temperature, difference in packed bed height for column format adsorbers, sealing pressure for column format adsorbers, fouling and reduced throughput. abilities leading to chromatogram shifts.

The control part of the method may include recording the signal values proportional to the concentration and/or the slope of this signal at the outlet of the upstream adsorber during phases I1, B1 and I2 and during the elution of compound "W" in zone 4 during phase B2 .

When monitoring the slope of a concentration-proportional signal, the method can be applied to initiate control action at the leading edge of the peak (the slope of the signal proportional to the concentration is greater than zero) or at the tail of the peak (the slope of the signal proportional to the concentration is less than zero). The evaluation part of the method continuously checks whether the concentration-proportional signal has reached a predetermined threshold and triggers a control action if the threshold is reached.

The threshold value may be predetermined (for example, based on information about the model chromatogram), or it is determined automatically using a control method based on information obtained during one or more previous cycles. For example, the threshold can be expressed as a percentage of the peak height of the main compound or the early eluting "W" compound.

In a further preferred embodiment of the present invention, a method for monitoring, evaluating and controlling a multi-adsorber countercurrent gradient purification process includes the following elements:

a. control of a signal proportional to the concentration, which is the absolute value of the signal, its integral, its slope, or the sign of the slope, or a combination thereof, and

b. evaluation of the chromatogram, including continuous comparison of the current value of at least one of the above parameters with a given value, and

c. regulation of the process, including the termination of the current running phase of the multi-adsorber countercurrent gradient purification process based on the coincidence of the current signal value with the set value and the beginning of a new phase,

moreover, the stages a.-s. performed at least once during the implementation of the cleaning process.

The term "signal proportional to concentration" in the context of the present invention may refer to the proportionality of the signal to the concentration of the product or impurities during the chromatographic analysis, but alternatively it may refer to the proportionality of the signal to the concentration of the modifier, including, but not limited to, at least one concentration selected from salt concentration or organic modifier concentration.

In a preferred embodiment of the present invention, two adsorbers are used in the multi-adsorber countercurrent gradient purification process. However, more than 2 adsorbers can be used, for example 3, 4, 5 or even 6 adsorbers.

In a preferred embodiment of the present invention, the method monitors the chromatogram during the internal recirculation and product elution phases and triggers a control action based on reaching a threshold.

Preferably exactly two adsorbers are used in the process.

In a preferred embodiment, the method includes:

(a) monitoring the slope and/or value of the signal proportional to the concentration at the outlet of the upstream adsorber during internal recirculation of weakly adsorbing impurities (phase I1);

(b) continuously comparing the value of the signal proportional to the concentration with a predetermined threshold during the period when the slope of the signal proportional to the concentration is positive and after reaching the threshold

(c) terminating the execution of phase I1 and starting the execution of phase B1, wherein the phase B1 has a fixed or variable duration depending on another threshold.

In another preferred embodiment, the method includes:

(a) monitoring the slope and/or value of the signal proportional to the concentration at the outlet of the upstream adsorber during internal recirculation of weakly adsorbing impurities (phase I1);

(b) continuously comparing the value of the signal proportional to the concentration with a predetermined threshold during the period when the slope of the signal proportional to the concentration is positive and after reaching the threshold

(c) continuing the execution of the I1 phase for a certain period of time (delay time) until the execution of the I1 phase is terminated and the execution of the B1 phase begins, wherein the B1 phase has a fixed duration, and the delay has a predetermined or variable duration depending on another threshold value.

In yet another preferred embodiment of the present invention, the method is modified such that phase B1 is started based on a threshold, which may be the same as or different from the threshold used to trigger the delay period during phase I1, and the delay period may have a minimum value. The reason for setting the minimum delay time is to avoid early start of the B1 phase due to impurities eluting before the main product and to reach the second threshold. The delay period can be set by time or volume.

In a further preferred embodiment, the method further comprises:

(a) monitoring the slope and/or value of the signal proportional to the concentration at the outlet of the adsorber eluting the product (phase B1), and

(b) continuously comparing the value of the signal proportional to the concentration with a predetermined threshold during the period when the slope of the signal proportional to the concentration is negative and after reaching the threshold

(c) stopping phase B1 and starting phase I2, with sample loading modulated such that sample loading occurs at the beginning of phase B1 and stops after a short period of time, while elution from the second adsorber continues until a threshold has been reached and phase I2 has a predetermined or variable duration that depends on another threshold.

In another preferred embodiment of the present invention, the end point of phase I2 is defined by a threshold, whereby when this threshold is reached, phase I2 is terminated and phase B2 begins.

In other preferred embodiments of the present invention, any of the above methods related to the beginning of phase B1 are combined with methods related to the completion of phase B1.

Thus, combinations of these methods are also possible with the start of the delay period during the I1 phase based on the first threshold, the start of the B1 harvest phase based on the second threshold, the slope of the UV signal during this period being positive, and the end of the B1 phase (and the start phase I2) based on the third threshold.

In other preferred embodiments of any of the above methods, additional information about the slope of the chromatogram is used to initiate control actions. In a preferred embodiment of the method in the above methods, instead of using a fixed duration delay volume, a slope reversal can be used as a criterion for terminating the delay and continuing to evaluate the signal until the second threshold is reached.

All methods may include continuing the run and extending the gradient of the eluting solvent into phases I1, B1, 12, preferably with the slope of the gradient used during the elution of the "W" components (phase 4 in a dual adsorber setup), which refers to the slope relative to the mobile volume. phase used in the process. This means that the elution gradient in the multi-adsorber process has a constant slope with respect to the volume of mobile phase used in the process during phases I1, B1, I2.

In the methods described, the thresholds may also be determined based on information obtained during the same run or cycle, ie they may be unknown at the beginning of the run or cycle. In this case, the first run may be partially or completely completed before thresholds are determined in the method based on an evaluation of the recorded signals that are significant for the remainder of the run (in case the run was partially completed by the time of the evaluation) or for the remainder of the chromatographic run. process. Accordingly, the threshold value for terminating a process phase and starting a new process phase is determined from a signal proportional to the concentration recorded during the same or a previous run of the chromatographic process.

The proposed method also includes starting the implementation of the regulatory action based on the failure to reach a certain threshold value within a given elution volume. This is possible, for example, if the chromatogram does not elute because one of the pumps used in the chromatographic process is not working properly. The preferred action is to stop the pumps used to supply the mobile phase. Reference values can be obtained from a simulated chromatogram.

In preferred embodiments of the present invention, the signal proportional to the concentration is based on the measurement of visible light, UV radiation, infrared radiation, fluorescence, Raman scattering, ionic strength, electrical conductivity or refractive index.

In the above description of the method, the durations are given in units of time or volume. The time t and the volume V can be converted to each other using the volumetric flow rate Q used in the corresponding phase according to the expression V=Q*t. The slope of a signal proportional to concentration can also be expressed in terms of volume or time.

Other embodiments of the present invention are described in dependent claims. The term "solvents" in the context of the present invention also includes buffer solutions and other types of mobile phases. The term "adsorber" in the context of the present invention includes chromatographic columns with a packed adsorbent bed or other devices containing a stationary chromatographic phase, including membranes, fibrous adsorbents and monolithic adsorbents.

BRIEF DESCRIPTION OF GRAPHICS

Hereinafter, the preferred embodiments of the present invention are described with reference to the accompanying drawings, which are only intended to illustrate the presented preferred embodiments of the present invention and do not limit it. As for graphic materials, then:

Fig. 1 schematically depicts a Multicolumn Countercurrent Solvent Gradient Purification (MCSGP) process; more specifically, it is an illustration of the first half of a cycle ("switch") of a dual-adsorber countercurrent gradient chromatographic separation process; dashed vertical lines separate the various tasks of the MCSGP process according to the areas of the offline schematic chromatogram shown at the bottom of the figure; phases I1, B1, I2, B2 are executed sequentially;

Fig. 2 shows: a) a chromatogram in which zone 6 is in the correct position; and b) a chromatogram in which zone 6 is misaligned and includes strongly adsorbing impurities; more specifically, the schematic chromatograms of one product elution from the MCSGP run are shown with the product elution window fixed, without the use of a control method; a) demonstrates a run under optimal operating conditions, during which the peak of the product was collected, and most of the impurities were excluded from the product pool; b) shows a run in which the chromatogram is shifted towards earlier elution times resulting in a product of lower concentration and purity;

Fig. 3 schematically depicts a control method (A) based on an MCGSP chromatogram showing the elution of one product from one of two adsorbers and phases I1, B1, I2 and B2;

Fig. 4 schematically depicts a control method (B) based on an MCGSP chromatogram showing the elution of one product from one of two adsorbers and phases I1, B1, I2 and B2;

Fig. 5 schematically depicts a control method (C) based on an MCGSP chromatogram showing the elution of one product from one of two adsorbers and phases I1, B1, I2 and B2;

Fig. 6 is a schematic representation of a control method (D) based on an MCGSP chromatogram showing the elution of one product from one of two adsorbers and phases I1, B1, I2 and B2;

Fig. 7 schematically depicts a control method (E) based on an MCGSP chromatogram showing the elution of one product from one of two adsorbers and phases I1, B1, I2 and B2;

Fig. 8 schematically depicts a control method (F) based on an MCGSP chromatogram showing the elution of one product from one of two adsorbers and phases I1, B1, I2 and B2;

Fig. 9 shows: a) chromatograms of 10 cycles of MCSGP run using two different adsorbers; b) superimposed chromatograms of the above 10 cycles showing threshold at 100 milliabsorbance units (mAU) and phases I1, B1, I2 and B2;

Fig. 10 schematically shows a process using 3 adsorbers (a) and a process using 4 adsorbers (b). The adsorbers used in the control method are highlighted in grey, while the adsorbers performing other tasks such as cleaning, balancing or performing a reaction step are not highlighted.

INTELLIGENCE. CONFIRMING THE POSSIBILITY OF CARRYING OUT THE INVENTION

A schematic diagram of an MCSGP process using two adsorbers (eg, a process involving two chromatographic columns or two membrane adsorbers) is shown in FIG. 1. Schematic chromatogram at the bottom of FIG. 1 is an offline chromatogram divided into different sections (by vertical dashed lines) according to the tasks that are performed during an offline chromatographic run (equilibration in zone 1, material loading in zone 2, washing in zone 3, elution in zones 4-7, cleaning and re-equilibration in zone 8). The elution phase is subdivided into additional zones according to the elution order of weakly adsorbing impurities (W), product (P), and strongly adsorbing impurities (S) in the chromatogram (elution W in zone 4, elution of overlapping W/P in zone 5, elution of pure P in zone 6, elution of the overlapping portion of P/S in zone 7). In the dual-adsorber MCSGP process, these particular tasks of zones 4-7 are performed in the same way as in the case of batch chromatography (intermittent chromatography), with the crucial difference that the W/P and P/S eluates are directed to a second adsorber to isolate P (zones 5 and 7). Accordingly, the process objectives of the single adsorber batch process and the MCSGP process are similar, and it is possible to determine the operating parameters for the MCSGP process from the operating parameters of the batch process and the corresponding chromatogram.

A full cycle of the dual adsorber MCSGP process includes two "switches" with four tasks (I1, B1, I2, B2) each, as shown in FIG. 1. In each switching tasks are identical; the only difference is in the position of the adsorbers: in the first switch, adsorber 1 is located downstream of adsorber 2, while in the second switch (not shown in Fig. 1), adsorber 2 is located downstream of adsorber 1.

The four phases include the following tasks:

Phase I1: the first phase of the operation of the adsorbers in the connected state. The overlapping portion of W/P is eluted from the upstream adsorber (zone 5 in FIG. 1) and internally recirculated in adsorber coupled mode to the downstream adsorber (zone 1). Between adsorbers, the liquid stream is usually diluted during the process with a buffer solution or re-adsorption solvent P (and overlapping part W) in an adsorber located downstream. At the end of phase I1, the pure product is ready to be eluted at the outlet of the upstream adsorber (zone 5).

On FIG. 28 of the publication EP-A-1877769 this phase is designated as stage "1".

Phase B1: The first phase of autonomous operation of adsorbers. The pure product P is eluted and collected from the adsorber in zone 5 (adsorber 2 in Fig. 1), while the overlapping portion of P/S and S is retained in the adsorber. At the same time, a fresh portion of the initial mixture is injected into the adsorber in zone 2.

On FIG. 28 of EP-A-1877769 this phase is designated as stage "2".

Phase I2: The second phase of the operation of the adsorbers in the connected state.

The overlapped portion of the P/S is eluted from the upstream adsorber (zone 7) and recycled internally to the downstream adsorber (zone 3). Between adsorbers, the stream is usually diluted during the process with a buffer solution or readsorption solvent P in a downstream adsorber. At the end of the step, all remaining product P has been eluted from the upstream adsorber and only S remains in the upstream adsorber.

On FIG. 28 of the publication EP-A-1877769 this phase is designated as stage "3".

Phase B2: The second phase of adsorber operation in autonomous state. The adsorber in zone 8 (adsorber 2 in Fig. 1) is cleaned to remove S and re-equilibrated. Simultaneously, W is eluted from another adsorber in zone 4.

On FIG. 28 of the publication EP-A-1877769 this phase is designated as stage "4".

After completing these tasks, the adsorbers change positions, and during the next phase 11 (not shown in Fig. 1), adsorber 2 is in the downstream position (zone 1) and adsorber 1 is in the upstream position (zone 5). At the beginning of this phase 11, adsorber 2 is cleaned and re-equilibrated and is ready to receive the W/P fraction from adsorber 1. After phases B1, I2 and B2 are completed again, the adsorbers return to their original positions and one cycle is completed. Adsorber 1 is now clean and ready to receive W/P from adsorber 2 during the next phase 11 (as shown in FIG. 1).

As with other countercurrent chromatographic processes, in practice, during the MCSGP process, adsorber movement is simulated by connecting and disconnecting adsorber inlets and outlets through valve switching, rather than by physically moving adsorbers.

The flowsheet of such multi-adsorber countercurrent processes is based on the separation of a "model chromatogram", such as the chromatogram shown in FIG. 1 and showing the elution of the product and impurities into essentially several different zones. The key to the technological scheme of the process are the first zone, in which only weakly adsorbing impurities W are present (zone 4 in Fig. 1), the second zone, in which weakly adsorbing impurities W overlap with the target compound P (zone 5 in Fig. 1), the third a zone in which pure product P is present (zone 6 in Fig. 1), a fourth zone in which product P and highly adsorbed impurities S overlap (zone 7 in Fig. 1), and a fifth zone in which only strongly adsorbed impurities are present S (zone 8 in Fig. 1).

As part of the process design, it is necessary to establish boundaries between different zones leading to the determination of process operating parameters (gradient concentrations, space velocities given by pumps) from a single adsorber chromatogram obtained offline. The delineation is performed based on the volume of elution, which is related to time and can be converted to time via volumetric flow rate. The position of the boundaries is critical to the efficiency of the process, i.e. product purity and throughput. For example, improper placement of the product elution zone boundary (zone 6 in FIG. 1) can result in the inclusion of weakly adsorbing impurities in the product pool and failure to meet purity specifications.

However, even with proper initial planning, external factors can have a detrimental effect on the purity of the product and the efficiency of the described multi-adsorber countercurrent process in the next step. Freshly prepared mobile phases may have slightly different compositions; ambient temperatures can vary, affecting the chromatographic adsorption process. The capacitance of the stationary phase can change over time.

In most cases, these factors lead to a shift in the chromatographic profile, although the resolution of the product and impurities remains similar. However, the shifted chromatogram may have different peak positions compared to the original model chromatogram used to design the multi-adsorber countercurrent process. This means that the placement of the boundaries between different zones based on the original simulated chromatogram is inaccurate and the purity of the product may be reduced as a result of peak shift.

An example is shown in FIG. 2: Although the product collection window is optimally placed (Fig. 2a), in the case of an earlier elution of the chromatogram by only about 1 minute, the zone 6 product elution interval (which has a fixed position based on the original chromatogram) does not correspond to the maximum of the product peak and includes a significant part of strongly adsorbed impurities (Fig. 2b). As a result, the concentration of the product in the collected fraction and, more importantly, the purity are reduced so that the product no longer meets the purity specifications. A 1 minute shift can be caused by a temperature change of several degrees Celsius.

Note that usually only a cumulative signal proportional to the concentration can be registered with a detector, for example, a cumulative UV signal (thick black line, UV). To visualize the incorporation of impurities into the product pool, the cumulative signal, proportional to concentration, was numerically expanded to show product and impurity peaks.

In order to take into account the change in environmental conditions, it is necessary to register a new model chromatogram with each new change in conditions, which can only be achieved with the application of extreme efforts, and which is practically meaningless.

One way to improve process design reliability is to narrow the product fraction (zone 6), resulting in an expansion of the internal recirculation zones (zones 5 and 7), but this has a negative effect on process performance and can only be done if the chromatogram shift is significant less than the width of the elution window. The preferred way to account for variations in operating parameters is to use real-time control.

One possibility is to use an estimate of the position of the peak maximum or the first peak moment to determine the control actions during the next product elution or the next cycle. Another possibility is to use real-time HPLC evaluation of the product eluate to determine yield and purity.

In these control methods, MCSGPs use complex control algorithms that can control and optimize the process based on cycle-to-cycle transitions. The advantage of control methods is the possibility of simultaneous control and process optimization. Their disadvantage is the delayed effect of the control actions, which do not become effective until the next elution of the product, since these methods require information about the complete phase of the elution of the product before the control action can be determined. Another disadvantage is the offline performance of analyzes and the complexity of the control algorithms. Typically, the control algorithms include evaluating product elutions to determine the actual value of a parameter related to process efficiency and/or product purity, and calculating an error based on the difference between the actual value and a target parameter value. Then, an action is performed based on the magnitude of the error.

Other methods are based on describing the MCSGP process using a chromatographic model and using the model to predict process performance and perform optimization (model-based predictive control). Although these methods are effective, they are difficult to use in practice, since they require an accurate description of the process and chromatographic separation based on numerous parameters related to the compounds to be separated, and to the stationary and mobile chromatographic phases used, which are difficult and time consuming to determine, and methods require considerable modeling experience.

Fig. 3 shows a chromatogram obtained using a method including:

(a) monitoring the slope and/or value of the signal proportional to the concentration at the outlet of the upstream adsorber during internal recirculation of weakly adsorbing impurities (phase I1);

(b) continuously comparing the value of the signal proportional to the concentration with a predetermined threshold at the time when the slope of the signal proportional to the concentration is positive and when the threshold is reached

(c) terminating the execution of phase I1 and starting the execution of phase B1, wherein the phase B1 has a fixed or variable duration depending on another threshold.

In this case, the signal proportional to the concentration is the UV signal, the UV threshold is 0.6 adsorption unit (AU), and the fixed duration of the B1 phase is 1.5 ml.

Fig. 4 shows a chromatogram obtained using a method including:

(a) monitoring the slope and/or value of the signal proportional to the concentration at the outlet of the upstream adsorber during internal recirculation of weakly adsorbing impurities (phase I1);

(b) continuously comparing the value of the signal proportional to the concentration with a predetermined threshold at the time when the slope of the signal proportional to the concentration is positive and when the threshold is reached

(c) continuing the execution of the I1 phase for a period of time or elution volume (delay) until the execution of the I1 phase is terminated and the execution of the B1 phase begins, wherein the B1 phase has a fixed duration, and the delay has a predetermined or variable duration depending on another threshold value.

In this example, the following is true: the signal proportional to the concentration is a UV signal, the threshold is 0.5 AU, the delay volume is 1.1 ml, the (fixed) duration of the product collection phase B1 is 1.5 ml.

Fig. 5 shows a chromatogram obtained using a method including a modification of the previous method in that phase B1 is started based on a threshold, which may be the same as the threshold used to start the delay period during phase I1, or it may different from it, and the delay period can have a minimum value. The reason for setting the minimum delay time is to avoid early start of the B1 phase due to impurities eluting before the main product and reaching the second threshold. The delay period can be expressed in terms of time or volume.

In this example, the following is true: the signal proportional to the concentration is a UV signal, threshold 1: 0.5 AU, minimum delay period: 1.0 ml, threshold 2: 0.6 AU, (fixed) collection duration: 1.5 ml.

Fig. 6 shows a chromatogram obtained using a method including:

(a) monitoring the slope and/or the value of the signal proportional to the concentration at the outlet of the adsorber eluting the product (phase B1); and

(b) continuously comparing the value of the signal proportional to the concentration with a predetermined threshold at the time when the slope of the signal proportional to the concentration is negative and when the threshold is reached

(c) stopping phase B1 and starting phase I2, with sample loading modulated such that sample loading occurs at the beginning of phase B1 and stops after a short period of time, while eluting from the other adsorber continues until the threshold is reached, and phase 12 has a predetermined or variable duration depending on another threshold value.

In this example, the concentration proportional signal is UV, the threshold is 0.2 AU, the loading interval is fixed at 0.5 ml, the I2 phase is fixed at 1.2 ml.

Fig. 7 shows a chromatogram obtained using a method in which the I2 endpoint is defined by a threshold value, and when this threshold value (c) is reached, the execution of the I2 phase is terminated and the execution of the B2 phase is started. In this example, the signal proportional to the concentration is a UV signal, threshold 1 for the start of the I2 phase is 0.2 AU, the fixed duration of the loading interval is 0.5 ml, the threshold 2 for the end of I2 is 0.1 AU.

Any of these methods related to the beginning of phase B1 can be combined with methods related to the completion of phase B1.

Fig. 8 shows a combination of the methods shown in FIG. 5 and FIG. 6, with the start of the delay period during the I1 phase based on the first threshold, the start of the product harvesting phase B1 based on the second threshold during the period when the UV slope is positive, and the end of the B1 phase (and the start of the I2 phase) based on the third threshold value.

In other preferred embodiments of any of the above methods, additional information about the slope of the chromatogram is used to start performing control actions. In a preferred embodiment of the method, the methods described that use a fixed duration delay volume may instead use a reversal of the sign of the slope as a criterion for terminating the delay and continuing to evaluate the signal until the second threshold is reached.

All methods include continuing the run and continuing the gradient elution during phases I1, B1, I2, preferably with the slope of the gradient used during phase 4 during the elution of Compound "W", which refers to the slope relative to the volume of mobile phase used in the process. This means that the elution gradient of the multi-adsorber process has a constant slope relative to the volume of mobile phase used in the process during phases I1, B1, I2, as illustrated in FIG. 3-8.

In the methods described, the thresholds may also be determined based on information obtained during the same run or cycle, thus it may be unknown at the start of the run or cycle. In this case, the first run may be partially or completely completed before thresholds are determined in the method based on an evaluation of the recorded signals that are significant for the rest of the run (in case the run was partially completed by the time of the evaluation) or for the rest of the chromatographic run. process. Accordingly, the threshold value for terminating a process phase and starting a new process phase is determined from a signal proportional to the concentration recorded during the same or a previous run of the chromatographic process. For example, the method monitors the UV signal during an MCSGP chromatographic run. The method is organized in such a way that the execution of phase B1 is stopped and the execution of phase I2 is started when the signal reaches 25% of the maximum value of the UV signal that should be obtained during phase B1. During the B1 phase, a peak with a maximum peak value of 0.80 AU is eluted (see Fig. 6). Once the method has reached 25% of the maximum peak value corresponding to 0.20 AU, phase B1 is stopped and the process continues with phase I2. In the next cycle, during phase B1, a maximum peak value of as little as 0.72 AU can be reached (eg due to variations in adsorbent quality). In the case of a method organized so that the action is performed when 25% of the maximum peak value reached during the B1 phase is reached, the process will continue until the threshold value of 0.18 AU is reached, after which the B1 phase will be terminated and the I2 phase will be started. . This type of method configuration, relating a threshold value to a concentration-proportional signal value obtained earlier during a run, balances variations in adsorbent quality, or detector quality, or detector calibration. The method also includes the use of several peaks found in the chromatogram to trigger the control action.

Example 1 [Fig. 9]:

The Contichrom system (manufactured by ChromaCon AG) was operated using control method (A). Two columns contained different cation exchange stationary phases (Fractoprep SO3(M) and Gigacap SO3) packed into columns with an inner diameter of 0.5 cm and a bed height of 10 cm. Two different resins were used to simulate columns with different packing qualities. columns. The system's live software was programmed to continuously monitor the A280 UV signal at the outputs of both columns, and the UV threshold to start the product collection phase was set to 0.1 AU (=100 mAU) based on simulated chromatogram data. The duration of the product elution phase was fixed at 5.5 minutes.

The loading material was a lysozyme solution and the buffers used were Buffer A: 25 mM phosphate, pH 6.0; Buffer B: 25 mM phosphate, pH 6.0, 1 M NaCl; wash solution: 1M NaOH. On FIG. 9 shows chromatograms of a 10 cycle run of the MCSGP process with repeated peaks of eluted product from each column. It can be seen that the product peaks had very different widths and heights (broad peaks for Fractoprep stationary phase, narrow peaks for Gigacap stationary phase) depending on which column they were eluted from. On FIG. 9B is an overlay of 10 run chromatograms which confirms that the product elutions from each column were highly reproducible when product elutions from the same column were compared. In addition, the figure shows the position of the used outlet valve (V1b, V2b) which is representative of the process phases. Valve position 4 corresponds to phase I1, position 3 corresponds to phase B1, position 4 corresponds to phase I2 and positions 1 and 5 correspond to phase B2.

The chromatograms show that, despite the very different peak shapes, the product collection starts at the set threshold of 0.1 AU and that the product collection is performed with a fixed duration, with in both cases the maximum of the peak is collected, which corresponds to the highest concentration of the product and the highest degree of purity.

Fig. 10 shows a process diagram similar to that shown in FIG. 1, with part a) showing a plant containing 3 adsorbers and part b) showing a plant containing 4 adsorbers. In part a) with 3 adsorbers, the top four rows I1 to B2 essentially correspond to the process illustrated in FIG. 1 as far as the tasks performed by adsorbers 1 and 2 are concerned. Adsorber 3 is passive as far as the actual separation process is concerned, including the W (weakly adsorbing fraction), P (product fraction) and S (strongly adsorbing fraction) components. In column 3 in this first block, purification, balancing or chemical reaction steps can be carried out.

After moving to the second block I1-B2 (the transition is shown by the upper arrow on the left), adsorber 1 of the first block takes over the function of adsorber 2 in the first block, adsorber 2 of the first block takes over the function of adsorber 3 in the first block (passive function), and adsorber 3 of the first block takes over the function of adsorber 1 in the first block.

After moving to the third block I1-B2 (the transition is shown by the bottom arrow on the left), adsorber 1 takes over the function of adsorber 3 in the first block (passive function), adsorber 2 of the first block takes over the function of adsorber 1 in the first block, and adsorber 3 of the first block takes over the function of adsorber 2 in the first block.

Concentration proportional signals in this triadsorber process depicted in FIG. 10a can be measured at the outputs of active columns, that is, in the first block from above - at the outputs of columns 1 and 2 (which is described in detail in the description of Fig. 1), in the second block from above - similarly at the outputs of columns 1 and 3, and in the lower block - similarly at the outlets of columns 2 and 3. Preferably, only the signal proportional to the concentration is measured at the outlet of the adsorber located upstream of the two active adsorbers in the operating mode of the adsorbers in the connected state (I1, I2), and in the autonomous phase work - at the outlet of the adsorber eluting the product (B1), for example - at the outlet of adsorber 2 during stages 1-4 (first block), at the outlet of adsorber 1 during stages 5-8 (second block) and at the outlet of adsorber 3 during stages 9-12 (block 3).

On FIG. 10b) in a process using 4 adsorbers, the top four rows I1 to B2 essentially correspond to the process illustrated in FIG. 1 in terms of the tasks performed by adsorbers 1 and 2. Adsorbers 3 and 4 are passive in terms of the actual separation process, including the components W (weakly adsorbing fraction), P (product fraction) and S (strongly adsorbing fraction ). In adsorbers 3 and 4, purification, balancing or chemical reaction steps can be carried out in this first block.

After moving to the second block I1-B2 (the transition is shown by the upper arrow on the left), adsorber 1 of the first block takes over the function of adsorber 2 in the first block, adsorber 2 of the first block takes over the function of adsorber 3 in the first block (passive function), adsorber 3 of the first block takes over the function of adsorber 4 in the first block (passive function), and adsorber 4 of the first block takes over the function of adsorber 1 in the first block.

After moving to the third block I1-B2 (the transition is shown by the middle arrow on the left), adsorber 1 takes over the function of adsorber 3 in the first block (passive function), adsorber 2 of the first block takes over the function of adsorber 4 in the first block (passive function), adsorber 3 takes over the function of adsorber 1 in the first block, and adsorber 4 takes over the function of adsorber 2 in the first block.

After moving to the fourth block I1-B2 (the transition is shown by the bottom arrow on the left), adsorber 1 takes over the function of adsorber 4 in the first block, adsorber 2 takes over the function of adsorber 1 in the first, adsorber 3 takes over the function of adsorber 2 in the first block, and adsorber 4 takes over the function of adsorber 3 in the first block.

The concentration proportional signals in this four adsorber process depicted in FIG. 10b can be measured at the outlets of the active columns, that is, in the first block from the top - at the outlets of adsorbers 1 and 2 (which is described in detail in the description of Fig. 1), in the second block from the top - similarly at the outlets of adsorbers 1 and 4, in in the third block, at the outlets of adsorbers 3 and 4, and in the lower block, similarly at the outlets of adsorbers 2 and 3. Again, only the signal proportional to the concentration is preferably measured at the outlet of the adsorber located upstream of the two active adsorbers , and in the phase of autonomous operation - at the outlet of the adsorber eluting the product (B1), for example - at the outlet of adsorber 2 during stages 1-4 (first block), at the outlet of adsorber 1 during stages 5-8 (second block ), at the outlet of the adsorber 4 during steps 9-12 (block 3) and at the outlet of the adsorber 3 during steps 13-16 (block 4).

Claims (32)

1. A method for monitoring, evaluating and regulating a cyclic chromatographic countercurrent gradient purification process, including at least two adsorbers, through which a liquid is passed with an initial mixture (Load) containing the target components of the product (P) and impurities (W, S), where impurities less adsorbed than the product (W) and impurities adsorbed more strongly than the product (S), wherein the purification process includes at least two different phases (I, B): - at least one phase (I) of operation of the adsorbers in a connected state, during which the two adsorbers are connected to each other so that the outlet of the upstream adsorber is in fluid communication with the inlet of the adsorber located downstream, and - at least one phase (B) of autonomous operation, during which at least one adsorber is not in liquid connection with other adsorbers, and during which the target components of the product (P) are isolated in purified form from the detached adsorber, wherein the method comprises at least the following steps: a) monitoring the chromatogram, including measuring in the liquid at least one current value of the signal proportional to the concentration, which is the absolute value of the signal, its integral, its slope or slope sign, or a combination thereof; b) evaluating the chromatogram, comprising continuously comparing at least one of the current signals proportional to the concentration measured in step a) with a threshold signal value; c) adjusting the chromatographic purification process by adapting the end of the current phase based on the comparison made in step b) and the start of the next phase; moreover, the sequence of steps a)-c) is performed in this order at least twice. 2. The method according to claim 1, characterized in that in the case of a signal proportional to the concentration, at least one parameter is taken into account, selected from its absolute value, its integral, its slope and the sign of the slope, and a combination of these parameters is taken into account, most preferably a combination absolute value and slope sign. 3. Method according to any one of the preceding claims, characterized in that the signal proportional to the concentration measured in step a) is measured at the outlet of at least one adsorber, preferably at the outlet of two adsorbers or at the outlet of all adsorbers, and also preferably a signal proportional to the concentration is measured at least in one of the following positions: at the outlet of the adsorber, which is in the phase (B1) of the autonomous elution of the target product, at the outlet of the adsorber, located upstream during the phase (I1, I2) of the operation of the adsorbers in connected state. 4. Method according to any one of the preceding claims, characterized in that the purification process comprises at least four different phases (I1, B1, I2, B2) in the following order: - at least one first phase (I1) of operation of the adsorbers in a connected state, during which the two adsorbers are connected to each other so that the outlet of the upstream adsorber is in fluid communication with the inlet of the adsorber located downstream , the solvent flows through the inlet to the upstream adsorber, and the target product components (P) and poorly adsorbed impurities (W) move from the upstream adsorber to the downstream adsorber, preferably until until essentially only the target components of the product (P) exit from the outlet of the upstream adsorber, preferably an intermediate dilution is carried out between the upstream adsorber and the downstream adsorber; - at least one first phase (B1) of autonomous operation, during which the adsorbers are not in liquid connection and during which the solvent enters the adsorber located upstream through the inlet during the first phase (I1) of operation of the adsorbers in a connected state , and through the outlet of this adsorber eluting the product, the target components of the product (P) are collected, while the liquid containing the initial mixture (Load) enters through the inlet into the adsorber located downstream during the first phase (I1) of the operation of the adsorbers in the connected state, and weakly adsorbable impurities are typically collected through the outlet of this adsorber; - at least one second phase (I2) of operation of the adsorbers in the connected state, during which the two adsorbers are connected to each other so that the outlet of the adsorber located upstream during the first phase (I1) of the operation of the adsorbers in the connected state is connected to the inlet of the adsorber located downstream during the first phase (I1) of the operation of the adsorbers in the connected state, the solvent enters through the inlet into the adsorber located upstream, and the target product components (P) and strongly adsorbed impurities (S) move from the upstream adsorber to the downstream adsorber, preferably until the outlet of the upstream adsorber substantially ceases to produce the desired product components (P), preferably between the adsorber located upstream, and an adsorber located downstream, an intermediate time is carried out addition; and - at least one second phase (B2) of autonomous operation, during which the adsorbers are not in liquid connection and during which the solvent enters the adsorber located upstream through the inlet during the second phase (I2) of operation of the adsorbers in a connected state , and through the outlet of this upstream adsorber, strongly adsorbed impurities (S) are collected, while the solvent enters through the inlet into the downstream adsorber during the second phase (I2) of operation of the adsorbers in the connected state, and through the outlet of this adsorber, previously located downstream, weakly adsorbing impurities are collected, moreover, the functions of the phases (I1, B1, I2, B2) are performed synchronously or, preferably, sequentially, and they are performed cyclically at least twice, and when the cycle is performed after or during the switching period, the adsorber previously located upstream during the second phase (B2) autonomous operation is moved so that it becomes the adsorber located downstream during the next first phase (I1) of operation of adsorbers in a connected state, and the adsorber previously downstream during the second phase (B2) autonomous operation, is moved so that it becomes the upstream adsorber during the subsequent first phase (I1) of operation of the adsorbers in the connected state. 5. The method according to claim 4, characterized in that step a) comprises measuring at least one current signal value proportional to the concentration in the liquid at the outlet of the upstream adsorber during the first phase (I1) of the operation of the adsorbers in the connected condition. 6. The method according to claim 4, characterized in that the absolute value of the signal proportional to the concentration is measured, and preferably also the sign of its slope, and when the threshold value of the absolute value is exceeded, the execution of the next first phase (B1) of autonomous operation of adsorbers is started, either in the form of the first phase of autonomous operation of adsorbers with a fixed duration, or in the form of the first phase of autonomous operation of adsorbers, which has a duration that varies depending on further monitoring, evaluation and regulation, or when the threshold value of the absolute value is exceeded, a fixed delay is provided until the start of the first phase (B1) of the autonomous operation of adsorbers, either in the form of the first phase of autonomous operation of adsorbers with a fixed duration, or in the form of the first phase of autonomous operation of adsorbers, which has a duration that varies depending on further control, evaluation and regulation, or when the first threshold value of the absolute value is exceeded, a minimum fixed delay is provided and after the second threshold value of the absolute value is exceeded, preferably with the additional condition checked, consisting in the fact that the signal slope is positive, the next first phase (B1) of autonomous operation of the adsorbers is started, either in the form of the first phases of autonomous operation of adsorbers with a fixed duration, or in the form of the first phase of autonomous operation of adsorbers, having a duration that varies depending on further monitoring, evaluation and regulation. 7. The method according to any one of paragraphs. 4-6, characterized in that stage a) includes measuring at least one current value of the signal proportional to the concentration in the liquid at the outlet of the adsorber eluting the product during the first phase (B1) of autonomous operation of the adsorbers, and at least one parameter selected from the absolute value of the signal and the sign of its slope, and preferably a combination of these two parameters is measured. 8. The method according to claim 7, characterized in that when the absolute value of the signal decreases below the threshold value, preferably under control of the additional condition that the signal slope is negative, the next second phase (I2) of the operation of the adsorbers in the connected state or in the form of the second phase of operation of the adsorbers in the connected state with a fixed duration, or in the form of the second phase of the operation of the adsorbers in the connected state, having a duration that varies depending on further monitoring, evaluation and regulation, moreover, it is preferable to load the adsorber that does not elute the product during the first phase (B1) of autonomous operation, start at the beginning of the first phase (B1) of autonomous operation and preferably stop it, in the typical case after a fixed loading time, before switching to the next phase ( I2) operation of adsorbers in the connected state. 9. The method according to any one of paragraphs. 7 and 8, characterized in that step a) comprises measuring at least one current signal value proportional to the concentration in the liquid at the outlet of the upstream adsorber during the second phase (I2) of operation of the adsorbers in the connected state, preferably at least one parameter is measured, selected from the absolute value of the signal and the sign of its slope, and preferably a combination of these two parameters is measured, moreover, after reducing the absolute value of the signal below the threshold value, preferably with checking the additional condition that the slope of the signal is negative, start the next second phase (B2) autonomous operation of the adsorbers, either in the form of a second phase of autonomous operation of the adsorbers with a fixed duration, or in the form of the second phase of autonomous operation of adsorbers, which has a duration that varies depending on further monitoring, evaluation and regulation. 10. The method according to any of the preceding claims, characterized in that at least two adsorbers are used in the cyclic chromatographic process, and each cycle includes at least two phases (I1, I2) of the operation of the adsorbers in the connected state, during which the two adsorbers are in the liquid connection for internal recirculation of the various partially purified fractions of by-products (W/P, P/S), and preferably during step a) at the outlet of the corresponding upstream adsorber, at least one parameter is measured, selected from the absolute the magnitude of the signal and its slope. 11. A method according to any one of the preceding claims, characterized in that the change in the sign of the slope of the signal is used as a criterion for performing the control action. 12. The method according to any one of the preceding claims, characterized in that the gradient of the eluting solvent in the process has a constant slope relative to the volume of the liquid mobile phase used in the process, during both phases of operation of the adsorbers in the connected state and the first phase of autonomous operation (I1, B1, I2) or has zero slope. 13. The method according to any one of the preceding claims, characterized in that the threshold value for terminating one phase of the process and starting a new phase of the process is determined from a signal proportional to the concentration recorded during the same or a previous cycle of the chromatographic process. 14. The method according to any one of the preceding claims, characterized in that the control action is started on the basis of failure to reach a predetermined threshold value within a predetermined elution volume, or time, or concentration gradient. 15. Method according to any one of the preceding claims, characterized in that the signal proportional to the concentration is based on the measurement of visible light, UV radiation, infrared radiation, fluorescence, Raman scattering, ionic strength, electrical conductivity or refractive index.

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