Material tracking and real-time release method for periodic countercurrent chromatography

Abstract

The invention discloses a material tracking and real-time release method for periodic countercurrent chromatography, and belongs to the field of chromatographic separation. The method comprises the following steps: carrying out parameter fitting and calibration on a chromatography residence time distribution model through static adsorption experiment and chromatography process data; on the basis of the calibration model, the actual operation parameters and the starting time and the duration time of the to-be-tracked material, predicting a residence time distribution curve of the to-be-tracked material in a single column, and inputting the result into a continuous flow chromatography model framework constructed on the basis of a periodic countercurrent chromatography mode to obtain complete residence time distribution of the tracked material in the system; by repeatedly calculating tracking materials with different time parameters, the distribution condition of the tracking materials in each elution peak is analyzed, and global material tracking is realized; and in combination with a preset product quality acceptance limit value, determining an elution peak needing to be shunted, and forming a real-time release scheme. According to the method, the influence of disturbance at the periodic countercurrent chromatography inlet on the outlet product can be predicted, and a product shunting strategy is formulated to meet the requirement of real-time release.

Description

Technical Field

[0001] This invention relates to protein chromatography separation technology in the fields of biochemical engineering and bioengineering, specifically to a material tracking and real-time release method for a periodic countercurrent chromatography separation process. Background Technology

[0002] Continuous biomanufacturing, as an important development direction in the biopharmaceutical industry, can effectively improve process efficiency, reduce production costs, ensure product quality stability, and significantly enhance the level of production intensification. To support the industry's transformation from traditional batch production to continuous manufacturing, the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) has published ICH Q13, a guideline for continuous manufacturing of active pharmaceutical ingredients (APIs) and formulations. This guideline outlines key considerations for continuous manufacturing, explicitly requires the tracking of materials throughout the entire process, and particularly emphasizes that Residence Time Distribution (RTD) is a core tool for achieving material traceability.

[0003] RTD (Probability Distribution Function) describes the probability distribution function of the time required for material to flow through a unit operation or a series of unit operations. It plays a crucial role in process design and system integration, providing important data for setting start-up and shutdown times and batch definition in continuous manufacturing systems. In actual production, process disturbances may occur at any stage with varying intensity and duration, leading to final product defects. In batch production, defective materials often result in the scrapping of the entire batch; however, in continuous manufacturing, a reasonable diversion strategy can be used to remove only the defective portion, ensuring the final product still meets quality standards. RTD can predict the propagation dynamics of defective materials within the system, thus supporting real-time decision-making on diversion timing. Therefore, based on RTD characterization, diversion schemes within the continuous manufacturing framework can be developed to meet real-time release requirements and ensure consistent product quality.

[0004] Periodic countercurrent chromatography is an important method for continuous antibody capture. The basic principle is to use a dual-column tandem loading system. The second column receives the protein flowing through the first column, and loading terminates at a higher breakthrough point on the first column. The other columns then perform elution and regeneration. This alternating column operation enables continuous operation, thereby improving process yield and media utilization while reducing buffer consumption and equipment size. A patent (US Patent 2012 / 0091063 A1) proposes a continuous flow device that can switch between three-column and four-column modes and applies it to the separation of mixtures containing monoclonal antibodies and bovine serum albumin. The specific process of three-column periodic countercurrent chromatography is as follows...Figure 2 As shown, the process mainly consists of two stages: connection mode loading and disconnect mode loading. First, the first and second chromatography columns are connected in series for loading. Proteins penetrating from the first column are captured by the second column, while the third column performs elution and regeneration. When the first column reaches the set loading endpoint and the third column completes all elution and regeneration, the process switches to disconnect mode loading. The first and second columns are disconnected, and the first and third columns are connected in series for connection washing. Unadsorbed proteins in the first column are captured by the third column, while the second column begins loading in disconnect mode. When the set connection washing volume is reached, one cycle ends. The three columns then switch their relative positions, with the second and third columns connected in series for loading, and the first column begins elution and regeneration. This connection and disconnect mode process is repeated three times, returning the columns to their initial positions, completing one cycle. The four-column periodic countercurrent chromatography process is similar to the three-column mode, specifically as follows: Figure 3 As shown. The difference between the three-column mode and the four-column mode is that in the four-column mode, the elution and regeneration steps are distributed to two chromatography columns and carried out simultaneously, and one cycle includes four switching operations.

[0005] To address the complexity of periodic countercurrent chromatography (PCC) processes, model-assisted methods have been widely applied in PCC process development. Patents (CN112451996A, CN110348090A, CN110348089A and US11958881 B2) utilize mathematical models to predict and analyze process yields and media utilization under different operating conditions, significantly reducing experimental workload and determining suitable process parameters. However, existing literature largely focuses on improving process yields and media utilization, with little attention paid to material traceability. Material traceability and availability are key requirements for drug regulatory authorities; failure to establish clear material traceability strategies in PCC will severely hinder the commercial application of this technology.

[0006] Periodic countercurrent chromatography employs multiple alternating columns to match loading and elution / regeneration times. While this method ensures uninterrupted loading and constant flow rate, it significantly increases process complexity, and the intricate pipeline switching patterns complicate material tracking. Although the paper (J Chromatogr A. 1683 (2022)463530.) used tracer experiments to investigate the residence time distribution behavior in two-column periodic countercurrent chromatography, the experimental scope is limited, failing to provide a comprehensive understanding of material tracking. Using mathematical models for reasonable residence time behavior characterization and predictive analysis can greatly reduce experimental trial and error, significantly improve process understanding, and enable tracking of different stages and material fragments. Once material traceability is achieved, a real-time diversion strategy can be formulated based on predetermined product quality requirements, thereby enabling real-time release for continuous manufacturing. Summary of the Invention

[0007] In view of the above-mentioned technical problems, the present invention provides a method for material tracking and real-time release in periodic countercurrent chromatography based on a chromatographic residence time distribution model, which is aimed at the periodic countercurrent chromatography operation mode. The method involves fitting adsorption data and chromatographic process curves to construct a residence time distribution model, and calculating and analyzing the chromatographic residence time distribution behavior of different tracked materials, thereby realizing material tracking and real-time release in periodic countercurrent chromatography.

[0008] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: A method for material tracking and real-time release in periodic countercurrent chromatography, characterized by comprising the following steps: Step 1: Fit the chromatographic residence time distribution model using the results of the static adsorption experiment and the chromatographic experiment process curve, calibrate the chromatographic residence time distribution model, and obtain the parameters of the chromatographic residence time distribution model. The model parameters include the adsorption parameters of the medium, the mass transfer related parameters, and the parameters related to the influence of the flow rate on adsorption. Step 2: Substitute the parameters of the chromatography residence time distribution model obtained in Step 1, the actual chromatography operation parameters, and the start and duration of the tracked material into the chromatography residence time distribution model to obtain the single-column residence time distribution curve of the specific tracked material; Step 3: Substitute the single-column residence time distribution curve, periodic countercurrent chromatography operating parameters, and mode predicted in Step 2 into the periodic countercurrent chromatography model framework to obtain the residence time distribution curve of the specific tracked material in periodic countercurrent chromatography; the periodic countercurrent chromatography model framework is compatible with different modes of periodic countercurrent chromatography, including three-column mode and four-column mode. Step 4: Define the start and duration of the tracking material, repeat steps 2 and 3 to obtain the residence time distribution curve of the tracking material in periodic countercurrent chromatography with different start and duration times, calculate the proportion of the tracking material in each elution peak, and realize material tracking in periodic countercurrent chromatography. Step 5: Based on the required product quality requirements, determine the acceptance limit for real-time release. Combined with the real-time release acceptance limit that meets the product quality requirements, determine the elution peaks in the elution peaks where the proportion of tracked material exceeds the release acceptance limit and divert them, thereby achieving real-time quality control and release of the product.

[0009] According to a preferred embodiment of the present invention, the calibration of the chromatography residence time distribution model further includes the following steps: Substitute the initial values ​​of the medium adsorption parameters in the chromatographic residence time distribution model into the Langmuir adsorption model, calculate the adsorption isotherm, and compare the calculated results with the experimentally obtained adsorption isotherm. Change the medium adsorption parameters to minimize the root mean square error of both, and obtain the medium adsorption parameters in the chromatographic residence time distribution model. Substitute the experimental operating parameters of the breakthrough and rinsing steps in the chromatography experiment, the calculated medium adsorption parameters, and the initial values ​​of the mass transfer related parameters in the chromatography residence time distribution model into the chromatography residence time distribution model. Compare the calculation results with the breakthrough and rinsing curves obtained from the chromatography experiment. Change the mass transfer related parameters to minimize the root mean square error of both, and obtain the mass transfer related parameters in the chromatography residence time distribution model. The experimental operating parameters of the elution step in the chromatography separation process, the calculated medium adsorption parameters and mass transfer related parameters, and the initial values ​​of the flow relative adsorption related parameters in the chromatography residence time distribution model were substituted into the chromatography residence time distribution model. The calculation results were compared with the experimentally obtained elution curves. The flow relative adsorption related parameters were changed to minimize the root mean square error of both, and the flow relative adsorption related parameters in the chromatography residence time distribution model were obtained.

[0010] According to a preferred embodiment of the present invention, the periodic countercurrent chromatography operating parameters in step 3 are process parameters that need to be set for performing periodic countercurrent chromatography, including the flow rate and time for connecting sample loading, disconnecting sample loading, elution, and each regeneration step.

[0011] According to a preferred embodiment of the present invention, the periodic countercurrent chromatography model framework established based on the three-column mode includes eleven calculation units, namely, residence time distribution models of the three chromatography columns, four inlet calculation units, and four outlet calculation units; the first calculation unit is the residence time distribution model of the first chromatography column, the second calculation unit is the residence time distribution model of the second chromatography column, the third calculation unit is the residence time distribution model of the third chromatography column, the fourth calculation unit is the sample pump inlet calculation unit, the fifth calculation unit is the regeneration pump inlet calculation unit, the sixth calculation unit is the connection rinsing inlet calculation unit, the seventh calculation unit is the elution pump inlet calculation unit, the eighth calculation unit is the first waste liquid outlet calculation unit, the ninth calculation unit is the second waste liquid outlet calculation unit, the tenth calculation unit is the third waste liquid outlet calculation unit, and the eleventh calculation unit is the collector outlet calculation unit.

[0012] Furthermore, in the periodic countercurrent tomography model framework established by the three-column model, the components adopt different connection sequences at different stages of the periodic countercurrent tomography process. The three-column periodic countercurrent tomography process includes multiple switching processes executed periodically, and a single switching process includes, in sequence: 1) The first and second columns are connected for sample loading, and the third column is connected for regeneration before elution. The connection order is as follows: the fourth calculation unit is connected to the first calculation unit, the first calculation unit is connected to the second calculation unit, the second calculation unit is connected to the eighth calculation unit, the fifth calculation unit is connected to the third calculation unit, the third calculation unit is connected to the ninth calculation unit, the sixth calculation unit is connected to the tenth calculation unit, and the seventh calculation unit is connected to the eleventh calculation unit. 2) The first and second columns are connected for the sample loading step, and the third column is connected for the elution step. The connection order is as follows: the fourth calculation unit is connected to the first calculation unit, the first calculation unit is connected to the second calculation unit, the second calculation unit is connected to the eighth calculation unit, the fifth calculation unit is connected to the ninth calculation unit, the sixth calculation unit is connected to the tenth calculation unit, the seventh calculation unit is connected to the third calculation unit, and the third calculation unit is connected to the eleventh calculation unit. 3) The first and second columns are connected for sample loading, and the third column is eluted and then regenerated. The connection order is as follows: the fourth calculation unit is connected to the first calculation unit, the first calculation unit is connected to the second calculation unit, the second calculation unit is connected to the eighth calculation unit, the fifth calculation unit is connected to the third calculation unit, the third calculation unit is connected to the ninth calculation unit, the sixth calculation unit is connected to the tenth calculation unit, and the seventh calculation unit is connected to the eleventh calculation unit. 4) The first and third columns are connected and flushed, and the second column is disconnected and loaded. The connection sequence is as follows: the fourth calculation unit is connected to the second calculation unit, the second calculation unit is connected to the eighth calculation unit, the fifth calculation unit is connected to the ninth calculation unit, the sixth calculation unit is connected to the first calculation unit, the first calculation unit is connected to the third calculation unit, the third calculation unit is connected to the tenth calculation unit, and the seventh calculation unit is connected to the eleventh calculation unit. The above describes the component connection sequence within an initial switch. The second switch replaces the first computing unit with the second computing unit, the second computing unit with the third computing unit, and the third computing unit with the first computing unit. The third switch replaces the first computing unit with the second computing unit, the second computing unit with the third computing unit, and the third computing unit with the first computing unit. The next switch returns to the component connection sequence within the initial switch, and the subsequent repeated switching process simulates the periodic countercurrent tomography process.

[0013] According to a preferred embodiment of the present invention, the periodic countercurrent chromatography operation model framework established based on the four-column model includes fourteen computational units, namely, four column residence time distribution models, five inlet computational units, and five outlet computational units. The first computational unit is the first column residence time distribution model, the second computational unit is the second column residence time distribution model, the third computational unit is the third column residence time distribution model, the fourth computational unit is the fourth column residence time distribution model, the fifth computational unit is the sample pump inlet computational unit, the sixth computational unit is the first regeneration pump inlet computational unit, the seventh computational unit is the second regeneration pump inlet computational unit, the eighth computational unit is the connection rinsing inlet computational unit, the ninth computational unit is the elution pump inlet computational unit, the tenth computational unit is the first waste liquid outlet computational unit, the eleventh computational unit is the second waste liquid outlet computational unit, the twelfth computational unit is the third waste liquid outlet computational unit, the thirteenth computational unit is the fourth waste liquid outlet computational unit, and the fourteenth computational unit is the collector outlet computational unit. In different operation steps, the first to fourteenth computational units are connected in a specific order.

[0014] Furthermore, in the model framework established by the four-column periodic countercurrent chromatography operation mode, the components adopt different connection sequences at different stages of the periodic countercurrent chromatography process. The four-column periodic countercurrent chromatography process includes multiple switching processes executed periodically, and a single switching process includes, in sequence: 1) The first and second columns are connected for sample loading, the third column is regenerated before elution, and the fourth column is regenerated after elution. The connection order is as follows: the fifth calculation unit is connected to the first calculation unit, the first calculation unit is connected to the second calculation unit, the second calculation unit is connected to the tenth calculation unit, the sixth calculation unit is connected to the third calculation unit, the third calculation unit is connected to the eleventh calculation unit, the seventh calculation unit is connected to the fourth calculation unit, the fourth calculation unit is connected to the twelfth calculation unit, the eighth calculation unit is connected to the thirteenth calculation unit, and the ninth calculation unit is connected to the fourteenth calculation unit. 2) The first and second columns are connected for sample loading, the third column is used for elution, and the fourth column is used for elution and regeneration. The connection order is as follows: the fifth calculation unit is connected to the first calculation unit, the first calculation unit is connected to the second calculation unit, the second calculation unit is connected to the tenth calculation unit, the sixth calculation unit is connected to the eleventh calculation unit, the seventh calculation unit is connected to the fourth calculation unit, the fourth calculation unit is connected to the twelfth calculation unit, the eighth calculation unit is connected to the thirteenth calculation unit, the ninth calculation unit is connected to the third calculation unit, and the third calculation unit is connected to the fourteenth calculation unit. 3) The first and third columns are connected and flushed, the second column is disconnected and loaded, and the fourth column is left to wait. The connection sequence is as follows: the fifth calculation unit is connected to the second calculation unit, the second calculation unit is connected to the tenth calculation unit, the sixth calculation unit is connected to the eleventh calculation unit, the seventh calculation unit is connected to the twelfth calculation unit, the eighth calculation unit is connected to the first calculation unit, the first calculation unit is connected to the third calculation unit, the third calculation unit is connected to the thirteenth calculation unit, and the ninth calculation unit is connected to the fourteenth calculation unit. The above describes the component connection sequence within an initial switch. The second switch replaces the first computing unit with the second computing unit, the second with the third computing unit, the third with the fourth computing unit, and the fourth with the first computing unit. The third switch replaces the first computing unit with the second computing unit, the second with the third computing unit, the third with the fourth computing unit, and the fourth with the first computing unit. The fourth switch replaces the first computing unit with the second computing unit, the second with the third computing unit, the third with the fourth computing unit, and the fourth with the first computing unit. The next switch returns to the component connection sequence within the initial switch, and this repeated switching process simulates a periodic countercurrent tomography process.

[0015] According to a preferred embodiment of the present invention, the input and output information of the input port calculation unit and the output port calculation unit are both curves showing the changes in the concentration of the tracked material, the concentration of the non-tracked material, and the pH of the mobile phase over time.

[0016] According to a preferred embodiment of the present invention, the chromatographic residence time distribution model includes two sub-models, namely a general rate model and a two-component Langmuir adsorption model modified by mobile phase pH. The present invention has the following beneficial effects: (1) The residence time distribution model is simple and efficient. Based on the characteristics of periodic countercurrent chromatography, a specific sequence is used to connect the residence time distribution model of the chromatography to construct the residence time distribution model of periodic countercurrent chromatography. This enables the direct prediction of the residence time distribution behavior of periodic countercurrent chromatography from the residence time distribution behavior of a single column, thereby greatly reducing the workload of tracer experiments. (2) The model is accurate and widely applicable. By fitting the chromatographic residence time distribution model with limited experimental data and inputting it into the periodic countercurrent chromatography model framework based on different operating modes, the material tracking situation of periodic countercurrent chromatography under different operating modes can be simulated and predicted, thus deepening the understanding of the process; (3) Real-time release and quality control based on model prediction. By using the chromatographic dwell time distribution model and based on the global understanding of material tracking, diversion schemes can be designed according to different product quality requirements to obtain real-time release strategies, providing a reliable theoretical basis and operational guidance for coping with fluctuations and ensuring product quality in continuous manufacturing. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of a material tracking and real-time release method for periodic countercurrent chromatography. Figure 2 This is a schematic diagram of the process of three-column periodic countercurrent chromatography; Figure 3 This is a schematic diagram of the four-column periodic countercurrent chromatography process; Figure 4 A framework for connecting the residence time distribution model in a switching three-column periodic countercurrent chromatography operation mode; Figure 5 A connection framework for the residence time distribution model of a four-column periodic countercurrent chromatography operation mode within a switching context; Figure 6 This is the fitting result of the adsorption isotherm; Figure 7 To compare the breakthrough and flushing curves of the chromatography experiment with the model calculation results; Figure 8 To compare the elution curves of the chromatography experiment with the calculation results of the model; Figure 9 The result is the prediction of the residence time distribution curve model for periodic countercurrent chromatography; Figure 10 A schematic diagram of a material tracking strategy for three-column periodic countercurrent chromatography; Figure 11 This is a schematic diagram of a real-time release scheme for three-column periodic countercurrent chromatography. Detailed Implementation

[0018] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. The present invention is applicable to periodic countercurrent chromatography in different operating modes, including but not limited to three-column and four-column periodic countercurrent chromatography. Obviously, the described embodiments are typical special cases of the present invention and do not include all implementation conditions. Based on the technical solution and embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] In this invention, the chromatographic residence time distribution model includes two sub-models: a general rate model and a two-component Langmuir adsorption model modified by mobile phase pH.

[0020] A general rate model is used to describe the convection and diffusion behavior of materials in a chromatography column. It is a general rate model that considers parallel diffusion, and its equations are as follows: in: c This represents the protein concentration in the bulk liquid phase, expressed in mg / mL. c p This represents the protein concentration in the liquid phase within the medium particles, expressed in mg / mL. c 0 This represents the concentration of the sampled protein, in mg / mL. q This represents the concentration of protein in the solid phase, expressed in mg / mL. t Time, in seconds; D ax The axial dispersion coefficient within the column is expressed in meters. 2 / s; x This represents the axial distance within the column, in meters. u Void velocity, in mL / min; ε The porosity within the column; ε p The porosity of the medium particles; k f is the liquid film mass transfer coefficient, with units of m / s; r This represents the radial distance within the medium particles, in meters (m). r p The particle radius is in meters (m). D p The liquid-phase diffusion coefficient within the medium particles is given in m. 2 / s; D sThe diffusion coefficient of the inner surface of the medium particles is given in m. 2 / s; L denoted as column length, in meters (m). i A value of 0 or 1 represents unlabeled protein components and labeled protein components, respectively. Unlabeled protein components are non-tracked materials, while labeled protein components are tracked materials.

[0021] A two-component Langmuir adsorption model with mobile phase pH modification is used to describe the exchange behavior between the material and the adsorbate in a chromatography column. The equation is as follows: in: q max This represents the saturated adsorption capacity, expressed in mg / mL. k a This is the adsorption constant of the protein in the equilibrium buffer, in units of m. 3 / (mol·s); k d This is the dissociation constant of the protein in the equilibrium buffer, in units of... s -1 ; c ( H + () represents the concentration of hydrogen ions in the buffer solution, in mol / m³. 3 ; c ( H + ) eq This represents the concentration of hydrogen ions in the equilibrium buffer solution, in mol / m³. 3 ; γ and β These are used to describe the effects of hydrogen ion concentration in solution on the adsorption constant and dissociation constant, respectively.

[0022] Media adsorption parameters of unlabeled and labeled proteins in the chromatography residence time distribution model ( q max , k a , k d Mass transfer related parameters () D ax , D p , D s , k f ) and the influence of flow on adsorption-related parameters ( γ , β The settings are all the same.

[0023] The initial and boundary conditions for the above equations are: in: c feed This refers to the protein concentration in the feed solution, expressed in mg / mL. c ( H + ) elution The concentration of hydrogen ions in the eluent is expressed in mol / m³. 3 ; t a , t b , t c , t d and t e These represent the start time of material tracking, the end time of material tracking, the end time of sample loading, the end time of rinsing, and the end time of elution, all in units of [unit missing]. s .

[0024] In practical applications, for the three-column operation mode of periodic countercurrent chromatography, this invention uses a connection matrix to connect three chromatographic residence time distribution models (the first calculation unit is the first chromatographic residence time distribution model, the second calculation unit is the second chromatographic column residence time distribution model, and the third calculation unit is the third chromatographic column residence time distribution model) with the input and output ports in a specific manner, constructing a retention time distribution model framework for three-column periodic countercurrent chromatography. The input ports include: a sample pump input calculation unit (fourth calculation unit), a regeneration pump input calculation unit (fifth calculation unit), a connection rinsing input calculation unit (sixth calculation unit), and an elution pump input calculation unit (seventh calculation unit). The output ports include: a first waste liquid output calculation unit (eighth calculation unit), a second waste liquid output calculation unit (ninth calculation unit), a third waste liquid output calculation unit (tenth calculation unit), and a collector output calculation unit (eleventh calculation unit). Both input and output information are curves showing the changes in tracked protein concentration, non-tracked protein concentration, and mobile phase pH over time. The inputs for tracked and non-tracked materials differ in the fourth calculation unit.

[0025] like Figure 4 As shown, in the connection matrix of the three-column operation mode of periodic countercurrent chromatography, the indices of the computational cells in the same row of the matrix represent mutual connections, specifically including: 1) The first and second columns undergo a connection loading step, and the third column undergoes a pre-elution regeneration step. At this point, the connection matrix is: Connection IL-1（3c-pcc） , means as follows: ; 2) The first and second columns undergo a connection loading step, while the third column undergoes an elution step. At this point, the connection matrix is: Connection IL-2 (3c-pcc) , means as follows: ; 3) The first and second columns undergo a connection loading step, and the third column undergoes a elution and regeneration step. At this point, the connection matrix is: Connection IL-3 (3c-pcc) , means as follows: ; 4) The first and third columns undergo a connection flushing step, while the second column undergoes a disconnection loading step. At this point, the connection matrix is: Connection DL (3c-pcc) , means as follows: ; The three-column periodic countercurrent tomography process includes multiple periodically executed switching processes. The above describes the component connection sequence within an initial switching process. The second switching process replaces the position of the first computational unit with the second computational unit, the position of the second computational unit with the third computational unit, and the position of the third computational unit with the first computational unit. The third switching process replaces the position of the first computational unit with the second computational unit, the position of the second computational unit with the third computational unit, and the position of the third computational unit with the first computational unit. The next switching process returns to the component connection sequence within the initial switching process, and the subsequent repeated switching processes simulate the periodic countercurrent tomography process.

[0026] In the simulation calculation of the residence time distribution model of three-column periodic countercurrent chromatography, the labeled protein enters the system from the fourth computational unit and leaves the system from the eleventh computational unit. The cumulative residence time distribution curve is shown in the figure. F ( t This is used to measure the proportion of labeled proteins that have left the system during the time period from 0 to t, out of the total labeled proteins. The specific formula is: in: c 1[Unit_11]( t( ) is the curve showing the change in concentration of the labeled protein at the eleventh computational unit over time, in mg / mL; c 1[Unit_4]( t () is the curve showing the change in concentration of the labeled protein over time at the fourth computational unit, in mg / mL; Q in ( t (This is a curve showing the elution flow rate over time at the eleventh calculation unit, in mL / min.) Q out ( t The curve shows the change in sample loading rate over time at the fourth calculation unit, in mL / min.

[0027] For the four-column operation mode of periodic countercurrent chromatography, this invention employs a connection matrix to connect four chromatographic residence time distribution models (calculation units 1, 2, 3, and 4) with the input and output ports in a specific manner, constructing a retention time distribution model framework for four-column periodic countercurrent chromatography. The input ports include: sample pump input (calculation unit 5), first regeneration pump input (calculation unit 6), second regeneration pump input (calculation unit 7), connection rinse input (calculation unit 8), and elution pump input (calculation unit 9). The output ports include: first waste liquid output (calculation unit 10), second waste liquid output (calculation unit 11), third waste liquid output (calculation unit 12), fourth waste liquid output (calculation unit 13), and collector output (calculation unit 14). Both input and output information are curves showing the changes in tracked protein concentration, non-tracked protein concentration, and mobile phase pH over time. The inputs for tracked and non-tracked materials differ in the fifth calculation unit.

[0028] like Figure 5 As shown, in the connection matrix of the four-column operation mode of periodic countercurrent chromatography, the indices of the computational units located in the same row of the matrix represent mutual connections, specifically including: 1) The first and second columns undergo a connection loading step, the third column undergoes a pre-elution regeneration step, and the fourth column undergoes a post-elution regeneration step. At this point, the connection matrix is: Connection IL-1（4c-pcc） , means as follows: ; 2) The first and second columns undergo a connection loading step, the third column undergoes an elution step, and the fourth column undergoes a post-elution regeneration step. At this point, the connection matrix is: Connection IL-2 (4c-pcc) , means as follows: ; 3) The first and third columns undergo a connection flushing step, the second column undergoes a disconnection loading step, and the fourth column waits. At this point, the connection matrix is: ConnectionDL (4c-pcc) , means as follows: ; Similarly, the four-column periodic countercurrent tomography process also includes multiple periodically executed switching processes. The aforementioned is the component connection sequence within an initial switch; the second switch replaces the position of the first computing unit with the second computing unit, the second computing unit with the third computing unit, the third computing unit with the fourth computing unit, and the fourth computing unit with the first computing unit; the third switch replaces the position of the first computing unit with the second computing unit, the second computing unit with the third computing unit, the third computing unit with the fourth computing unit, and the fourth computing unit with the first computing unit; the fourth switch replaces the position of the first computing unit with the second computing unit, the second computing unit with the third computing unit, the third computing unit with the fourth computing unit, and the fourth computing unit with the first computing unit; the next switch returns to the component connection sequence within the initial switch, and the subsequent repeated switching processes realize the simulation of the periodic countercurrent tomography process.

[0029] In the simulation calculation of the residence time distribution model of four-column periodic countercurrent chromatography, the labeled protein enters the system from the fifth computational unit and leaves the system from the fourteenth computational unit. The cumulative residence time distribution curve is shown in the figure. F ( t This is used to measure the proportion of labeled proteins that have left the system during the time period from 0 to t, out of the total labeled proteins. The specific formula is: in: c 1[Unit_11]( t ( ) is the curve showing the change in concentration of the labeled protein at the fourteenth computational unit over time, in mg / mL; c 1[Unit_5]( t ( ) is the curve showing the change in concentration of the labeled protein at the fifth computational unit over time, in mg / mL; Q in ( t (This is a curve showing the elution flow rate over time at the fourteenth calculation unit, in mL / min.) Q out ( t The curve shows the change in sample loading rate over time at the fifth calculation unit, in mL / min.

[0030] The retention time distribution models for single-column and periodic countercurrent chromatography, as described above, can be used to construct material tracking methods and formulate and implement diversion schemes, specifically as follows: Figure 1As shown, it includes the following steps: Step 1: Fit the chromatographic residence time distribution model using the results of the static adsorption experiment and the chromatographic experiment process curve, calibrate the chromatographic residence time distribution model, and obtain the parameters of the chromatographic residence time distribution model. The model parameters include the adsorption parameters of the medium, the mass transfer related parameters, and the parameters related to the influence of the flow rate on adsorption. Step 2: Substitute the parameters of the chromatography residence time distribution model obtained in Step 1, the actual chromatography operation parameters, and the start and duration of the tracked material into the chromatography residence time distribution model to obtain the single-column residence time distribution curve of the specific tracked material; Step 3: Substitute the single-column residence time distribution curve, periodic countercurrent chromatography operating parameters, and mode predicted in Step 2 into the periodic countercurrent chromatography model framework to obtain the residence time distribution curve of the specific tracked material in periodic countercurrent chromatography; the periodic countercurrent chromatography model framework is compatible with different modes of periodic countercurrent chromatography, including three-column mode and four-column mode. Step 4: Define the start and duration of the tracking material, repeat steps 2 and 3 to obtain the residence time distribution curve of the tracking material in periodic countercurrent chromatography with different start and duration times, calculate the proportion of the tracking material in each elution peak, and realize material tracking in periodic countercurrent chromatography. Step 5: Based on the required product quality requirements, determine the acceptance limit for real-time release. Combined with the real-time release acceptance limit that meets the product quality requirements, determine the elution peaks in the elution peaks where the proportion of tracked material exceeds the release acceptance limit and divert them, thereby achieving real-time quality control and release of the product.

[0031] To make the technical effects of the embodiments of the present invention more apparent, the following description, in conjunction with figures and illustrations, illustrates the implementation process of the invention.

[0032] Example 1: Calibration of Chromatographic Residence Time Distribution Model The chromatographic residence time distribution model of this invention is applicable to different chromatographic media, including but not limited to Protein A affinity chromatographic media. The following description uses Cytiva's MabSelect SuRe LX medium as an example, and the chromatographic column is a Cytiva Tricon 5 / 50 chromatographic column. The chromatographic residence time distribution model is applicable to all proteins, and the following description uses IgG protein as an example.

[0033] (1) Fitting of medium adsorption parameters Static adsorption equilibration experiments were conducted on proteins with concentrations ranging from 0.5 mg / mL to 10 mg / mL. The adsorption capacities of the medium corresponding to different liquid-phase equilibrium protein concentrations were obtained. Langmuir adsorption isotherm equations were fitted, yielding a dissociation equilibrium constant of 4.83 × 10⁻⁶ for the protein in the equilibration buffer.-3 g / L.

[0034] Figure 6 This is the fitting result of the adsorption isotherm.

[0035] (2) Fitting of mass transfer related parameters Protein chromatography separation experiments were performed with a sample protein concentration of 2 mg / ml, a sample loading flow rate and a wash flow rate of 150 cm / h, a sample protein volume of 43 times the column volume, and a wash volume of 5 times the column volume.

[0036] The initial values ​​for the mass transfer-related parameters are: axial diffusion coefficient 1×10⁻⁶. -7 m 2 / s, liquid film mass transfer coefficient 20×10 -6 m / s, solid-phase mass transfer coefficient within particles 4×10 -15 m 2 / s, intraparticle liquid phase mass transfer coefficient 1×10 -12 m 2 / s, saturated adsorption capacity 130 mg / mL, adsorption constant of protein in equilibrium buffer 10 m 3 / (mol·s).

[0037] As known from Example 1, the dissociation equilibrium constant of the protein in the equilibration buffer is 4.83 × 10⁻⁶. -3 g / L. Substituting the initial model parameters and experimental operating parameters into the chromatography residence time distribution model, the protein breakthrough and rinsing curves were calculated and compared with the experimental curves. Using the root mean square error of both as the objective function, a genetic algorithm was used to minimize the objective function, and the fitted mass transfer related parameters were obtained as follows: axial diffusion coefficient 3.57 × 10⁻⁶ g / L. -7 m 2 / s, liquid film mass transfer coefficient 13.8×10 -6 m / s, intraparticle solid-phase mass transfer coefficient 2.34×10 -15 m 2 / s, intraparticle liquid phase mass transfer coefficient 5.68×10 -12 m 2 / s, saturated adsorption capacity 129.3 mg / mL, protein adsorption constant in equilibrium buffer 9.12 m 3 / (mol·s).

[0038] Figure 7 To compare the breakthrough and flushing curves of the chromatography experiment with the model calculation results.

[0039] (3) Fitting of pH-related parameters Protein chromatography separation experiments were performed. First, protein was loaded and washed. The protein concentration was 2 mg / ml, and both the loading and washing flow rates were 150 cm / h. The loading protein volume was 43 times the column volume, and the washing volume was 5 times the column volume. Then, elution was performed with the following parameters: flow rate 150 cm / h, elution volume 5 times the column volume, and pH of the elution buffer 3.2.

[0040] The initial values ​​for pH-related parameters are: the effect of hydrogen ion concentration on the adsorption constant ( γ The value of ) is 1, and the effect of hydrogen ion concentration on the dissociation constant is ( β The value is -1.

[0041] As known from Example 1, the dissociation equilibrium constant of the protein in the equilibration buffer is 4.83 × 10⁻³ g / L, and as known from Example 2, the axial diffusion coefficient is 3.57 × 10⁻³ g / L. -7 m 2 / s, liquid film mass transfer coefficient 13.8×10 -6 m / s, intraparticle solid-phase mass transfer coefficient 2.34×10 -15 m 2 / s, intraparticle liquid phase mass transfer coefficient 5.68×10 -12 m 2 / s, saturated adsorption capacity 129.3 mg / mL, protein adsorption constant in equilibrium buffer 9.12 m 3 / (mol·s). Substituting the initial model parameters and relevant experimental parameters into the chromatographic residence time distribution model, the protein elution curve was calculated and compared with the experimental data. Using the root mean square error of both as the objective function, a genetic algorithm was used to minimize the objective function, and the pH-related parameters were fitted as follows: the effect of hydrogen ion concentration on the adsorption constant ( γ The value is 0.93, and the effect of hydrogen ion concentration on the dissociation constant is ( β The value is -1.87.

[0042] Figure 8 To compare the elution curves of the chromatography experiment with the results calculated by the model.

[0043] Example 2: Calculation of residence time distribution curve in periodic countercurrent chromatography The residence time distribution curves of the tracking material in the chromatography column with different start times and durations were simulated and calculated to obtain the residence time distribution curve of periodic countercurrent chromatography. The model calculation results were verified using a tracer experiment. A fluorescently labeled protein was injected at the inlet of the periodic countercurrent chromatography column as the tracking material, and the fluorescence intensity was detected at the outlet. The residence time distribution curve of the periodic countercurrent chromatography was experimentally measured. The model of this invention can predict and calculate the residence time distribution curves of the tracking material with different start times and durations in periodic countercurrent chromatography under different operating modes, including but not limited to three-column and four-column periodic countercurrent chromatography. The following uses three-column periodic countercurrent chromatography as an example: (1) The chromatographic column used in Example 1 was used to conduct a tracer experiment of three-column periodic countercurrent chromatography. The operating parameters of periodic countercurrent chromatography were as follows: the retention time for connecting the sample was 2 min, the retention time for disconnecting the sample was 2 min, the duration of connecting the sample and media cleaning and regeneration was 49 min, and the duration of disconnecting the sample and connecting the rinse was 8 min. The starting time for injecting the tracking material into the sample pump inlet was the moment when the sample was first loaded, and the duration was 1 min.

[0044] The model parameters calibrated in Example 1 were used: protein dissociation equilibrium constant in equilibration buffer 4.83 × 10⁻³ g / L, axial diffusion coefficient 3.57 × 10⁻³ g / L. -7 m 2 / s, liquid film mass transfer coefficient 13.8×10 -6 m / s, intraparticle solid-phase mass transfer coefficient 2.34×10 -15 m 2 / s, intraparticle liquid phase mass transfer coefficient 5.68×10 -12 m 2 / s, saturated adsorption capacity 129.3 mg / mL, protein adsorption constant in equilibrium buffer 9.12 m 3 / (mol·s), the effect of hydrogen ion concentration in the buffer solution on the adsorption constant ( γ The value is 0.93, and the effect of hydrogen ion concentration in the buffer solution on the dissociation constant is ( β The value is -1.87. The residence time distribution curve for this specific tracked material was calculated, and the prediction results are as follows: Figure 9 As shown in (a) of the diagram.

[0045] (2) The chromatographic column used in Example 1 was used to conduct a tracer experiment of three-column periodic countercurrent chromatography. The operating parameters of periodic countercurrent chromatography were as follows: retention time for sample loading was 2 min, retention time for sample unloading was 2 min, duration of sample loading and media cleaning and regeneration was 49 min, and duration of sample unloading and rinsing was 8 min. The starting time for injecting the tracking material at the sample pump inlet was when the sample volume was 100 times the column volume, and the duration was 1 min.

[0046] The model parameters calibrated in Example 1 were used: protein dissociation equilibrium constant in equilibration buffer 4.83 × 10⁻³ g / L, axial diffusion coefficient 3.57 × 10⁻³ g / L. -7 m 2 / s, liquid film mass transfer coefficient 13.8×10 -6 m / s, intraparticle solid-phase mass transfer coefficient 2.34×10 -15 m 2 / s, intraparticle liquid phase mass transfer coefficient 5.68×10 -12 m 2 / s, saturated adsorption capacity 129.3 mg / mL, protein adsorption constant in equilibrium buffer 9.12 m 3 / (mol·s), the effect of hydrogen ion concentration in the buffer solution on the adsorption constant ( γ The value is 0.93, and the effect of hydrogen ion concentration in the buffer solution on the dissociation constant is ( β The value is -1.87. The residence time distribution curve for this specific tracked material was calculated, and the prediction results are as follows: Figure 9 As shown in (b) of the diagram.

[0047] (3) The chromatographic column used in Example 1 was used to conduct a tracer experiment of three-column periodic countercurrent chromatography. The operating parameters of periodic countercurrent chromatography were as follows: retention time for sample loading was 2 min, retention time for sample unloading was 2 min, duration of sample loading and media cleaning and regeneration was 49 min, and duration of sample unloading and rinsing was 8 min. The starting time for injecting the tracking material at the sample pump inlet was when the sample volume was 220 times the column volume, and the duration was 2 min.

[0048] The model parameters calibrated in Example 1 were used: protein dissociation equilibrium constant in equilibration buffer 4.83 × 10⁻³ g / L, axial diffusion coefficient 3.57 × 10⁻³ g / L. -7 m 2 / s, liquid film mass transfer coefficient 13.8×10 -6 m / s, intraparticle solid-phase mass transfer coefficient 2.34×10 -15 m 2 / s, intraparticle liquid phase mass transfer coefficient 5.68 × 10⁻⁶-12 m 2 / s, saturated adsorption capacity 129.3 mg / mL, protein adsorption constant in equilibrium buffer 9.12 m 3 / (mol·s), the effect of hydrogen ion concentration in the buffer solution on the adsorption constant ( γ The value is 0.93, and the effect of hydrogen ion concentration in the buffer solution on the dissociation constant is ( β The value is -1.87. The residence time distribution curve for this specific tracked material was calculated, and the prediction results are as follows: Figure 9 As shown in (c) in the figure.

[0049] Example 3: Material Tracking Strategy for Periodic Countercurrent Chromatography Substituting the tracking materials with different start time parameters (0-3420 s) and duration parameters (0-30780 s) into the residence time distribution model of three-column periodic countercurrent chromatography, and repeating the calculation process in Example 2, the distribution of different tracking materials in the 12 elution peaks can be obtained, thus achieving material tracking. The material tracking results are as follows: Figure 10 As shown in the examples below: (1) When the start time parameter of the tracked material is 1000 s and the duration parameter is 10000 s, the proportions of the tracked material in the 12 elution peaks are 17.5%, 66.8%, 72.9%, 82.78%, 26.4%, 4.8%, 0.7%, 0.07%, 0.005%, 0%, 0% and 0%, respectively.

[0050] (2) When the start time parameter of the tracked material is 1000 s and the duration parameter is 20000 s, the proportions of the tracked material in the 12 elution peaks are 21.9%, 83.5%, 92.8%, 99.8%, 100%, 100%, 27.9%, 3.8%, 0.8%, 0.07%, 0.007% and 0%, respectively.

[0051] (3) When the start time parameter of the tracked material is 2000 s and the duration parameter is 20000 s, the proportions of the tracked material in the 12 elution peaks are 10.5%, 74.6%, 100%, 100%, 98.4%, 96.5%, 46.3%, 6.8%, 0.7%, 0.12%, 0.008% and 0.002%, respectively.

[0052] Example 4: Real-time release strategy for periodic countercurrent chromatography Based on the acceptance limit for real-time release and in conjunction with the material tracking strategy, the number of elution peaks that need to be diverted to meet the real-time release requirements is determined. In this example, the material tracking strategy in Example 3 is used to track disturbances and non-conforming materials. The following further explains how to achieve real-time release using the material tracking strategy in Example 3: (1) When the start time parameter of the disturbance or nonconforming material is 1000 s and the duration parameter is 10000 s: at the acceptance limit ( T When the value is 0.1, which requires the final product to contain less than 10% non-conforming materials, it is necessary to split the elution peaks into 5 separate peaks; at the acceptable limit ( T When the value is 0.01, which requires the final product to contain less than 1% non-conforming material, six elution peaks need to be split; at the acceptable limit ( T When the value is 0.001, which requires the final product to contain less than 0.1% non-conforming material, it is necessary to split 7 elution peaks; at the acceptable limit ( T When the value is 0.0001, it is required that the final product contains less than 0.01% non-conforming material, which requires splitting into 8 elution peaks.

[0053] (2) When the start time parameter of the disturbance or nonconforming material is 1000s and the duration parameter is 20000s: at the acceptance limit ( T When the value is 0.1, which requires the final product to contain less than 10% non-conforming materials, it is necessary to split 7 elution peaks; at the acceptable limit ( T When the value is 0.01, which requires the final product to contain less than 1% non-conforming material, it is necessary to split the elution peaks into 8 segments; at the acceptable limit ( T When the value is 0.001, which requires the final product to contain less than 0.1% non-conforming material, it is necessary to split 9 elution peaks; at the acceptable limit ( T When the value is 0.0001, it is required that the final product contains less than 0.01% non-conforming material, which necessitates the diversion of 10 elution peaks.

[0054] (3) When the start time parameter of the disturbance or nonconforming material is 1000 s and the duration parameter is 20000 s: at the acceptance limit ( T When the value is 0.1, which requires the final product to contain less than 10% non-conforming materials, it is necessary to split 7 elution peaks; at the acceptable limit ( T When the value is 0.01, which requires the final product to contain less than 1% non-conforming material, it is necessary to split the elution peaks into 8 segments; at the acceptable limit ( T When the value is 0.001, which requires the final product to contain less than 0.1% non-conforming material, it is necessary to split 9 elution peaks; at the acceptable limit ( TWhen the value is 0.0001, it is required that the final product contains less than 0.01% non-conforming material, which necessitates the diversion of 10 elution peaks.

[0055] For other disturbances or non-conforming materials that occur at a specific time and last for a specific duration, a real-time release strategy can be formulated based on acceptance limits. The results of the real-time release strategy are as follows: Figure 11 As shown.

[0056] It should be understood that the exemplary embodiments described herein are illustrative and not restrictive. Although one or more embodiments of the invention have been described in conjunction with the accompanying drawings, those skilled in the art will understand that various changes in form and detail may be made without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. A method for material tracking and real-time release in periodic countercurrent chromatography, characterized in that, Includes the following steps: Step 1: Fit the chromatographic residence time distribution model using the results of the static adsorption experiment and the chromatographic experiment process curve, calibrate the chromatographic residence time distribution model, and obtain the parameters of the chromatographic residence time distribution model. The model parameters include the adsorption parameters of the medium, the mass transfer related parameters, and the parameters related to the influence of the flow rate on adsorption. Step 2: Substitute the parameters of the chromatography residence time distribution model obtained in Step 1, the actual chromatography operation parameters, and the start and duration of the tracked material into the chromatography residence time distribution model to obtain the single-column residence time distribution curve of the specific tracked material; Step 3: Substitute the single-column residence time distribution curve, periodic countercurrent chromatography operating parameters, and mode predicted in Step 2 into the periodic countercurrent chromatography model framework to obtain the residence time distribution curve of the specific tracked material in periodic countercurrent chromatography; the periodic countercurrent chromatography model framework is compatible with different modes of periodic countercurrent chromatography, including three-column mode and four-column mode. Step 4: Define the start and duration of the tracking material, repeat steps 2 and 3 to obtain the residence time distribution curve of the tracking material in periodic countercurrent chromatography with different start and duration times, calculate the proportion of the tracking material in each elution peak, and realize material tracking in periodic countercurrent chromatography. Step 5: Based on the required product quality requirements, determine the acceptance limit for real-time release. Combined with the real-time release acceptance limit that meets the product quality requirements, determine the elution peaks in the elution peaks where the proportion of tracked material exceeds the release acceptance limit and divert them, thereby achieving real-time quality control and release of the product.

2. The material tracking and real-time release method for periodic countercurrent chromatography as described in claim 1, characterized in that, The calibration of the chromatography residence time distribution model further includes the following steps: Substitute the initial values ​​of the medium adsorption parameters in the chromatographic residence time distribution model into the Langmuir adsorption model, calculate the adsorption isotherm, and compare the calculated results with the experimentally obtained adsorption isotherm. Change the medium adsorption parameters to minimize the root mean square error of both, and obtain the medium adsorption parameters in the chromatographic residence time distribution model. Substitute the experimental operating parameters of the breakthrough and rinsing steps in the chromatography experiment, the calculated medium adsorption parameters, and the initial values ​​of the mass transfer related parameters in the chromatography residence time distribution model into the chromatography residence time distribution model. Compare the calculation results with the breakthrough and rinsing curves obtained from the chromatography experiment. Change the mass transfer related parameters to minimize the root mean square error of both, and obtain the mass transfer related parameters in the chromatography residence time distribution model. The experimental operating parameters of the elution step in the chromatography separation process, the calculated medium adsorption parameters and mass transfer related parameters, and the initial values ​​of the flow relative adsorption related parameters in the chromatography residence time distribution model were substituted into the chromatography residence time distribution model. The calculation results were compared with the experimentally obtained elution curves. The flow relative adsorption related parameters were changed to minimize the root mean square error of both, and the flow relative adsorption related parameters in the chromatography residence time distribution model were obtained.

3. The material tracking and real-time release method for periodic countercurrent chromatography as described in claim 1, characterized in that, The periodic countercurrent chromatography operating parameters mentioned in step 3 are the process parameters that need to be set for periodic countercurrent chromatography, including the flow rate and time for connecting and disconnecting the sample, elution, and each regeneration step.

4. The material tracking and real-time release method for periodic countercurrent chromatography as described in claim 1, characterized in that, The periodic countercurrent chromatography model framework established based on the three-column model includes eleven computational units. These eleven computational units are: residence time distribution models for the three chromatography columns, four inlet computational units, and four outlet computational units. The first computational unit is for the residence time distribution model of the first chromatography column, the second computational unit is for the residence time distribution model of the second chromatography column, the third computational unit is for the residence time distribution model of the third chromatography column, the fourth computational unit is for the sample pump inlet computational unit, the fifth computational unit is for the regeneration pump inlet computational unit, the sixth computational unit is for the connection rinsing inlet computational unit, the seventh computational unit is for the elution pump inlet computational unit, the eighth computational unit is for the first waste liquid outlet computational unit, the ninth computational unit is for the second waste liquid outlet computational unit, the tenth computational unit is for the third waste liquid outlet computational unit, and the eleventh computational unit is for the collector outlet computational unit.

5. The material tracking and real-time release method for periodic countercurrent chromatography as described in claim 4, characterized in that, In the three-column periodic countercurrent tomography model framework, the components adopt different connection sequences at different stages of the periodic countercurrent tomography process. The three-column periodic countercurrent tomography process includes multiple switching processes executed periodically. A single switching process includes, in sequence: 1) The first and second columns are connected for sample loading, and the third column is connected for regeneration before elution. The connection order is as follows: the fourth calculation unit is connected to the first calculation unit, the first calculation unit is connected to the second calculation unit, the second calculation unit is connected to the eighth calculation unit, the fifth calculation unit is connected to the third calculation unit, the third calculation unit is connected to the ninth calculation unit, the sixth calculation unit is connected to the tenth calculation unit, and the seventh calculation unit is connected to the eleventh calculation unit. 2) The first and second columns are connected for the sample loading step, and the third column is connected for the elution step. The connection order is as follows: the fourth calculation unit is connected to the first calculation unit, the first calculation unit is connected to the second calculation unit, the second calculation unit is connected to the eighth calculation unit, the fifth calculation unit is connected to the ninth calculation unit, the sixth calculation unit is connected to the tenth calculation unit, the seventh calculation unit is connected to the third calculation unit, and the third calculation unit is connected to the eleventh calculation unit. 3) The first and second columns are connected for sample loading, and the third column is eluted and then regenerated. The connection order is as follows: the fourth calculation unit is connected to the first calculation unit, the first calculation unit is connected to the second calculation unit, the second calculation unit is connected to the eighth calculation unit, the fifth calculation unit is connected to the third calculation unit, the third calculation unit is connected to the ninth calculation unit, the sixth calculation unit is connected to the tenth calculation unit, and the seventh calculation unit is connected to the eleventh calculation unit. 4) The first and third columns are connected and flushed, and the second column is disconnected and loaded. The connection sequence is as follows: the fourth calculation unit is connected to the second calculation unit, the second calculation unit is connected to the eighth calculation unit, the fifth calculation unit is connected to the ninth calculation unit, the sixth calculation unit is connected to the first calculation unit, the first calculation unit is connected to the third calculation unit, the third calculation unit is connected to the tenth calculation unit, and the seventh calculation unit is connected to the eleventh calculation unit. The above describes the component connection sequence within an initial switch. The second switch replaces the first computing unit with the second computing unit, the second computing unit with the third computing unit, and the third computing unit with the first computing unit. The third switch replaces the first computing unit with the second computing unit, the second computing unit with the third computing unit, and the third computing unit with the first computing unit. The next switch returns to the component connection sequence within the initial switch, and the subsequent repeated switching process simulates the periodic countercurrent tomography process.

6. The material tracking and real-time release method for periodic countercurrent chromatography as described in claim 1, characterized in that, The periodic countercurrent chromatography operation model framework established based on the four-column model comprises fourteen computational units: four column residence time distribution models, five inlet computational units, and five outlet computational units. The first computational unit is for the first column residence time distribution model, the second for the second column residence time distribution model, the third for the third column residence time distribution model, the fourth for the fourth column residence time distribution model, the fifth for the sample pump inlet, the sixth for the first regeneration pump inlet, the seventh for the second regeneration pump inlet, the eighth for the connection rinse inlet, the ninth for the elution pump inlet, the tenth for the first waste liquid outlet, the eleventh for the second waste liquid outlet, the twelfth for the third waste liquid outlet, the thirteenth for the fourth waste liquid outlet, and the fourteenth for the collector outlet. In different operational steps, the first through fourteenth computational units are connected in a specific order.

7. The material tracking and real-time release method for periodic countercurrent chromatography as described in claim 6, characterized in that, In the model framework established by the four-column periodic countercurrent chromatography operation mode, the components adopt different connection sequences at different stages of the periodic countercurrent chromatography process. The four-column periodic countercurrent chromatography process includes multiple switching processes executed periodically. A single switching process includes, in sequence: 1) The first and second columns are connected for sample loading, the third column is regenerated before elution, and the fourth column is regenerated after elution. The connection order is as follows: the fifth calculation unit is connected to the first calculation unit, the first calculation unit is connected to the second calculation unit, the second calculation unit is connected to the tenth calculation unit, the sixth calculation unit is connected to the third calculation unit, the third calculation unit is connected to the eleventh calculation unit, the seventh calculation unit is connected to the fourth calculation unit, the fourth calculation unit is connected to the twelfth calculation unit, the eighth calculation unit is connected to the thirteenth calculation unit, and the ninth calculation unit is connected to the fourteenth calculation unit. 2) The first and second columns are connected for sample loading, the third column is used for elution, and the fourth column is used for elution and regeneration. The connection order is as follows: the fifth calculation unit is connected to the first calculation unit, the first calculation unit is connected to the second calculation unit, the second calculation unit is connected to the tenth calculation unit, the sixth calculation unit is connected to the eleventh calculation unit, the seventh calculation unit is connected to the fourth calculation unit, the fourth calculation unit is connected to the twelfth calculation unit, the eighth calculation unit is connected to the thirteenth calculation unit, the ninth calculation unit is connected to the third calculation unit, and the third calculation unit is connected to the fourteenth calculation unit. 3) The first and third columns are connected and flushed, the second column is disconnected and loaded, and the fourth column is left to wait. The connection sequence is as follows: the fifth calculation unit is connected to the second calculation unit, the second calculation unit is connected to the tenth calculation unit, the sixth calculation unit is connected to the eleventh calculation unit, the seventh calculation unit is connected to the twelfth calculation unit, the eighth calculation unit is connected to the first calculation unit, the first calculation unit is connected to the third calculation unit, the third calculation unit is connected to the thirteenth calculation unit, and the ninth calculation unit is connected to the fourteenth calculation unit. The above describes the component connection sequence within an initial switch. The second switch replaces the first computing unit with the second computing unit, the second with the third computing unit, the third with the fourth computing unit, and the fourth with the first computing unit. The third switch replaces the first computing unit with the second computing unit, the second with the third computing unit, the third with the fourth computing unit, and the fourth with the first computing unit. The fourth switch replaces the first computing unit with the second computing unit, the second with the third computing unit, the third with the fourth computing unit, and the fourth with the first computing unit. The next switch returns to the component connection sequence within the initial switch, and this repeated switching process simulates a periodic countercurrent tomography process.

8. A method for material tracking and real-time release in periodic countercurrent chromatography as described in claim 4 or 6, characterized in that, The input and output information of both the input and output calculation units are curves showing the changes in the concentration of the tracked material, the concentration of the non-tracked material, and the pH of the mobile phase over time.

9. A method for material tracking and real-time release in periodic countercurrent chromatography as described in claim 4 or 6, characterized in that, In the chromatographic residence time distribution model, the parameters related to medium adsorption, mass transfer, and the influence of flow relative adsorption on the tracked and non-tracked materials are set the same. However, in the periodic countercurrent chromatography model framework, the inputs of the tracked and non-tracked materials in the sample pump inlet calculation unit are different.

10. The material tracking and real-time release method for periodic countercurrent chromatography as described in claim 1, characterized in that, The chromatography residence time distribution model includes two sub-models: a general rate model and a two-component Langmuir adsorption model modified by mobile phase pH. The general rate model is used to describe the convection and diffusion behavior of materials in a chromatography column, and its equations are as follows: ； ； in: c This represents the protein concentration in the bulk liquid phase. c p This represents the protein concentration in the liquid phase within the media particles. c 0 This refers to the concentration of the sampled protein. q This refers to the concentration of solid-phase protein. t For time, D ax denoted as the axial dispersion coefficient within the column. x This represents the axial distance within the column. u Void velocity, ε The porosity within the column; ε p The porosity of the medium particles; k f The mass transfer coefficient of the liquid film is . r The radial distance within the medium particles. r p Where is the particle radius, D p The liquid-phase diffusion coefficient within the medium particles. D s The diffusion coefficient of the inner surface of the medium particles is denoted as . L Let be the length of the column, where the subscript is . i A value of 0 or 1 represents unlabeled protein components and labeled protein components, respectively. Unlabeled protein components are non-tracked materials, while labeled protein components are tracked materials. The pH-modified two-component Langmuir adsorption model is used to describe the exchange behavior between the material and the adsorbate in the chromatography column, and the equation is as follows: ； in: q max This represents the saturation adsorption capacity. k a This is the adsorption constant of the protein in the equilibrium buffer. k d This is the dissociation constant of the protein in the equilibrium buffer. c ( H + () represents the concentration of hydrogen ions in the buffer solution. c ( H + ) eq This represents the concentration of hydrogen ions in the equilibrium buffer solution. γ and β These are used to describe the effects of hydrogen ion concentration in solution on the adsorption constant and dissociation constant, respectively. The mass transfer related parameters are: D ax , D p , D s and k f ; Media adsorption-related parameters include q max , k a and k d The relevant parameters for the influence of flow on adsorption are: γ and β .

Images