Methods for cross-scale process development of isolation processes

Abstract

A method for optimizing at least one process parameter of a separation process for at least one target substance from at least one impurity using a chromatography system, comprising the steps of: a) providing a set of preliminary system-specific parameters suitable for describing the concentration transport of at least one target substance and at least one impurity in a preliminary chromatography system using a system-specific transport model, wherein the preliminary chromatography system does not include any chromatography column but includes a column shortcut; b) providing a set of preliminary column-specific parameters suitable for describing the concentration transport of at least one target substance and at least one impurity in only a preliminary chromatography column containing a preliminary chromatography matrix using a column-specific transport model; c) using an sorption model, the set of preliminary system-specific parameters, and the set of preliminary column-specific parameters to optimize at least one process parameter of the separation of at least one target substance into or from a preliminary chromatography matrix contained in a preliminary chromatography column. A step of determining a set of sorption-specific parameters suitable for describing the adsorption and / or desorption of one target substance and at least one impurity, wherein the preliminary chromatography system includes the preliminary chromatography column of step b) instead of the column shortcut of step a), d) a set of system-specific parameters suitable for describing the concentration transport of at least one target substance and at least one impurity in the chromatography system using a system-specific transport model, wherein the chromatography system does not include any chromatography column but includes a column shortcut, e) a set of column-specific parameters suitable for describing the concentration transport of at least one target substance and at least one impurity in chromatography columns including a chromatography matrix using a column-specific transport model, f) the following parameters: using the set of system-specific parameters of step d), the set of column-specific parameters of step e), and the set of sorption-specific parameters of step c),A step for optimizing at least one process parameter in silico, wherein the chromatography system includes a step of step e) with a chromatography column instead of a column shortcut in step d), and the sum of the volume of the chromatography system without any chromatography column and the volume of the chromatography column is different from the sum of the volume of the spare chromatography system without any chromatography column and the volume of the spare chromatography column.

Description

[Technical Field]

[0001] The present invention relates to a method for optimizing the separation process of a target substance from impurities using a chromatography system. [Background technology]

[0002] Chromatography, particularly liquid chromatography, plays a crucial role in the downstream bioprocessing of target substances, especially in the purification of therapeutic proteins, antibiotics, hormones (e.g., insulin and human growth hormone), blood products, antibodies, vaccines, enzymes, nucleic acids, viruses, viral vectors, virus-like particles, cells, natural fragrances and flavor compounds, and nutrient proteins. Liquid chromatography separation techniques provide mild conditions favorable to such sensitive molecules. Furthermore, chromatographic separation techniques offer a wide range of possible interaction modes, making them applicable to diverse separation problems. Moreover, chromatographic separation techniques have a long history in industry and regulatory bodies. Therefore, they are widely adopted and commonly used. The global chromatography market size was valued at $8.706 billion in 2020 and is projected to reach $15.339 billion by 2030.

[0003] Downstream bioprocessing involves complex setups of various separation processes, including chromatography. Therefore, it is desirable to optimize such setups to achieve the highest possible purity and yield. In particular, the chromatography process included in downstream bioprocessing has been a subject of such consideration. Because optimization involves the execution of multiple test processes, optimizing a chromatography setup on an industrial scale results in unreasonably large amounts of materials, such as matrix, diluents, tracers, target substances, and test molecules, as well as long execution times. Therefore, a well-established solution to this problem is to optimize the chromatography setup on a laboratory scale and then scale up this optimized process to industrial production scale. To further minimize the need for materials and time, it has become established in recent years to move as much of the optimization work of such setups as possible to virtual environments such as in silico simulations.

[0004] While scaling up requires fewer runs than optimization, the process of scaling up results optimized at the laboratory scale to an industrial scale involves experimental execution of industrial-scale systems. Therefore, scaling up liquid chromatography separation processes from laboratory scale to industrial scale remains time-consuming and expensive, because the volumes of chromatography columns used at industrial scale can reach several hundred liters, each requiring large quantities of materials such as matrix, diluents, tracers, target substances, and test molecules.

[0005] Scale-up is typically achieved linearly by increasing the column diameter while keeping other parameters constant, such as bed height, flow rate, and the ratio of diluent volume to column bed volume applied at various stages of the process (loading, elution, washing). However, industrial-scale chromatography columns are usually only available in discontinuous sizes with respect to diameter. Therefore, linear scale-up methods have the further drawback of being less flexible. To address this lack of flexibility, a scaling process based on a constant contact time (residence time) has been proposed (see Hansen, EB, "Chromatographic Scale-Up on a Volume Basis." Preparative Chromatography for Separation of Proteins, 2017, https: / / doi.org / 10.1002 / 9781119031116.ch7), in which the column bed height is introduced as a process design variable rather than a fixed parameter. Since bed height is a continuous variable, in contrast to diameter, it can be used to obtain the exact volume required at any scale. However, such methods still introduce complexity into the scale-up process.

[0006] A third drawback of the approach of optimizing at the laboratory level and scaling up to industrial scale is that the optimization of the separation process is not carried out at the industrial scale. It is well known that process performance, i.e., separation efficiency, generally improves as the scale increases because dead volume becomes relatively smaller. However, when experimental workflows are used for process development, this positive effect cannot be accurately predicted. As a result, the scale-up process is often over-optimized with respect to purity, and consequently, results in suboptimal yields.

[0007] In light of these issues, it has been shown that cross-scale quality assessment of chromatographic separations using mechanistic modeling is feasible (see: Saleh D et al., "Cross-scale quality assessment of a mechanistic cation exchange chromatography model", Biotechnology Prog.2021, 37(1):e3081, doi:10.1002 / btpr.3081). [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Hansen, EB, “Chromatographic Scale-Up on a Volume Basis.” Preparative Chromatography for Separation of Proteins, 2017, https: / / doi.org / 10.1002 / 9781119031116.ch7 [Non-Patent Document 2] Saleh D et al., "Cross-scale quality assessment of a mechanistic cation exchange chromatography model", Biotechnology Prog.2021, 37(1):e3081, doi:10.1002 / btpr.3081) [Non-Patent Document 3] “Preparative Chromatography” edited by Henner Schmidt-Traub (ISBN978-3-527-34486-4) [Non-Patent Document 4] T. Hahn et al., "Predictive scaling of fiber-based protein A capture chromatography using mechanistic modeling," Biotechnology and Bioengineering, May 20, 2023, pp. 1-12 (https: / / doi.org / 10.1002 / bit.28434) [Overview of the project] [Problems that the invention aims to solve]

[0009] However, scaling procedures to industrial production scale must still be implemented, especially in experiments, and as outlined above, this results in demands for large amounts of materials and time, low flexibility, and a high likelihood of errors. [Means for solving the problem]

[0010] Therefore, an object of the present invention is to provide a method for cross-scale process development in which the optimization of a chromatography process is carried out directly at a desired scale without requiring prior optimization at a laboratory scale.

[0011] Now, remarkably, a method for optimizing at least one process parameter of a separation process for at least one target substance from at least one impurity using a chromatography system, comprising the following steps: a) A step of providing a set of preliminary system-specific parameters suitable for describing the concentration transport of at least one target substance and at least one impurity in a preliminary chromatography system using a system-specific transport model, wherein the preliminary chromatography system does not include any chromatography column but includes a column shortcut. b) A step of providing a set of pre-column specific parameters suitable for describing the concentration transport of at least one target substance and at least one impurity in a pre-chromatographic column containing a pre-chromatographic matrix, using a column-specific transport model. c) A step of determining a set of sorption-specific parameters suitable for describing the adsorption and / or desorption of at least one target substance and at least one impurity to and from a pre-chromatographic matrix contained in a pre-chromatographic column, using an sorption model, a set of pre-system-specific parameters, and a set of pre-column-specific parameters, wherein the pre-chromatographic system includes the pre-chromatographic column of step b) instead of the column shortcut of step a), d) A step of providing a set of system-specific parameters suitable for describing the concentration transport of at least one target substance and at least one impurity in a chromatography system using a system-specific transport model, wherein the chromatography system does not include any chromatography column but includes a column shortcut. e) A step of providing a set of column-specific parameters suitable for describing the concentration transport of at least one target substance and at least one impurity in a chromatography column containing a chromatography matrix, using a column-specific transport model. f) A step of optimizing at least one process parameter in silico using the following parameters: a set of system-specific parameters from step d), a set of column-specific parameters from step e), and a set of sorption-specific parameters from step c), wherein the chromatography system includes the chromatography column from step e) instead of the column shortcut from step d), It was found that the above objective was achieved in a different way than the sum of the volume of a chromatography system without any chromatography columns and the volume of a chromatography column, compared to the sum of the volume of a spare chromatography system without any chromatography columns and the volume of a spare chromatography column.

[0012] This method, surprisingly, allows for rapid process development because it eliminates the time-consuming process of initial small-scale optimization. Furthermore, since the optimization of step f) is performed in silico, fewer consumables, such as disposable columns, resins, microtiter plates, chemicals, or water, are required for process development. Assuming equal transport rates for target molecules and / or impurities, the parameters of the preliminary chromatography system can be used for further separation problems, thus reducing the need for additional consumables. In general, because it eliminates tedious and time-consuming optimization experiments, the method leads to reduced labor and therefore more economical process development.

[0013] definition As used herein, the term “process parameter” refers to a parameter that affects the separation process. Preferably, the process parameter is selected from a list consisting of the flow rate of at least one loading step, the pH value of the loading fluid, the temperature of the loading fluid, the conductivity of the loading fluid, the composition of the loading fluid, the amount of at least one target substance bound to the chromatography matrix per volume of the chromatography column, the amount of at least one impurity bound to a defined volume of the chromatography matrix, the concentration of at least one target substance, the concentration of at least one impurity in the loading fluid, and / or the duration of at least one loading step, the flow rate of at least one washing step, the pH value of the washing fluid, the temperature of the washing fluid, the conductivity of the washing fluid, the composition of the washing fluid, the flow rate of at least one elution step, the duration of at least one elution step, the initial or final concentration of the elution fluid that induces the desorption of at least one target substance and / or at least one impurity from the chromatography matrix of the chromatography column, and / or the composition of the elution fluid.

[0014] As used herein, the term “separation process” refers to a process comprising a stationary phase, preferably a chromatographic matrix, and a mobile phase, preferably at least one solvent, for separating a target substance from impurities. Preferably, the separation process is a chromatographic process. More preferably, the separation process is a chromatographic process selected from the list consisting of affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, reversed-phase chromatography, multimodal, or mixed-mode chromatography.

[0015] As used herein, the term “target substance” refers to a substance to be separated from a mixture of components containing the target substance. Preferably, the target substance is a substance of biological origin, or is produced by a biological process, or is intended for use in a biological environment. More preferably, the target substance is selected from a list consisting of therapeutic proteins, antibiotics, hormones (e.g., insulin and human growth hormone), blood products, antibodies, vaccines, enzymes, nucleic acids, viruses, viral vectors, virus-like particles, cells, natural fragrances, and flavor compounds, and nutritional proteins. Most preferably, at least one target substance is a polypeptide.

[0016] As used herein, the term "impurity" refers to a substance that reduces the purity of the target substance. Preferably, at least one of the impurities is a polypeptide.

[0017] As used herein, the term “column shortcut” refers to a device having low dead volume. Preferably, a column shortcut is a zero dead volume connector. However, a column shortcut may also be a tube or two connected column ends. It should be understood that the dead volume of a column shortcut depends on the scale of the system. Therefore, a column shortcut in a laboratory-scale system usually has a lower dead volume than a column shortcut in an industrial-scale system. However, more preferably, a column shortcut has a dead volume in the range of 0.00001 to 100 mL, more preferably 0.0001 to 10 mL, even more preferably 0.001 to 0.5 mL, and most preferably 2 to 5 μL.

[0018] As used herein, the term “chromatography system” refers to a system comprising a pump, a first tube, and a chromatography column or column shortcut, the first tube fluidly connecting the pump to the chromatography column or column shortcut. Preferably, the chromatography system further comprises a second tube, and a detector and / or fraction collector, the second tube fluidly connecting the column or column shortcut to the detector and / or fraction collector. The chromatography system typically implements input and output streams by connecting each vessel to the pump and / or detector by tubes. The chromatography system may further comprise at least one valve and / or at least one bubble trap and / or at least one mixing chamber. Exemplary chromatography systems are the AKTA start, AKTA go, AKTA pure, AKTA avant, AKTA pilot, or AKTA ready, available from Cytiva.

[0019] The term "unit" refers to a device included in a chromatography system, and different units of a chromatography system are generally connected by tubes. Preferably, a unit is selected from a list consisting of a chromatography column, column shortcut, detector, fraction collector, valve, bubble trap, or mixing chamber.

[0020] As used herein, the term “preliminary chromatography system” refers to a chromatography system in which sorption-specific parameters suitable for describing the adsorption and / or desorption of a target substance and at least one impurity are determined.

[0021] As used herein, the term "detector" may refer to any device suitable for detecting substances in a fluid passing through the device. Preferably, the detector is selected from the list of ultraviolet absorption (UV) detectors, visible light absorption (VIS) detectors, photodiode array (PDA) detectors, refractive index detectors, evaporative light scattering detectors, multi-angle light scattering detectors, mass spectrometers, conductivity detectors, fluorescence detectors, chemiluminescence detectors, optical rotation detectors, and electrochemical detectors. Preferably, the detector is located downstream of the column.

[0022] As used herein, the term "chromatographic system volume" refers to the system volume of a chromatography system. The system volume is the total volume of the units contained within the chromatography system.

[0023] As used herein, the term "delayed volume" refers to dead volume in a system that causes a shift in the x-axis (time or volume) of a sensor signal without altering the shape of the sensor signal. In particular, it is assumed that no mixing or dispersion effects occur in the delayed volume.

[0024] As used herein, the term "chromatographic column" refers to a device for separating substances that include a chromatographic matrix as a stationary phase.

[0025] As used herein, the term “interaction” refers to an attractive force between at least two structural units, and the attractive force is based on chemical and / or physical principles. Preferably, the interaction is selected from a list consisting of ionic interactions, hydrophobic interactions, preferably ionic and hydrophobic interactions, partially specific interactions, hydrogen bonding interactions, or combinations thereof.

[0026] The term "chromatographic matrix" refers to the stationary phase in a chromatographic separation process. Preferably, the chromatographic matrix is ​​suitable for interacting with the target substance and / or at least one impurity. More preferably, the chromatographic matrix comprises at least one active portion that interacts with the target substance and / or impurity by one or more physical or chemical interactions. The chromatographic matrix may be fibrous, monolithic, membrane-like, or specific. Preferably, the chromatographic matrix is ​​a compound selected from a list of natural polymers and / or synthetic polymers, preferably a compound selected from a list of polysaccharides, polystyrene, polyacrylamide, polymethacrylate, or mixtures thereof.

[0027] As used herein, the term "in silico" refers to a process performed by computer modeling or computer simulation.

[0028] As used herein, the term “fitting” refers to the process of constructing a mathematical function that best fits a set of data points, such as data points measured by a detector in the form of a measured chromatogram. Preferably, the term fitting refers to finding the parameter values ​​of a mathematical function that minimize the sum of the squared differences between the data points and the values ​​obtained by evaluating the mathematical function at the same coordinates as the data points (e.g., by the usual least squares method).

[0029] As used herein, the term "porous material" refers to a material containing micropores. Preferably, the term porous material refers to a material having micropores in which any mass transport processes to and from these pores are diffusion-dominant, i.e., the influence of convective mass transport is negligible.

[0030] As used herein, the term "micropore" refers to a cavity on the surface of a material, which obstructs the convection of fluids through the material. More preferably, the micropores have a diameter of 2 nm to 50 nm. Due to the small pore size, the mass transport of target substances and / or impurities within the micropores of porous materials is diffusion-dominated, while convective mass transport is inhibited.

[0031] As used herein, the term "tracer" refers to a substance that can be processed in a chromatography system and detected by a detector. Preferably, the tracer does not interact with any components of the chromatography system, for example, by adsorption. Preferably, the tracer is soluble in the mobile phase of the chromatography system. Depending on the size of the tracer, the effect of pores on mass transport in the chromatography matrix can be explained. Therefore, preferably, non-bulky tracers are selected to determine the effect of pores on mass transport, preferably micropores in porous materials, on the separation process.

[0032] Accordingly, as used herein, the term "non-bulky tracer" refers to a tracer having a size suitable for penetrating the pores of a stationary phase, preferably a chromatography matrix, the chromatography matrix preferably comprising, and more preferably consisting of, a porous material.

[0033] As used herein, the term "dextran" refers to a large polymer of anhydroglucose having a molecular weight in the range of 3 kDa to 5 MDa. Dextrans having a molecular weight of 2 MDa or greater cannot penetrate the micropores of a chromatography matrix, including, preferably made of, a porous material.

[0034] As used herein, the term “active moiety” refers to a molecule or substance that interacts with the target substance and / or impurities by one or more physical or chemical interactions. Preferably, the active moiety is sulfopropyl, diethylaminoethyl, diethylaminopropyl, diethyl-(2-hydroxypropyl)aminoethyl, octylamine, N-benzyl-n-methylethanolamine, methylsulfonate, quaternary ammonium, carboxymethyl, alkyl, preferably octyl, hexyl, butyl, phenyl, biphenyl, pentafluorophenyl, phenyl-hexyl, ethyl, benzyl, or isopropyl, silanol, peptide, nucleotide, protein, immunoglobulin-binding protein. The following are selected from the list: proteins, preferably protein A, protein G, protein L; metal ions, preferably cations of Cu, Ni, Co, or Zn; nitrilotriacetate; domain antibodies or domain antibody fragments; CHT; polydeoxythymidine (e.g., 25mer of dT); C4, C6, C8, C10, C12, C14, C16; octadecyl carbon (C18); C20, C22, C24; heparin; dextran sulfate; 2-mercaptopyridine; hydroxyl apatite; fluoroapatite; or combinations thereof.

[0035] The term “partially specific interaction,” also known as affinity interaction, as used herein, refers to a specific interaction between a target substance and / or impurity and an interaction partner in the form of an active moiety. Preferably, the target molecule and / or impurity binds specifically to the active moiety. Partially specific interactions are preferably: an interaction between an enzyme as a target substance and / or impurity and a substrate analog as a part; an interaction between an antigen as a target substance and / or impurity and an antibody as a part; an interaction between a polysaccharide as a target substance and / or impurity and a lectin as a part; an interaction between a complementary nucleotide sequence as a target substance and / or impurity and a nucleic acid as a part; an interaction between a hormone receptor as a target substance and / or impurity and a hormone as a part; an interaction between biotin or a biotin conjugate substance, preferably a biotin conjugate protein, and avidin as a part; The interaction is selected from the following: the interaction between a calmodulin-binding partner as a target substance and / or impurity and calmodulin as a part; the interaction between a glutathione S-transferase fusion substance, preferably a glutathione S-transferase fusion protein, as a target substance and / or impurity and glutathione as a part; the interaction between an immunoglobulin as a target substance and / or impurity and an immunoglobulin-binding substance, preferably an immunoglobulin-binding protein, as a part; and the interaction between a complex of a polyhistidine fusion substance, preferably a polyhistidine fusion protein, as a target substance and / or impurity and a metal cation-binding protein as a part. [Modes for carrying out the invention]

[0036] The present invention relates to a method for optimizing at least one process parameter of a separation process. The present invention will be described in more detail below.

[0037] The present invention relates to a method for optimizing at least one process parameter of a separation process for at least one target substance from at least one impurity using a chromatography system, comprising the steps of: a) providing a set of preliminary system-specific parameters suitable for describing the concentration transport of at least one target substance and at least one impurity in a preliminary chromatography system using a system-specific transport model, wherein the preliminary chromatography system does not include any chromatography column but includes a column shortcut; b) providing a set of preliminary column-specific parameters suitable for describing the concentration transport of at least one target substance and at least one impurity in only a preliminary chromatography column containing a preliminary chromatography matrix using a column-specific transport model; c) using an sorption model, a set of preliminary system-specific parameters, and a set of preliminary column-specific parameters to optimize at least one process parameter of the separation process for at least one target substance and at least one impurity in a preliminary chromatography column containing a preliminary chromatography matrix. A step of determining a set of sorption-specific parameters suitable for describing the adsorption and / or desorption of one target substance and at least one impurity, wherein the preliminary chromatography system includes the preliminary chromatography column of step b) instead of the column shortcut of step a), d) using a system-specific transport model to provide a set of system-specific parameters suitable for describing the concentration transport of at least one target substance and at least one impurity in the chromatography system, wherein the chromatography system does not include any chromatography column but includes a column shortcut, e) using a column-specific transport model to provide a set of column-specific parameters suitable for describing the concentration transport of at least one target substance and at least one impurity in chromatography columns including a chromatography matrix only, f) using the following parameters: using the set of system-specific parameters of step d), the set of column-specific parameters of step e), and the set of sorption-specific parameters of step c),The present invention relates to a method for optimizing at least one process parameter in silico, wherein the chromatography system includes a step e) with a chromatography column instead of a column shortcut in step d), and the sum of the volume of a chromatography system without any chromatography column and the volume of a chromatography column is different from the sum of the volume of a spare chromatography system without any chromatography column and the volume of a spare chromatography column.

[0038] The remarkable technical effect of performing optimization in step f), combined with the requirement that the sum of the volume of the chromatography system without any chromatography column and the volume of the chromatography column differs from the sum of the volume of the preliminary chromatography system without any chromatography column and the volume of the preliminary chromatography column, is that it enables direct process optimization at the desired scale. This particularly solves the problem of over-optimization of purity and, as a result, suboptimal yields achieved by optimization and scale-up methods known from the prior art. Furthermore, the fact that the optimization is performed in silico helps in optimizing the chromatography system regardless of the availability of chromatography columns with discontinuous diameters. Thus, the method according to the present invention allows for finer selection of the chromatography column diameter compared to optimization and scale-up methods known from the prior art.

[0039] Preferably, the volume of the chromatography system without any chromatography column is different from the volume of the preliminary chromatography system without any chromatography column, and / or the volume of the chromatography column is different from the volume of the preliminary chromatography column, preferably only the volume of the chromatography column is different from the volume of the preliminary chromatography column.

[0040] Preferably, the volume of the chromatography system without any chromatography column is greater than the volume of the preliminary chromatography system without any chromatography column, preferably 10 to 100,000 times greater, more preferably 100 to 70,000 times greater, and most preferably 1,000 to 10,000 times greater, and / or the volume of the chromatography column is greater than the volume of the preliminary chromatography column, preferably 10 to 100,000 times greater, more preferably 100 to 10,000 times greater, and most preferably 1,000 to 5,000 times greater. More preferably, the volume of the chromatography system including the chromatography column is in the range of more than 1.5 l to 1,000 l, preferably 13 l to 800 l, and the volume of the preliminary chromatography system including the preliminary chromatography column is in the range of 0.0003 l to 1.5 l, preferably 0.001 l to 1.0 l. This ensures that the chromatography system is larger than the preliminary chromatography system, and preferably industrial in scale.

[0041] Process a) Preferably, in step a), the set of preliminary system-specific parameters is the preliminary system internal dispersion (D) of the preliminary chromatography system. ax_pre_sys ), and / or preferably the pre-system mixing rate (MR pre_sys The preliminary chromatography system includes, preferably, a set of preliminary system-specific parameters, and preferably comprises a preliminary tube internal dispersion (D ax_pre_tub ), and one pre-unit mixing rate (MR) for each unit included in the pre-chromatography system. pre_unt ) comprises, preferably, a set of preliminary system-specific parameters, further comprising the preliminary system delay volume of the preliminary chromatography system, wherein the preliminary chromatography system does not include any chromatography column, but rather a column shortcut (DV). pre_sys) is included. The delay volume of the preliminary system is particularly useful when the information about the pipe is not fully described.

[0042] In a first preferred embodiment of the method of the present invention, step a) comprises the following steps: aa) Measuring at least one measured preliminary system-specific chromatogram of the tracer in a preliminary chromatography system, wherein the preliminary chromatography system does not include any chromatography columns but includes a column shortcut; ab) Based on at least one measured preliminary system-specific chromatogram, determining the internal dispersion (D ax_pre_sys ) in the preliminary system, and / or preferably and determining the mixing rate (MR pre_sys ) in the preliminary system, preferably determining the internal dispersion (D ax_pre_tub ) in the preliminary pipe and one preliminary unit mixing rate (MR pre_unt ) for each unit included in the preliminary chromatography system; comprising, preferably consisting of, a tracer that does not interact with any components of the chromatography system.

[0043] The internal dispersion (D ax_pre_tub ) in the preliminary pipe may range from 0.0001 mm 2 / s to 1000 mm 2 / s, preferably from 0.001 mm 2 / s to 100 mm 2 / s, most preferably from 0.1 mm 2 / s to 10 mm 2 / s.

[0044] Since the tracer does not interact with any components of the chromatography system, the resulting chromatogram is not affected by the adsorption mechanism and reflects only the mixing and dispersion effects within the chromatography system.

[0045] Preferably, step a) of the first preferred embodiment of the present invention involves fitting a simulated preliminary system-specific chromatogram obtained from a system-specific transport model to at least one measured preliminary system-specific chromatogram, thereby reducing the preliminary system internal variance (D ax_pre_sys ), and / or preferably the pre-system mixing rate (MR pre_sys A step to determine the internal dispersion (D) of the reserve tube, preferably D ax_pre_tub ), and one pre-unit mixing rate (MR) for each unit included in the pre-chromatography system. pre_unt The system-specific transport model includes a step to determine the preliminary system internal distribution (D ax_pre_sys ) and the pre-system mixing rate (MR pre_sys ) contains preferably internal dispersion in the reserve tube (D ax_pre_tub ), and one pre-unit mixing rate (MR) for each unit included in the pre-chromatography system. pre_unt The system-specific transport model includes ) as a parameter, and describes a preliminary chromatography system that does not include any chromatography columns but includes column shortcuts.

[0046] Preferably, the system-specific transport model of step a) of the first preferred embodiment of the present invention comprises, and preferably comprises, at least one continuous stirred tank reactor (CSTR) model and / or at least one dispersed plug flow reactor (DPFR) model.

[0047] The CSTR model is a common model of chemical reactors in chemical and environmental engineering. This mathematical model works for liquids, gases, and slurries and can be used as a tool to model the mixing of substances and fluids in compartments of a chromatography system, such as bubble traps or mixing chambers, using the mixing rate MR.

[0048] The DPFR model has an effective variance coefficient D axThis is used to describe the dispersion of a substance in a fluid within a tubular compartment of a chromatography system, such as a tube or hose.

[0049] Preferably, the adaptation of step a) of the first preferred embodiment of the present invention involves setting the parameter of the system-specific transport model, the preliminary system internal variance (D), until the simulated preliminary system-specific chromatogram fits to at least one preliminary system-specific chromatogram to be measured. ax_pre_sys ) and / or pre-system mixing rate (MR pre_sys ) to adjust, preferably internal dispersion in the reserve tube (D ax_pre_tub ) and one pre-unit mixing rate (MR) for each unit included in the pre-chromatography system. pre_unt This is done by adjusting the chromatogram. Preferably, the preliminary system-specific chromatogram to be measured is measured by injecting the tracer into the preliminary chromatography system and then measuring the tracer concentration downstream of the column shortcut. Preferably, the tracer concentration is measured by ultraviolet spectroscopy, visible light spectroscopy, near-infrared spectroscopy, refractive index measurement, mass spectrometry, conductivity measurement, fluorescence spectroscopy, chemiluminescence spectroscopy, and / or electrochemistry. Preferably, the tracer is selected from the group consisting of dextran, preferably dextran having a molecular weight of 2 MDa or more, glucose, acetone, chloride, at least one target substance, at least one impurity, protein, peptide, nanoparticles, preferably metal nanoparticles, more preferably gold nanoparticles.

[0050] It should be understood that the selection of the tracer in step aa) of the first preferred embodiment of the present invention depends on the selection of the pre-chromatography system. The tracer should have little to no interaction with any unit or tube of the pre-chromatography system, or should not have any interaction at all. Since the pre-chromatography system according to the first preferred embodiment of the present invention does not include a pre-chromatography column, the interaction between the tracer and the pre-chromatography matrix can be ignored in this embodiment.

[0051] Step b) Preferably, the column-specific transport model in step b) is selected from a list consisting of an ideal model, an equilibrium dispersion model, a transport dispersion model, a concentrated velocity model, a concentrated dynamics model, a general velocity model, or a combination thereof.

[0052] The ideal model considers only convective transport and assumes a permanently established local equilibrium between the mobile and stationary phases. Compared to the ideal model, the equilibrium dispersion model additionally includes a term describing axial dispersion in the mass balance of the mobile phase. The transport dispersion model is characterized by a second parameter that describes the rate-limiting factor, separate from axial dispersion. This second parameter subdivides the model into those where either mass transport or a dynamical term is rate-limiting. The condensed velocity model uses a single condensed transport coefficient (k) to represent the internal and external mass transport resistances. eff) To summarize: The condensed dynamics model assumes that adsorption dynamics are rate-limiting. General velocity models incorporate at least two other parameters that describe mass transport effects in addition to axial dispersion. These two parameters can be a combination of mass transfer within liquid films and pores, as well as surface diffusion and various types of adsorption dynamics. Further information on these models can be found in "Preparative Chromatography" edited by Henner Schmidt-Traub (ISBN 978-3-527-34486-4).

[0053] In a second preferred embodiment of the present invention, the set of preliminary column-specific parameters in step b) is the preliminary column axial dispersion (D ax_pre_col ), and / or preferably, the pre-column void ratio (ε p_pre_col ) includes, preferably consists of, Step b) is the following: ba) A step of measuring a preliminary column-specific chromatogram of at least one tracer in a preliminary chromatography system, wherein the preliminary chromatography system includes the preliminary chromatography column of step b) instead of the column shortcut of step a), bb) Based on at least one measured preliminary column-specific chromatogram, the preliminary column axial dispersion (D ax_pre_col ), and / or preferably, the pre-column void ratio (ε p_pre_col The process of determining ) It includes, and preferably consists of, The tracer does not interact with any components of the pre-chromatography system or pre-chromatography column.

[0054] Alternatively, the pre-column void ratio (ε p_pre_col ) may be derived from the preliminary system pressure curve using the Kozeny-Carman equation.

[0055] Preferably, step bb) of the second preferred embodiment of the present invention involves fitting a simulated preliminary column-specific chromatogram obtained from a column-specific transport model to at least one measured preliminary column-specific chromatogram, thereby reducing the preliminary column axial dispersion (D ax_pre_col ), and / or preferably, the pre-column void ratio (ε p_pre_col This includes determining the column-specific transport model, preferably consisting of a preliminary column axial dispersion (D ax_pre_col ) and the void ratio of the preliminary column (ε p_pre_col The column-specific transport model includes ) as a parameter and describes the preliminary chromatography column.

[0056] Pre-column axial dispersion (D ax_pre_col ) is 0.001 mm 2 / s~100mm 2 / s, preferably 0.01 mm 2 / s~10mm 2 / s, most preferably 0.1 mm 2 / s~1mm 2 The range may be / s. Preliminary column void ratio (ε p_pre_col ) may be in the range of 0 to 1, preferably 0.1 to 0.8, and most preferably 0.2 to 0.7.

[0057] Preferably, the adaptation of step bb) of the second preferred embodiment of the present invention involves setting the preliminary column axial dispersion (D) as a parameter of the column-specific transport model until the simulated preliminary column-specific chromatogram fits at least one preliminary column-specific chromatogram to be measured. ax_pre_col ), and / or preferably, the pre-column void ratio (ε p_pre_col This is done by adjusting the following: Preferably, at least one preliminary column-specific chromatogram to be measured is measured by injecting a tracer into a preliminary chromatography system and then measuring the concentration of at least one tracer downstream of the preliminary chromatography column. Preferably, the measurement of the concentration of at least one tracer is performed by ultraviolet spectroscopy, visible light spectroscopy, near-infrared spectroscopy, refractive index measurement, mass spectrometry, conductivity measurement, fluorescence spectroscopy, chemiluminescence spectroscopy, and / or electrochemistry. Preferably, the tracer is selected from the group consisting of dextran, preferably dextran having a molecular weight of 2 MDa or more, glucose, acetone, sodium chloride, at least one target substance, at least one impurity, protein, peptide, nanoparticles, preferably metal nanoparticles, more preferably gold nanoparticles.

[0058] It should be understood that the selection of the tracer in step ba) of the second preferred embodiment of the present invention depends on the selection of the pre-chromatography system and the pre-chromatography column, in particular the pre-chromatography matrix. The tracer should have little to no interaction with any unit or tube of the pre-chromatography system, including the pre-chromatography matrix. Preferred combinations of pre-chromatography matrix and tracer are dextran with a molecular weight of 2 MDa or greater in beads, and NaCl in membranes or monoliths without any micropores. Furthermore, the tracer should not be permeable to porous materials.

[0059] In a second particularly preferred embodiment of the second preferred embodiment of the present invention, the pre-chromatographic matrix comprises a porous material, preferably consisting of a set of pre-column specific parameters, where the total porosity of the pre-column (ε) tot_pre_col ), Pre-column effective mass transfer coefficient (k eff_pre_col ), and / or preferably, the preliminary column membrane transfer coefficient (k film_pre_col ) and preliminary column pore diffusion coefficient (D p_pre_col ) further includes both of the above, and step b) is the following step: bc) A step of measuring at least one preliminary matrix-specific chromatogram of a non-bulky tracer in a preliminary chromatography system that includes a preliminary chromatography column instead of the column shortcut of step a), bd) Based on at least one measured preliminary matrix-specific chromatogram, the total porosity of the preliminary column (ε tot_pre_col ), Pre-column effective mass transfer coefficient (k eff_pre_col ), and / or preferably, the preliminary column membrane transfer coefficient (k film_pre_col ) and preliminary column pore diffusion coefficient (D p_pre_col This further includes the process of determining both of the following: The tracer cannot penetrate the porous material of the preliminary chromatography matrix of the preliminary chromatography column. Non-bulky tracers do not interact with any components of the pre-chromatography system or pre-chromatography column. Non-bulky tracers can penetrate the porous material of the preliminary chromatography matrix of the preliminary chromatography column. Steps ba) and bc) can be performed simultaneously, thereby providing a composite chromatogram including a preliminary column-specific chromatogram and a preliminary matrix-specific chromatogram, or they can be performed sequentially, thereby providing two separate chromatograms: a preliminary column-specific chromatogram and a preliminary matrix-specific chromatogram.

[0060] Total porosity of the preliminary column (ε tot_pre_col) may be in the range of 0 to 1, preferably 0.5 to 0.95, most preferably 0.6 to 0.9. Preliminary column effective mass transfer coefficient (k eff_pre_col The ) may be in the range of 0.0001 mm / s to 10 mm / s, preferably 0.001 mm / s to 1 mm / s, and most preferably 0.001 mm / s to 0.01 mm / s. Preliminary column membrane transfer coefficient (k film_pre_col The pore diffusion coefficient (D) of the preliminary column may be in the range of 0.0001 mm / s to 10 mm / s, preferably 0.001 mm / s to 1 mm / s, and most preferably 0.01 mm / s to 0.1 mm / s. p_pre_col ) is 1x10 -7 mm 2 / s~0.01mm 2 / s, preferably 1x10 -6 mm 2 / s~0.001mm 2 / s, most preferably 1x10 -5 mm 2 / s~1x10 -4 mm 2 It can also be within the range of / s.

[0061] Preferably, step bd) of the second particularly preferred embodiment of the second preferred embodiment of the present invention involves fitting a simulated preliminary matrix-specific chromatogram obtained from a column-specific transport model to at least one measured preliminary matrix-specific chromatogram, thereby determining the preliminary column total porosity (ε tot_pre_col ), Pre-column effective mass transfer coefficient (k eff_pre_col ), and / or preferably, the preliminary column membrane transfer coefficient (k film_pre_col ) and preliminary column pore diffusion coefficient (D p_pre_col This includes determining both of the following, preferably consisting of the column-specific transport model, which is the preliminary column axial dispersion (D) determined in step bb). ax_pre_col ) and the void ratio of the preliminary column (ε p_pre_col ), and the total porosity of the preliminary column (ε tot_pre_col ), Pre-column effective mass transfer coefficient (k eff_pre_col ), and / or preferably, the preliminary column membrane transfer coefficient (k film_pre_col) and the preliminary column pore diffusion coefficient (D p_pre_col ) as parameters, the column-specific transport model describes the preparative chromatography column. Preferably, at least one measured preparative matrix-specific chromatogram is measured by injecting an unswollen tracer into the preparative chromatography system and then measuring the concentration of the unswollen tracer downstream of the preparative chromatography column. Preferably, the measurement of the concentration of the unswollen tracer is performed by ultraviolet spectroscopy, visible light spectroscopy, near-infrared spectroscopy, refractive index measurement, mass spectrometry, conductivity measurement, fluorescence spectroscopy, chemiluminescence spectroscopy, and / or electrochemistry.

[0062] Alternatively, step bd) determines the preliminary column effective mass transfer coefficient (k eff_pre_col ), and / or preferably also, the preliminary column pore diffusion coefficient (D p_pre_col ) and both of the preliminary column film transfer coefficients (k film_pre_col ) based on already determined values and / or correlations obtained from the literature.

[0063] Preferably, the adaptation for the determination of the parameters in step bd) of the second particularly preferred embodiment of the second preferred embodiment of the present invention is such that the column-specific transport model parameters, the preliminary column axial dispersion (D ax_pre_col ), the preliminary column interstitial void fraction (ε p_pre_col ), the preliminary column total void fraction (ε tot_pre_col ), the preliminary column effective mass transfer coefficient (k eff_pre_col ), and / or preferably also, the preliminary column film transfer coefficient (k film_pre_col ) and both of the preliminary column pore diffusion coefficients (D p_pre_col ) are adjusted until the simulated preparative matrix-specific chromatogram fits at least one measured preparative matrix-specific chromatogram.

[0064] Preferably, the non-bulky tracer in step bd) of the second particularly preferred embodiment of the second preferred embodiment of the present invention is selected from the group consisting of acetone, glucose, at least one target substance, at least one impurity, protein, and peptide.

[0065] It should be understood that the selection of the tracer in step ba) of the second particularly preferred embodiment of the second preferred embodiment of the present invention depends on the selection of the pre-chromatography system and the pre-chromatography column, in particular the pre-chromatography matrix. The tracer should have little to no interaction with any unit or tube of the pre-chromatography system, including the pre-chromatography matrix. A preferred combination of pre-chromatography matrix and tracer is, in the case of beads, dextran with a molecular weight of 2 MDa or greater. Furthermore, the tracer should not be permeable to porous materials.

[0066] Similarly, it should be understood that the selection of a non-bulky tracer in step bc) of the second particularly preferred embodiment of the second preferred embodiment of the present invention depends on the selection of the pre-chromatography system and the pre-chromatography column, in particular the pre-chromatography matrix. The non-bulky tracer should have little to no interaction with any unit or tube of the pre-chromatography system, including the pre-chromatography matrix. A preferred tracer is NaCl. However, the non-bulky tracer should be permeable to porous materials.

[0067] Process c) In a third preferred embodiment of the present invention, step c) is the following step: ca) A step of measuring at least one pre-target-specific chromatogram of at least one target substance in a pre-chromatography system, wherein the pre-chromatography system includes the pre-chromatography column of step b) instead of the column shortcut of step a), cb) A step of measuring at least one pre-impurity-specific chromatogram of at least one impurity to be measured in a pre-chromatography system, wherein the pre-chromatography system includes the pre-chromatography column of step b) instead of the column shortcut of step a), cc) The process preferably includes the step of determining a set of sorption-specific parameters based on at least one measured preliminary target-specific chromatogram and at least one measured preliminary impurity-specific chromatogram, Steps ca) and cb) can be performed simultaneously, thereby providing a composite chromatogram including a target-specific chromatogram and an impurity-specific chromatogram, or they can be performed sequentially, thereby providing two separate chromatograms: a target-specific chromatogram and an impurity-specific chromatogram.

[0068] In the first particularly preferred embodiment of the third preferred embodiment of the present invention, step cc) is the following step: cca) A step of determining the sorption-specific parameters of a target substance by fitting a simulated preliminary target-specific chromatogram of at least one target substance in a preliminary chromatography system including the preliminary chromatography column of step b) to at least one preliminary target-specific chromatogram to be measured, wherein the simulated preliminary target-specific chromatogram is - An sorption model for step c), which includes sorption-specific parameters of the target substance as parameters, and describes the adsorption / desorption behavior of at least one target substance by a pre-chromatographic matrix contained in a pre-chromatographic column. - System-specific transport model for step a), and - Column-specific transport model for step b) Process obtained from, A step in which, instead of the column shortcut in step a), the sorption-specific parameters of an impurity are determined by fitting a simulated preliminary impurity-specific chromatogram of at least one impurity in a preliminary chromatography system including a preliminary chromatography column to at least one preliminary impurity-specific chromatogram to be measured, wherein the simulated preliminary impurity-specific chromatogram is - An sorption model for step c), which includes an sorption-specific parameter for impurities as a parameter, and describes the adsorption / desorption behavior of at least one impurity by the pre-chromatographic matrix contained in the pre-chromatographic column. - System-specific transport model for step a), and - Column-specific transport model for step b) This process includes obtaining the results from, and preferably consists of, The set of sorption-specific parameters preferably includes sorption-specific parameters for the target substance and sorption-specific parameters for at least one impurity.

[0069] Preferably, in step cc) of the third preferred embodiment of the present invention, the sorption-specific parameter of at least one target substance is the pre-equilibrium constant (k) of the at least one target substance with respect to the pre-chromatographic matrix. eq_target ), and / or preferably the preliminary kinetic constant (k) of at least one target substance from the preliminary chromatography matrix. kin_target Preferably consists of including ).

[0070] Similarly, preferably in step cc) of the third preferred embodiment of the present invention, the sorption-specific parameter of at least one impurity is the pre-equilibrium constant (k) of at least one impurity relative to the pre-chromatographic matrix. eq_imp ), and / or preferably the preliminary kinetic constant (k) of at least one impurity from the preliminary chromatography matrix. kin_imp Preferably consists of including ).

[0071] Preferably, the sorption model in step c) describes the interaction between at least one target substance and a pre - chromatography matrix, preferably at least one active moiety comprised in the pre - chromatography matrix, and the interaction between at least one impurity and the pre - chromatography matrix, preferably at least one active moiety comprised in the pre - chromatography matrix. Preferably, the interaction is selected from the list consisting of ionic interactions, hydrophobic interactions, preferably ionic and hydrophobic interactions, partial - specific interactions, hydrogen - bonding interactions, or combinations thereof.

[0072] In a second particularly preferred embodiment of the third preferred embodiment of the present invention, the interaction is an ionic interaction, and the set of sorption - specific parameters further includes the ion capacity of the pre - chromatography matrix comprised in the pre - chromatography column. The set of sorption - specific parameters of the target substance includes the charge (v ads_target ) of at least one target substance, the steric shielding (σ ads_target ) of at least one target substance, and / or preferably and the lateral charge (z lat_target ) of at least one target substance. The set of sorption - specific parameters of at least one impurity includes the charge (v ads_imp ) of at least one target substance, the steric shielding (σ ads_imp ) of at least one target substance, and / or preferably and the lateral charge (z lat_imp ) of at least one target substance.

[0073] In a third particularly preferred embodiment of the third preferred embodiment of the present invention, the interaction is a hydrophobic interaction. The set of sorption - specific parameters of the target substance includes the activity parameter K salt_target that describes the change in the thermodynamic activity of the target substance due to the presence of salt ions, the stoichiometric parameter n i_target that describes the number of hydrophobic ligands occupied by the target substance, and / or preferably and q max,targetThe set of parameters specific to impurity sorption, including the target substance-specific saturation capacity, describes the change in the thermodynamic activity of the impurity due to the presence of salt ions, and includes the activity parameter K. salt_imp , a stoichiometric parameter n that describes the number of hydrophobic ligands occupied by impurities i_imp , and / or preferably and q max,imp , including impurity-specific saturation capacity.

[0074] In a fourth particularly preferred embodiment of a third preferred embodiment of the present invention, the interaction is a hydrogen bonding interaction, and the set of sorption-specific parameters includes parameters describing the hydrogen bonding interaction between the target substance and the pre-chromatographic matrix, and / or preferably a set of sorption-specific parameters describing the hydrogen bonding interaction between the impurity and the pre-chromatographic matrix.

[0075] In a fifth particularly preferred embodiment of the third preferred embodiment of the present invention, the interaction is a combination of ionic interactions, hydrophobic interactions, and / or preferably hydrogen bonding interactions.

[0076] In a sixth particularly preferred embodiment of the third preferred embodiment of the present invention, the interaction is a partially specific interaction, and the set of sorption-specific parameters is a pre-equilibrium constant (k) of at least one target substance that describes the specific interaction between the target substance and the portion contained in the pre-chromatographic matrix. eq_target_moiety ), a preliminary dynamics constant (k) of at least one target substance that describes the specific interaction between the target substance and the portion contained by the preliminary chromatography matrix. kin_target_moiety ), a pre-equilibrium constant (k) for at least one impurity that describes the specific interaction between the impurity and the portion contained by the pre-chromatographic matrix. eq_imp_moiety ), and / or preferably, a preliminary dynamics constant (k) of at least one impurity that describes the specific interaction between the impurity and the portion included by the preliminary chromatography matrix. kin_imp_moiety ) further includes.

[0077] Step d) Preferably, the set of system-specific parameters in step d) is the system internal dispersion (D) of the chromatography system. ax_sys ), and / or preferably the system mixing rate (MR sys ), preferably internal dispersion (D ax_tub ) and one unit mixing rate (MR) for each unit included in the chromatography system. unt ) comprises, preferably consisting of, and the chromatography system does not include any chromatography column but includes column shortcuts. Most preferably, the system-specific set of parameters further includes the system delay volume of the chromatography system, and the chromatography system does not include any chromatography column but includes column shortcuts (DV sys ) includes. System delay volume is particularly useful when the internal dispersion of the tube is not fully described.

[0078] In a fourth preferred embodiment of the present invention, step d) is the following step: da) A step of measuring at least one system-specific chromatogram of a tracer in a chromatography system, wherein the chromatography system does not include any chromatography column but includes a column shortcut. Based on at least one measured system-specific chromatogram, the system internal dispersion (D) of the chromatography system is determined. ax_sys ), and / or preferably the system mixing rate (MR sys A process to determine the internal dispersion (D) of the tube, preferably D ax_tub ), and one unit mixing rate (MR) for each unit included in the chromatography system. unt This includes a step of determining the following, preferably consisting of: The tracer does not interact with any of the components of the chromatography system.

[0079] Dispersion inside the pipe (D ax_tub ) is 0.0001 mm2 / s~1000mm 2 / s, preferably 0.001 mm 2 / s~100mm 2 / s, most preferably 0.1 mm 2 / s~10mm 2 It can also be within the range of / s.

[0080] Preferably, step db) of the fourth preferred embodiment of the present invention involves fitting a simulated system-specific chromatogram obtained from a system-specific transport model to at least one measured system-specific chromatogram, thereby determining the system internal dispersion (D) of the chromatographic system. ax_sys ), and / or preferably the system mixing rate (MR sys A process to determine the internal dispersion (D) of the tube, preferably D ax_tub ), and one unit mixing rate (MR) for each unit included in the chromatography system. unt The system-specific transport model includes a step to determine the system internal dispersion (D ax_sys ), and / or preferably the system mixing rate (MR sys ), preferably internal dispersion (D ax_tub ), and optionally, one unit mixing rate (MR) for each unit included in the chromatography system. unt The system-specific transport model includes ) as a parameter, describes a chromatography system, and the chromatography system does not include any chromatography columns but includes column shortcuts.

[0081] Preferably, in step db) of the fourth preferred embodiment of the present invention, the system-specific transport model comprises, and preferably comprises, at least one continuous stirred tank reactor (CSTR) model and / or at least one dispersed plug flow reactor (DPFR) model.

[0082] The CSTR model is a common model of chemical reactors in chemical and environmental engineering. This mathematical model works for liquids, gases, and slurries and can be used as a tool to model the mixing of substances and fluids in compartments of a chromatography system, such as bubble traps or mixing chambers, using the mixing rate MR.

[0083] The DPFR model has an effective variance coefficient D ax This is used to describe the dispersion of a substance in a fluid within a tubular compartment of a chromatography system, such as a tube or hose.

[0084] Preferably, the adaptation of step db) of the fourth preferred embodiment of the present invention is performed until the simulated chromatogram conforms to at least one system-specific chromatogram to be measured, with the system internal dispersion (D) of the chromatographic system as a parameter of the system-specific transport model. ax_sys ), and / or preferably the system mixing rate (MR sys ), preferably internal dispersion (D ax_tub ), and one unit mixing rate (MR) for each unit included in the chromatography system. unt This is done by adjusting the chromatogram. Preferably, the system-specific chromatogram to be measured is measured by injecting the tracer into the chromatography system and then measuring the tracer concentration downstream of the column shortcut. Preferably, the tracer concentration is measured by ultraviolet spectroscopy, visible light spectroscopy, near-infrared spectroscopy, refractive index measurement, mass spectrometry, conductivity measurement, fluorescence spectroscopy, chemiluminescence spectroscopy, and / or electrochemistry. Preferably, the tracer is selected from the group consisting of dextran, preferably dextran having a molecular weight of 2 MDa or more, glucose, acetone, chloride, at least one target substance, at least one impurity, protein, peptide, nanoparticles, preferably metal nanoparticles, more preferably gold nanoparticles.

[0085] It should be understood that the selection of the tracer in step da) of the fourth preferred embodiment of the present invention depends on the selection of the chromatography system. The tracer should have little to no interaction with any unit or tube of the chromatography system, or should not have any interaction at all. Since the chromatography system according to the fourth preferred embodiment of the present invention does not include a chromatography column, the interaction between the tracer and the chromatography matrix can be ignored in this embodiment.

[0086] Process e) Preferably, the column-specific transport model in step e) is selected from a list consisting of an ideal model, an equilibrium dispersion model, a transport dispersion model, a condensed velocity model, a condensed dynamics model, a general velocity model, or a combination thereof.

[0087] Preferably, the chromatography matrix contained in the chromatography column is suitable for the same interactions as the pre-chromatography matrix of the pre-chromatography column in steps b) and c). More preferably, the chromatography matrix and the pre-chromatography matrix each contain an active site, and each active site enables the same interactions. Even more preferably, the chromatography matrix and the pre-chromatography matrix each contain the same active site. Also preferably, the chromatography matrix and the pre-chromatography matrix contain the same compound. Most preferably, the chromatography matrix and the pre-chromatography matrix each contain the same active site, and the chromatography matrix and the pre-chromatography matrix contain the same compound.

[0088] In a fifth preferred embodiment of the present invention, the set of column-specific parameters is the axial column dispersion (D ax_col ), and / or preferably, the intercolumnar porosity (ε p_col ) includes, preferably consisting of, and step e) is the following step: ea) A step of measuring the column-specific chromatogram of at least one tracer in a chromatography system, wherein the chromatography system includes the chromatography column of step e) instead of the column shortcut of step d), eb) Based on at least one measured column-specific chromatogram, the axial column dispersion (D ax_col ), and / or preferably, the intercolumnar porosity (ε p_col The process of determining ) It includes, and preferably consists of, The tracer does not interact with any components of the chromatography system or chromatography column.

[0089] Alternatively, the intercolumnar porosity (ε p_col ) may be derived from the system pressure curve using the Kozeny-Carman equation.

[0090] Columnar axial dispersion (D ax_col ) is 0.001 mm 2 / s~100mm 2 / s, preferably 0.01 mm 2 / s~10mm 2 / s, most preferably 0.1 mm 2 / s~1mm 2 The range may be / s. Column void ratio (ε p_col ) may be in the range of 0 to 1, preferably 0.1 to 0.8, and most preferably 0.2 to 0.7.

[0091] Preferably, step eb) according to a fifth preferred embodiment of the present invention, by fitting the simulated column-specific chromatogram obtained from the column-specific transport model of step e) to at least one measured column-specific chromatogram, thereby reducing the axial column dispersion (D ax_col ), and / or preferably, the intercolumnar porosity (ε p_col This includes determining the column-specific transport model, preferably consisting of the column axial dispersion (D ax_col), and / or preferably, the intercolumnar porosity (ε p_col The column-specific transport model includes ) as a parameter and describes a chromatography column.

[0092] Preferably, the adaptation of step eb) of the fifth preferred embodiment of the present invention is performed by setting the column axial dispersion (D) as a parameter of the column-specific transport model in step e) until the simulated column-specific chromatogram fits at least one measured column-specific chromatogram. ax_col ), and / or preferably, the intercolumnar porosity (ε p_col This is done by adjusting the following: Preferably, at least one column-specific chromatogram to be measured is measured by injecting the tracer into the chromatography system and then measuring the concentration of at least one tracer downstream of the chromatography column. Preferably, the concentration of the non-bulky tracer is measured by ultraviolet spectroscopy, visible light spectroscopy, near-infrared spectroscopy, refractive index measurement, mass spectrometry, conductivity measurement, fluorescence spectroscopy, chemiluminescence spectroscopy, and / or electrochemistry. Preferably, the tracer is selected from the group consisting of dextran, preferably dextran having a molecular weight of 2 Mda or more, glucose, acetone, sodium chloride, at least one target substance, at least one impurity, protein, peptide, nanoparticles, preferably metal nanoparticles, more preferably gold nanoparticles.

[0093] In the first particularly preferred embodiment of the fifth preferred embodiment of the present invention, the chromatography matrix of the chromatography column comprises, and preferably comprises, a non-porous material.

[0094] In a second particularly preferred embodiment of the fifth preferred embodiment of the present invention, the chromatography matrix of the chromatography column comprises a porous material, preferably consisting of The set of column-specific parameters is the total porosity of the column (ε tot_col ), column effective mass transfer coefficient (k eff_col ), and / or preferably, the column membrane transfer coefficient (kfilm_col ) and column pore diffusion coefficient (D p_col ) further includes both, Step e) is the following: In a chromatography system comprising the chromatography column of step e) instead of the column shortcut of step d), the step of measuring at least one matrix-specific chromatogram of a non-bulky tracer, Based on at least one measured matrix-specific chromatogram, the total porosity of the column (ε tot_col ), column effective mass transfer coefficient (k eff_col ), and / or preferably, the column membrane transfer coefficient (k film_col ) and column pore diffusion coefficient (D p_col This further includes the process of determining both of the following: The tracer cannot penetrate the porous material of the chromatography matrix of the chromatography column. Non-bulky tracers do not interact with any components of the chromatography system or chromatography column. Non-bulky tracers can penetrate the porous material of the chromatography matrix in the chromatography column. Steps ea) and ec) can be performed simultaneously, thereby providing a composite chromatogram including a column-specific chromatogram and a matrix-specific chromatogram, or they can be performed sequentially, thereby providing two separate chromatograms: a column-specific chromatogram and a matrix-specific chromatogram.

[0095] Column total porosity (ε tot_col ) may be in the range of 0 to 1, preferably 0.5 to 0.95, most preferably 0.6 to 0.9. Column effective mass transfer coefficient (k eff_col ) may be in the range of 0.0001 mm / s to 10 mm / s, preferably 0.001 mm / s to 1 mm / s, and most preferably 0.001 mm / s to 0.01 mm / s. Column membrane transfer coefficient (k film_colThe pore diffusion coefficient (D) may be in the range of 0.0001 mm / s to 10 mm / s, preferably 0.001 mm / s to 1 mm / s, and most preferably 0.01 mm / s to 0.1 mm / s. p_col ) is 1x10 -7 mm 2 / s~0.01mm 2 / s, preferably 1x10 -6 mm 2 / s~0.001mm 2 / s, most preferably 1x10 -5 mm 2 / s~1x10 -4 mm 2 It can also be within the range of / s.

[0096] Preferably, step ed) of the second particularly preferred embodiment of the fifth preferred embodiment of the present invention involves fitting a simulated matrix-specific chromatogram obtained from a column-specific transport model to at least one measured matrix-specific chromatogram, thereby determining the total column porosity (ε tot_col ), column effective mass transfer coefficient (k eff_col ), and / or preferably, the column membrane transfer coefficient (k film_col ) and column pore diffusion coefficient (D p_col The process includes determining both of the following, preferably comprising the column-specific transport model, and the column axial dispersion (D) determined in step eb) ax_col ) and intercolumnar porosity (ε p_col ), and the total porosity of the column (ε tot_col ), column effective mass transfer coefficient (k eff_col ), and / or preferably, the column membrane transfer coefficient (k film_col ) and column pore diffusion coefficient (D p_colThe column-specific transport model includes both of the parameters and describes the chromatographic column. Preferably, at least one matrix-specific chromatogram to be measured is determined by injecting a non-bulky tracer into the chromatographic system and then measuring the concentration of a second tracer downstream of the chromatographic column. Preferably, the concentration of the non-bulky tracer is determined by ultraviolet spectroscopy, visible light spectroscopy, near-infrared spectroscopy, refractive index measurement, mass spectrometry, conductivity measurement, fluorescence spectroscopy, chemiluminescence spectroscopy, and / or electrochemistry.

[0097] Alternatively, the process (ed) determines the effective mass transfer coefficient of the column (k) based on already determined values ​​and / or correlations obtained from the literature. eff_col ), and / or preferably, the column pore diffusion coefficient (D p_col ) and column membrane transfer coefficient (k film_col This includes the process of determining both of the following:

[0098] Preferably, the fitting for parameter determination in step ed) of the second particularly preferred embodiment of the fifth preferred embodiment of the present invention is performed until the simulated matrix-specific chromatogram fits to at least one measured matrix-specific chromatogram, with the total column porosity (ε) as a parameter of the column-specific transport model. tot_col ), column effective mass transfer coefficient (k eff_col ), and / or preferably, the column membrane transfer coefficient (k film_col ) and column pore diffusion coefficient (D p_col This is done by adjusting both of the following:

[0099] Preferably, the non-bulky tracer of step ed) of the second particularly preferred embodiment of the fifth preferred embodiment of the present invention is selected from the group consisting of acetone, glucose, at least one target substance, at least one impurity, protein, and peptide.

[0100] It should be understood that the selection of the tracer in step ea) of the second particularly preferred embodiment of the fifth preferred embodiment of the present invention depends on the selection of the chromatography system and the chromatography column, in particular the chromatography matrix. The tracer should have little to no interaction with any unit or tube of the chromatography system, including the chromatography matrix. A preferred combination of chromatography matrix and tracer is, in the case of beads, dextran with a molecular weight of 2 MDa or greater. Furthermore, the tracer should not be permeable to porous materials.

[0101] Similarly, it should be understood that the selection of a non-bulky tracer in step ec) of the second particularly preferred embodiment of the fifth preferred embodiment of the present invention depends on the selection of the chromatography system and the chromatography column, in particular the chromatography matrix. The non-bulky tracer should have little to no interaction with any unit or tube of the chromatography system, including the chromatography matrix. A preferred tracer is NaCl. However, the non-bulky tracer should be permeable to porous materials.

[0102] Process f) In a sixth preferred embodiment of the present invention, step f) includes optimizing at least one process parameter in silico with respect to at least one objective function using at least one optimization algorithm.

[0103] Preferably, the optimization step of the sixth preferred embodiment of the present invention preferably includes a step of adjusting at least one process parameter by an optimization algorithm until the objective function reaches an extremum, preferably a minimum or maximum value.

[0104] Preferably, at least one process parameter optimized by the method according to the present invention is used as an input parameter for a model comprising a set of system-specific parameters in step d), a set of column-specific parameters in step e), and a set of sorption-specific parameters in step c), in a sixth preferred embodiment of the present invention, where the model output provides an input for an objective function. Preferably, the model output is a chromatogram.

[0105] Preferably, at least one objective function of the optimization step in the sixth preferred embodiment of the present invention represents at least one performance criterion of the separation process. Preferably, the performance criterion is selected from a list consisting of the purity of at least one target substance, the recovery rate of at least one target substance, the dilution of at least one target substance, the duration of the separation process, the productivity of the separation process, the volume of at least one collected fraction of at least one target substance, and / or at least one impurity after passing through the chromatography column.

[0106] Preferably, the optimization algorithm for the optimization step of the sixth preferred embodiment of the present invention is a heuristic optimization algorithm or a deterministic optimization algorithm. Preferably, the heuristic optimization algorithm is selected from a list consisting of simulated annealing and genetic algorithms. Preferably, the deterministic optimization algorithm is selected from a list consisting of steepest descent and Levenberg-Marquardt simulation.

[0107] In the first particularly preferred embodiment of the sixth preferred embodiment of the present invention, the separation process includes at least one loading step in which a loading fluid containing at least one target substance and at least one impurity is applied to a chromatographic column by a chromatographic system, and at least one target substance and / or at least one impurity are bound to the chromatographic matrix of the chromatographic column by adsorption. Preferably, at least one process parameter is the flow rate of the at least one loading step, the pH value of the loading fluid, the conductivity of the loading fluid, the composition of the loading fluid, the amount of at least one target substance bound to the chromatographic matrix per volume of the chromatographic column, the amount of at least one impurity bound to a defined volume of the chromatographic matrix, the concentration of at least one target substance, the concentration of at least one impurity in the loading fluid, and / or preferably the duration of the at least one loading step.

[0108] In a second particularly preferred embodiment of the sixth preferred embodiment of the present invention, the separation process further includes at least one washing step, after at least one loading step fluid, to apply washing to a chromatography column by a chromatography system to remove at least one target substance and / or at least one impurity that is not bound to the chromatography matrix of the chromatography column from the chromatography column. Preferably, at least one process parameter is the flow rate of the at least one washing step, the pH value of the washing fluid, the conductivity of the washing fluid, and / or preferably the composition of the washing fluid.

[0109] In a third particularly preferred embodiment of the sixth preferred embodiment of the present invention, the separation process further comprises at least one elution step, in which an elution fluid is applied to the chromatographic column of the chromatography system after at least one loading step or at least one washing step, to induce the desorption of at least one target substance and / or at least one impurity from the chromatographic matrix of the chromatographic column. Preferably, the desorption of at least one target substance and / or at least one impurity from the chromatographic matrix of the chromatographic column is induced by pH value, conductivity, composition, and the concentration of at least one eluent suitable for influencing the adsorption or desorption of at least one target substance and / or at least one impurity to or from the matrix of the chromatographic column. Preferably, at least one process parameter is the flow rate of at least one elution step, the duration of at least one elution step, the initial or final concentration of the elution fluid, the initial or final concentration of at least one eluent, and / or the composition of the elution fluid. A preferred process parameter is the ratio of the concentrations of at least two eluents.

[0110] In a seventh preferred embodiment of the present invention, at least one of the parameters of steps a), b), d), and / or e) is predetermined.

[0111] In the eighth preferred embodiment of the present invention, step a) is step a) of the first preferred embodiment of the present invention, step b) is step b) of the second preferred embodiment of the present invention, step c) is step c) of the third preferred embodiment of the present invention, step d) is step d) of the fourth preferred embodiment of the present invention, step e) is step e) of the fifth preferred embodiment of the present invention, and step f) is step f) of the sixth preferred embodiment of the present invention.

[0112] In the ninth preferred embodiment of the present invention, step a) is step a) of the first preferred embodiment of the present invention, step b) is step b) of the second particularly preferred embodiment of the second preferred embodiment of the present invention, step c) is step c) of the third preferred embodiment of the present invention, step d) is step d) of the fourth preferred embodiment of the present invention, step e) is step e) of the second particularly preferred embodiment of the fifth preferred embodiment of the present invention, and step f) is step f) of the sixth preferred embodiment of the present invention.

[0113] In a tenth preferred embodiment of the present invention, the pre-chromatographic matrix of the pre-chromatographic column is a porous material, and the chromatography matrix of the chromatography column is a porous material.

[0114] In an eleventh preferred embodiment of the present invention, the pre-chromatographic matrix of the pre-chromatographic column is a non-porous material, and the chromatography column is a non-porous material. A partial implementation of this eleventh preferred embodiment, including steps a), b), c), d), and e), is shown in T. Hahn et al., "Predictive scaling of fiber-based protein: A capture chromatography using mechanistic modelling," Biotechnology and Bioengineering, May 20, 2023, pp. 1-12 (https: / / doi.org / 10.1002 / bit.28434). The contents of this publication are incorporated more fully herein by reference to the greatest extent possible.

[0115] In a twelfth preferred embodiment of the present invention, the pre-chromatographic matrix of the pre-chromatographic column is a porous material, and the chromatography column is a non-porous material.

[0116] In a thirteenth preferred embodiment of the present invention, the pre-chromatography matrix of the pre-chromatography column is a non-porous material, and the chromatography column is a porous material.

Claims

1. A method for optimizing at least one process parameter of a separation process for at least one target substance from at least one impurity using a chromatography system, comprising the following steps: a) A step of providing a set of preliminary system-specific parameters suitable for describing the concentration transport of at least one target substance and at least one impurity in a preliminary chromatography system using a system-specific transport model, wherein the preliminary chromatography system does not include any chromatography column but includes a column shortcut. b) A step of providing a set of pre-column specific parameters suitable for describing the concentration transport of at least one target substance and at least one impurity in a pre-chromatographic column containing a pre-chromatographic matrix, using a column-specific transport model. c) A step of determining a set of sorption-specific parameters suitable for describing the adsorption and / or desorption of at least one target substance and at least one impurity to or from a pre-chromatographic matrix contained in a pre-chromatographic column, using an sorption model, a set of pre-system-specific parameters, and a set of pre-column-specific parameters, wherein the pre-chromatographic system includes the pre-chromatographic column of step b) instead of the column shortcut of step a), d) A step of providing a set of system-specific parameters suitable for describing the concentration transport of at least one target substance and at least one impurity in a chromatography system using a system-specific transport model, wherein the chromatography system does not include any chromatography column but includes a column shortcut. e) A step of providing a set of column-specific parameters suitable for describing the concentration transport of at least one target substance and at least one impurity in a chromatography column containing a chromatography matrix, using a column-specific transport model. f) A step of optimizing at least one process parameter in silico using the following parameters: a set of system-specific parameters from step d), a set of column-specific parameters from step e), and a set of sorption-specific parameters from step c), wherein the chromatography system includes the chromatography column from step e) instead of the column shortcut from step d), Includes, A method wherein the sum of the volume of a chromatography system without any chromatography columns and the volume of chromatography columns is different from the sum of the volume of a spare chromatography system without any chromatography columns and the volume of spare chromatography columns.

2. The method according to claim 1, wherein the volume of the chromatography system without any chromatography column is greater than the volume of the pre-chromatographic system without any chromatography column, preferably 10 to 100,000 times greater, more preferably 100 to 70,000 times greater, most preferably 1,000 to 10,000 times greater, and / or the volume of the chromatography column is greater than the volume of the pre-chromatographic column, preferably 10 to 100,000 times greater, more preferably 100 to 10,000 times greater, most preferably 1,000 to 5,000 times greater, preferably the volume of the chromatography system including the chromatography column is in the range of more than 1.5 l to 1,000 l, preferably 13 l to 800 l, and the volume of the pre-chromatographic system including the pre-chromatographic column is in the range of 0.0003 l to 1.5 l, preferably 0.001 l to 1.0 l.

3. In step a), a set of preliminary system-specific parameters is used to determine the preliminary system internal dispersion (D) of the preliminary chromatography system. ax_pre_sys ) and / or pre-system mixing rate (MR pre_sys ) preferably comprising, and the preliminary chromatography system does not include any chromatography column but includes a column shortcut, Step a) is the following step: aa) A step of measuring at least one preliminary system-specific chromatogram of a tracer in a preliminary chromatography system, wherein the preliminary chromatography system does not include any chromatography column but includes a column shortcut. ab) Based on at least one measured preliminary system-specific chromatogram, the preliminary system internal variance (D ax_pre_sys ) and / or pre-system mixing rate (MR pre_sys The process of determining ) It includes, and preferably consists of, The method according to claim 1 or 2, wherein the tracer does not interact with any component of the chromatography system.

4. A set of preliminary column-specific parameters determines the preliminary column axial dispersion (D ax_pre_col ) and / or preliminary column void ratio (ε p_pre_col ) includes, preferably consists of, Step b) is the following step: ba) A step of measuring at least one pre-column-specific chromatogram of a tracer in a pre-chromatography system, wherein the pre-chromatography system includes the pre-chromatography column of step b) instead of the column shortcut of step a), bb) Based on at least one measured preliminary column-specific chromatogram, the preliminary column axial dispersion (D ax_pre_col ) and / or preliminary column void ratio (ε p_pre_col The process of determining ) It includes, and preferably consists of, The method according to any one of claims 1 to 3, wherein the tracer does not interact with any component of the pre-chromatography system or pre-chromatography column.

5. The pre-chromatographic matrix of the pre-chromatographic column contains a porous material, preferably composed of A set of pre-column specific parameters includes the total void fraction of the pre-column (ε tot_pre_col ), the effective mass transfer coefficient of the pre-column (k eff_pre_col ), and / or both the film transfer coefficient of the pre-column (k film_pre_col ) and the pore diffusion coefficient of the pre-column (D p_pre_col ). Step b) is the following step: bc) A step of measuring at least one preliminary matrix-specific chromatogram of a non-bulky tracer in a preliminary chromatography system that includes a preliminary chromatography column instead of the column shortcut of step a), bd) Based on at least one measured preliminary matrix-specific chromatogram, the total porosity of the preliminary column (ε tot_pre_col ), Pre-column effective mass transfer coefficient (k eff_pre_col ), and / or the preliminary column membrane transfer coefficient (k film_pre_col ) and preliminary column pore diffusion coefficient (D p_pre_col The process of determining both of the following It further includes, The tracer is unable to penetrate the porous material of the preliminary chromatography matrix of the preliminary chromatography column. The non-bulky tracer does not interact with any component of the pre-chromatography system or pre-chromatography column. Non-bulky tracers can penetrate the porous material of the preliminary chromatography matrix of the preliminary chromatography column. The method according to any one of claims 1 to 4, wherein steps ba) and bc) can be performed simultaneously to provide a composite chromatogram including a preliminary column-specific chromatogram and a preliminary matrix-specific chromatogram, or can be performed sequentially to provide two separate chromatograms, a preliminary column-specific chromatogram and a preliminary matrix-specific chromatogram.

6. Step c) is the following step: ca) A step of measuring at least one pre-target-specific chromatogram of at least one target substance in a pre-chromatography system, wherein the pre-chromatography system includes the pre-chromatography column of step b) instead of the column shortcut of step a), cb) A step of measuring at least one pre-impurity-specific chromatogram of at least one impurity to be measured in a pre-chromatography system, wherein the pre-chromatography system includes the pre-chromatography column of step b) instead of the column shortcut of step a), cc) A step of determining a set of sorption-specific parameters based on at least one measured preliminary target-specific chromatogram and at least one measured preliminary impurity-specific chromatogram. It includes, and preferably consists of, The method according to any one of claims 1 to 5, wherein steps ca) and cb) can be performed simultaneously to provide a composite chromatogram including a target-specific chromatogram and an impurity-specific chromatogram, or can be performed sequentially to provide two separate chromatograms, a target-specific chromatogram and an impurity-specific chromatogram.

7. Process cc) is the following process: cca) A step of determining the sorption-specific parameters of a target substance by fitting a simulated preliminary target-specific chromatogram of at least one target substance to at least one preliminary target-specific chromatogram to be measured in a preliminary chromatography system that includes a preliminary chromatography column in step b) instead of a column shortcut in step a), wherein the simulated preliminary target-specific chromatogram is - An sorption model for step c), which includes sorption-specific parameters of the target substance as parameters, and describes the adsorption / desorption behavior of at least one target substance by a pre-chromatographic matrix contained in a pre-chromatographic column, - System-specific transport model for step a), and - Column-specific transport model for step b) The process obtained from A step in which the sorption-specific parameters of an impurity are determined by fitting a simulated preliminary impurity-specific chromatogram of at least one impurity to at least one measurable preliminary impurity-specific chromatogram in a preliminary chromatography system that includes a preliminary chromatography column instead of the column shortcut of step a), wherein the simulated preliminary impurity-specific chromatogram is - An sorption model for step c), which includes sorption-specific parameters for impurities as parameters, and describes the adsorption / desorption behavior of at least one impurity by the pre-chromatographic matrix contained in the pre-chromatographic column, - System-specific transport model for step a), and - Column-specific transport model for step b) The process obtained from It includes, and preferably consists of, The method according to claim 6, wherein the set of sorption-specific parameters preferably includes sorption-specific parameters of the target substance and sorption-specific parameters of at least one impurity.

8. The sorption-specific parameter of at least one target substance is the pre-equilibrium constant (k) of at least one target substance relative to the pre-chromatographic matrix. eq_target ), and / or the preliminary kinetic constant (k) of at least one target substance from the preliminary chromatography matrix. kin_target The method according to claim 7, preferably comprising )

9. The sorption-specific parameter of at least one impurity is the pre-equilibrium constant (k) of at least one impurity relative to the pre-chromatographic matrix. eq_imp ), and / or the preliminary kinetic constant (k) of at least one impurity from the preliminary chromatography matrix. kin_imp The method according to claim 7 or 8, preferably comprising )

10. In step d), the system-specific set of parameters determines the system internal dispersion (D) of the chromatography system. ax_sys ) and / or system mixing rate (MR sys ) preferably comprising, and the chromatography system does not include any chromatography column but includes a column shortcut, Step d) is the following step: da) A step of measuring at least one system-specific chromatogram of a tracer in a chromatography system, wherein the chromatography system does not include any chromatography column but includes a column shortcut. Based on at least one measured system-specific chromatogram, the system internal variance (D) is calculated. ax_sys ) and / or system mixing rate (MR sys The process of determining ) It includes, and preferably consists of, The method according to any one of claims 1 to 9, wherein the tracer does not interact with any component of the chromatography system.

11. A set of column-specific parameters determines the axial column dispersion (D ax_col ) and / or intercolumnar porosity (ε p_col ) includes, preferably consists of, Step e) is the following step: ea) A step of measuring the column-specific chromatogram of at least one tracer in a chromatography system, wherein the chromatography system includes the chromatography column of step e) instead of the column shortcut of step d), eb) Based on at least one measured column-specific chromatogram, the axial column dispersion (D ax_col ) and / or intercolumnar porosity (ε p_col The process of determining ) It includes, and preferably consists of, The method according to any one of claims 1 to 10, wherein the tracer does not interact with any component of the chromatography system or chromatography column.

12. The chromatography matrix of the chromatography column comprises a porous material, preferably consisting of A set of column-specific parameters determines the total column porosity (ε tot_col ), column effective mass transfer coefficient (k eff_col ), and / or the column membrane transfer coefficient (k film_col ) and column pore diffusion coefficient (D p_col ) further includes both, Step e) is the following step: In a chromatography system comprising the chromatography column of step e) instead of the column shortcut of step d), the step of measuring at least one matrix-specific chromatogram of a non-bulky tracer, Based on at least one measured matrix-specific chromatogram, the total porosity of the column (ε tot_col ), column effective mass transfer coefficient (k eff_col ), and / or the column membrane transfer coefficient (k film_col ) and column pore diffusion coefficient (D p_col The process of determining both of the following It further includes, The tracer is unable to penetrate the porous material of the chromatography matrix of the chromatography column. Non-bulky tracers do not interact with any components of the chromatography system or chromatography column. Non-bulky tracers can penetrate the porous material of the chromatography matrix in the chromatography column. The method according to any one of claims 1 to 11, wherein steps ea) and ec) can be performed simultaneously, thereby providing a composite chromatogram including a column-specific chromatogram and a matrix-specific chromatogram, or can be performed sequentially, thereby providing two separate chromatograms, a column-specific chromatogram and a matrix-specific chromatogram.

13. Step f) includes a step of optimizing at least one process parameter in silico with respect to at least one objective function using at least one optimization algorithm, The method according to any one of claims 1 to 12, preferably comprising the step of adjusting at least one process parameter by an optimization algorithm until the objective function reaches an extremum, preferably a minimum or maximum value.

14. The method according to claim 13, wherein at least one process parameter is used as an input parameter to a model comprising a set of system-specific parameters for step d), a set of column-specific parameters for step e), and a set of sorption-specific parameters for step c), and the model output, preferably a chromatogram, provides the input to an objective function.

15. At least one objective function represents at least one performance criterion for the separation process, The method according to claim 13 or 14, wherein the performance criteria are preferably selected from a list consisting of the purity of at least one target substance, the recovery rate of at least one target substance, the dilution of at least one target substance, the duration of the separation process, the productivity of the separation process, the volume of at least one collected fraction of at least one target substance, and / or at least one impurity after passing through the chromatography column.

Images