Multi modular chromatography system

Abstract

A chromatography system comprising a mobile phase origin A, an eluent phase origin B, a sample origin C, a waste sink D, a target sink E, a central control unit F, and at least two chromatography units Gx, wherein each chromatography unit Gx comprises an inlet valve ax, a pump bx, a chromatography column cx having an inlet and an outlet, an outlet valve dx, and a client control unit fx, wherein the inlet valve is in fluid connection with the pump via an inlet conduit, wherein the pump is in fluid connection with the inlet of the chromatography column via a column conduit, wherein the outlet of the chromatography column is in fluid connection with the outlet of the chromatography unit via an outlet conduit, wherein the mobile phase origin and the eluent phase origin are in fluid connection with each inlet valve of each of the at least two chromatography units, preferably via separate flow paths, wherein the sample origin is in fluid connection with the mobile phase origin, each inlet valve of each of the at least two chromatography units, preferably via a separate flow path, each inlet conduit of each of the at least two chromatography units, preferably via a sample-inlet joint, each column conduit of each of the at least two chromatography units, preferably via a sample-column joint sjx, or mixtures thereof, wherein each outlet of each of the at least two chromatography units is in fluid connection with the waste sink via a waste conduit, wherein each outlet of each of the at least two chromatography units is in fluid connection with the target sink via a target conduit, and wherein each client control unit of each of the at least two chromatography units cx is in communication with the central control unit F, wherein the outlet valve dx is in fluid connection with the inlet valve of a subsequent chromatography unit of the at least two chromatography units, preferably via a separate flow path, the inlet conduit of the subsequent chromatography unit, preferably via the sample-inlet joint of the subsequent chromatography unit, the column conduit of the subsequent chromatography unit, preferably via the sample-column joint of the subsequent chromatography unit, or mixtures thereof, wherein, if the chromatography unit is the last chromatography unit Gx, the subsequent chromatography unit is the first chromatography unit G1.

Description

[0001] Multi Modular Chromatography System

[0002] Technical Field

[0003] The present invention is concerned with a multi modular chromatography system.

[0004] Background

[0005] In the manufacturing of biopharmaceuticals, such as vaccines, antibodies, recombinant proteins, and gene therapy vectors, several chromatographic separation steps are usually needed to remove various contaminants and impurities from the product. These separation steps add significant cost and process time. Hence, there is a general interest in improving the yield and throughput of each of the steps. Furthermore, in downstream bioprocessing, many of the chromatography steps used are carried out in a non-continuous manner, i.e., in batch-wise mode. Thus, downstream bioprocessing is usually tried to be carried out in semi-continuous mode. Thereby, it is beneficial that each batch-wise step can reach maximum throughput.

[0006] Normally, in a chromatography process, one separation device such as a column or a membrane is used. To solve the above-mentioned problems, multicolumn continuous chromatography (MCC) has been developed, in which more than one separation device are connected to each other. Usually, the separation devices are of the same type. Hence, e.g., in MCC mode, a chromatography process comprises the usage of three chromatography columns having the same dimensions and the same stationary phase. If the columns are not identical, the theoretical calculations typically used to design continuous chromatography process will not be correct, and it will become difficult to design an efficient and robust continuous chromatography process. The same argument applies if feed concentration and flow rates vary with time in an unexpected manner.

[0007] In MCC, the feed is applied to the first column and is then diverted to one or more subsequent columns as the first columns approaches saturation. The first column is subsequently eluted and regenerated to be loaded again during elution and regeneration of the subsequent column(s). Such processes are usually denoted as periodic counter-current chromatography (PCC) or simulated moving bed (SMB) and are of considerable interest for separation of biopharmaceuticals (cf. US 7,901 ,581 B1 , US 2013 / 0248451 A1 , US 2013 / 0280788 A1 and US 7,220,356 B1 , the contents of which are hereby incorporated by reference to the maximum permissible extent possible). PCC / SMB processes can significantly increase the productivity, but the increases achieved can depend quite strongly on the design of the process and apparatus. Thus, in MCC all columns can be run in principle simultaneously, but with the method steps shifted in time. The procedure can be repeated, so that each column is loaded, cleaned, and regenerated several times in the process. Compared to ‘conventional’ chromatography in continuous chromatography based on multiple identical columns all steps, such as sample loading, stripping, cleaning in place (CIP), and re-equilibration, occur simultaneously, but on different columns each.

[0008] Continuous chromatography operation results in better utilization of the chromatography resin, i.e. , the stationary phase, reduced processing time, decreased amount of resin needed, and reduced buffer requirements, all of which benefits process economy.

[0009] Summary of the Invention

[0010] However, commercially available PCC systems, while being very efficient, tend to be very complex. Furthermore, such systems have certain limitations, cannot be easily extended, such as the number of columns to be used, and also often cannot be easily scaled up or down.

[0011] Hence, the object of the present invention is to provide a modular, simple, and easily extendable multi column chromatography system, which can be run in periodic countercurrent chromatography used.

[0012] It has now surprisingly been found out that above-mentioned object is achieved by a chromatography system comprising a mobile phase origin, an eluent phase origin, a sample origin, a waste sink, a target sink, a central control unit, and at least two chromatography units, wherein each chromatography unit comprises an inlet valve, a pump, a chromatography column having an inlet and an outlet, an outlet valve, and a client control unit, wherein the inlet valve is in fluid connection with the pump via an inlet conduit, wherein the pump is in fluid connection with the inlet of the chromatography column via a column conduit, wherein the outlet of the chromatography column is in fluid connection with the outlet of the chromatography unit via an outlet conduit, wherein the mobile phase origin and the eluent phase origin are in fluid connection with each inlet valve of each of the at least two chromatography units, preferably via separate flow paths, wherein the sample origin is in fluid connection with the mobile phase origin, each inlet valve of each of the at least two chromatography units, preferably via a separate flow path, each inlet conduit of each of the at least two chromatography units, preferably via a sample-inlet joint, each column conduit of each of the at least two chromatography units, preferably via a sample-column joint, or mixtures thereof, wherein each outlet of each of the at least two chromatography units is in fluid connection with the waste sink via a waste conduit, wherein each outlet of each of the at least two chromatography units is in fluid connection with the target sink via a target conduit, and wherein each client control unit of each of the at least two chromatography units is in communication with the central control unit, wherein the outlet valve is in fluid connection with the inlet valve of a subsequent chromatography unit of the at least two chromatography units, preferably via a separate flow path, the inlet conduit of the subsequent chromatography unit, preferably via the sample-inlet joint of the subsequent chromatography unit, the column conduit of the subsequent chromatography unit, preferably via the sample-column joint of the subsequent chromatography unit, or mixtures thereof, wherein, if the chromatography unit is the last chromatography unit, the subsequent chromatography unit is the first chromatography unit.

[0013] Brief Description of the Drawings

[0014] Figure 1 shows a schematic view of the chromatography system according to the present invention including two chromatography columns including a mobile phase (A), an eluent phase (B), and a sample phase (C).

[0015] Figure 2 shows a schematic view of a preferred embodiment of the chromatography system according to the present invention including two chromatography columns, wherein the mobile phase (A) and the eluent phase (B) are each provided by an active phase origin (A1) and an inversely active phase origin (B1).

[0016] Figure 3 shows a schematic view of a preferred embodiment of the chromatography system according to the present invention including three chromatography columns including a mobile phase (A), an eluent phase (B), and a sample phase (C).

[0017] Figure 4 shows a schematic view of a preferred embodiment of the chromatography system according to the present invention including three chromatography columns, wherein the mobile phase (A) and the eluent phase (B) are each provided by an active phase origin (A1) and an inversely active phase origin (B1).

[0018] Figure 5 shows a schematic view of a preferred embodiment of the chromatography system according to the present invention including three chromatography columns including a mobile phase (A), an eluent phase (B), and a sample phase (C), further having an additional connection of the outlet of a column with the inlet of a subsequent-to-subsequent column (enabling post load washing).

[0019] Figure 6 shows a schematic view of a preferred embodiment of the chromatography system according to the present invention including three chromatography columns, wherein the mobile phase (A) and the eluent phase (B) are each provided by an active phase origin (A1) and an inversely active phase origin (B 1 ) , further having an additional connection of the outlet of a column with the inlet of a subsequent-to-subsequent column (enabling post load washing).

[0020] Figure 7 shows a schematic view of the steps the central control unit (F) of a chromatography system according to Figure 1 is configured to carry out to achieve a bind / elute mode periodic counter-current chromatography without a post load washing step.

[0021] Figure 8 shows a schematic view of the steps the central control unit (F) of a chromatography system according to Figure 1 is configured to carry out to achieve a flow-through mode periodic counter-current chromatography without a post load washing step.

[0022] Figure 9 shows a schematic view of the steps the central control unit (F) of a chromatography system according to Figure 5 is configured to carry out to achieve a bind / elute mode periodic counter-current chromatography including post load washing steps.

[0023] Definitions

[0024] The term “chromatography system" as used herein denotes a system comprising a pump, a first tubing, and a chromatography column or a column shortcut, wherein the first tubing fluidly connects the pump with the chromatography column or the column shortcut. Preferably, the chromatography system further comprises a second tubing and a detector and / or fraction collector, wherein the second tubing fluidly connects the column or the column shortcut with the detector and / or fraction collector. The chromatography system usually implements input and output streams by connecting respective vessels by tubing to the pump and / or the detector. 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 AKTA start, AKTA go, AKTA pure, AKTA avant, AKTA pilot, or AKTA ready available by Cytiva.

[0025] The term “unit’ denotes a device comprised in the chromatography system, wherein different units of the chromatography system are usually connected by tubing. Preferably, the unit is selected from the list consisting of a chromatography column, a column shortcut, a detector, a fraction collector, a valve, a bubble trap, or a mixing chamber.

[0026] The term “detector"’ as used herein may be any device suitable for detecting substances in a fluid passing the device. Preferably, the detector is selected from the list consisting of an ultraviolet light absorption (UV) detector, a visible light absorption (VIS) detector, a photo diode array (PDA) detector, a refractive-index detector, an evaporative light scattering detector, a multi-angle light scattering detector, a mass spectrometer, a conductivity detector, a fluorescence detector, a chemiluminescence detector, an optical rotation detector, and an electrochemical detector. Preferably the detector is located downstream of the column.

[0027] The term “chromatography column" as used herein denotes a device for the separation of substances comprising a chromatography matrix as stationary phase.

[0028] The term “chromatography matrix" denotes the stationary phase in a chromatography separation process. Preferably, the chromatography matrix comprises at least one active moiety that interacts by one or more physical or chemical interactions with the target substance and / or the impurity. The chromatography matrix may be fibrous, monolithic, membranous, or particular. Preferably, the chromatography matrix is a compound selected from the list consisting of natural polymers and / or synthetic polymers, preferably a compound selected from the list consisting of polysaccharides, polystyrene, polyacrylamide, polymethacrylate, or mixtures thereof.

[0029] The term “dextran” as used herein denotes a large polymer of anhydroglucose having a molecular weight in the range of from 3 kDa to 5 MDa. Dextrans having a molecular weight of more than or equal to 2 MDa are not able to penetrate the micro pores of chromatography matrix comprising, preferably consisting of, a porous material.

[0030] The term “active moiety” as used herein denotes a molecule or substance that interacts by one or more physical or chemical interactions with the target substance and / or the impurity. Preferably, the active moiety is selected from the list consisting of sulfopropyl, diethylaminoethyl, diethylaminopropyl, diethyl-(2-hydroxy-propyl)aminoethyl, octylamine, N-benzyl-n-methyl ethanolamine, methyl sulfonate, quarternary ammonium, carboxymethyl, alkyl, preferably octyl, hexyl, butyl, phenyl, bi-phenyl, pentafluorophenyl, phenyl-hexyl, ethyl, benzyl, or isopropyl, silanol, peptides, nucleotides , protein, an immunoglobulin binding protein, preferably protein A, protein G, protein L, metal ions, preferably cations of Cu, Ni, Co, Zn, nitrilotriacetic acid, domain antibody or domain antibody fragment, CHT, poly deoxythymidine (e.g. 25 mer for dT), C4, Ce, Cs, C10, C12, C14, C16, octadecyl carbon (Cis), C20, C22, C24, heparin, dextran sulphate, 2-mercaptopyridine, hydroxylapatite, fluoroapatite, or combinations thereof. The term “moiety-specific interaction" , also known as affinity interaction, as used herein denotes a specific interaction between the target substance and / or the impurity, and an interaction partner in form of the active moiety. Preferably, the target molecule / and or impurity binds specifically to the active moiety. The moiety specific interaction is preferably selected from the following: an interaction between an enzyme as target substance and / or impurity with a substrate analogue as moiety, an interaction between an antigen as target substance and / or impurity with an antibody as moiety, an interaction between a polysaccharide as target substance and / or impurity with a lectin as moiety, an interaction between a complementary base sequence as target substance and / or impurity with a nucleic acid as moiety, an interaction between a hormone receptor as target substance and / or impurity with a hormone as moiety, an interaction between biotin or biotin-conjugated substance, preferably a biotin-conjugated protein, as target substance and / or impurity with avidin as moiety, an interaction between a calmodulin binding partner as target substance and / or impurity with calmodulin as moiety, an interaction between a glutathione S-transferase fused substance, preferably a glutathione S-transferase fused protein, as target substance and / or impurity with glutathione as moiety, an interaction between an immunoglobulin as target substance and / or impurity with an immunoglobulin binding substance, preferably an immunoglobulin binding protein as moiety, an interaction between a polyhistidine fused substance, preferably a polyhistidine fused protein, as target substance and / or impurity with a complex of a metal cation binding protein as moiety

[0031] The term “fluid volume” as used herein denotes a volume comprising a fluid and preferably the target species. Preferably, the fluid is a liquid. More preferably, the fluid is a liquid comprising, preferably consisting of, a solvent. Even more preferably, the solvent is selected from water-based buffer solutions, such as phosphate, acetate, citrate, or tris(hydroxymethyl)aminomethane, and mixtures thereof with other solvents such as ethanol. The solvents can comprise additives such as polymers, e.g., PEG and / or dextran, salts, e.g., ammonium sulphate, sodium chloride etc., detergents, and / or stabilizers. Some affinity stationary phases (membranes or beads) may require more complex mixtures. For instance, Immobilized Metal Chelate Affinity Chromatography (IMAC) requires the addition of an organic compound such as imidazole.

[0032] The term “target species" as used herein denotes the product to be isolated. Usually, the target species is a substance naturally occurring in animal or plant tissue, is produced by naturally occurring microorganisms or enzymes, and / or is produced by modified, preferably genetically modified, microorganisms or enzymes. As such, the target species can be, but is not limited to, antibodies, proteins, viruses, organic substances, DNA molecules, RNA molecules, shorter nucleotides molecules, exosomes, cells (such as used in cell therapy), or specific target molecules.

[0033] The term “raw mixture" as used herein denotes a mixture of various contaminant, solvents, and at least one target species. The mixture can be a natural product, such as animal or plant tissues, which already has been brought in contact with at least one solvent, or a mixture from a bioreactor. Preferably, the raw mixture is a mixture from a bioreactor comprising microorganisms, such as yeasts, bacteria, and / or fungi, cells, viruses, tissue, target species, and / or solvents.

[0034] The term “flow-through mode" as used herein denotes a chromatography method, in which impurities in a raw mixture are bound to the chromatography matrix, while the target species is not bound, thereby separating the target species in the dilute.

[0035] The term “bind / elute mode" as used herein denotes a chromatography method, in which the target species in a raw mixture is bound to the chromatography matrix, while the impurities are not bound. The impurities are washed from the column with the dilute and the column is flushed with an elute, which removes the target species from the chromatography column, thereby separating the target species in the elute.

[0036] The term “bioprocessing unit" as used herein denotes a unit, which receives a fluid volume comprising a target species and usually contaminants and which outputs a fluid volume comprising the target species and usually less contaminants. Preferably, the bioprocessing unit is selected from the list consisting of chromatography devices, including columns and membranes, filters, centrifuges, extraction devices, and two- phase separation devices.

[0037] The term “off-line analysis" as used herein denotes an analysis step, wherein the sample is taken out of the bioprocessing unit or a transportation line in sterile conditions and analysed in a laboratory after physical pre-treatments (e.g., filtration and dilution). Preferably, the preparation and handling require clear Standard Operating Procedures (SOPs) as well as skilled personnel. Together with the complexity involved in manual handling, the major disadvantage of off-line measurement is the time delay, which results in lower measurement frequency. Due to these issues off-line measurements are usually not considered true PAT (Process Analytical Technology) unless there are no other measurement possibilities (e.g., HPLC for product titter or mass spectroscopy for product quality). Off-line laboratory measurements are commonly used to monitor and validate the accuracy of the in-line / on-line process analysers.

[0038] The term “at-line analysis" as used herein denotes an analysis step, wherein the sample is removed from the bioprocessing unit or a transportation line and is analysed in close proximity to the production process, either manually or by using automated sampling devices. Similar to off-line measurement, sterile conditions must be maintained for accurate results. At-line measurement is most common for parameters which cannot be measured accurately in-line or on-line. Advantages of at-line measurement include shortened time delay (relative to off-line), and the possibility for automated control.

[0039] The term "on-line analysis" as used herein denotes an analysis step, wherein the sample is diverted from the bioprocessing unit or a transportation line by a branching and may be returned to the bioprocessing process after analysis. The sample is automatically measured in the by-pass by process sensors. The advantages of this method can be found in the simple sterilization and the straightforward access to the sample in stationary conditions. The implementation of such a solution requires a specifically designed or modified bioprocessing process, in particular bioprocessing unit and / or transportation line.

[0040] The term “in-line analysis" as used herein denotes an analysis step, wherein the measurement occurs directly in the bioprocessing unit or the transportation line with a process sensor. Preferably, the generated measurements are sent in real-time to the control system. Process parameters such as pH, ORP (redox potential), dissolved oxygen, dissolved CO2, temperature, and / or conductivity are commonly measured by in-line analysis. Moreover, also measurements allowing for detection of specific molecules can be measured by in-line analysis, such as LIV / VIS absorbance / transmittance, fluorescence emission, and refractive index.

[0041] The term “post load washing" (PLW) as used herein denotes a technique in bind / elute mode, wherein after loading of a column has been finished, the loaded column is washed with the dilute, i.e., without eluting the target species from the column, thereby removing target species, which is not bound to the chromatography matrix. Preferably, the washing dilute is directed towards the subsequent column, which will be loaded in the next cycle. Thereby, the target species, which has not been bound to the chromatography matrix being subject to the post load wash will be subjected to a binding process to the chromatography matrix of the subsequent column. This ensures the maximum binding of non-bound target species after loading of the first column.

[0042] As used in this specification and in the appended claims, the singular forms of “a“ and “an" also include the respective plurals unless the context clearly dictates otherwise. In the context of the present invention, the terms “about" and “approximately" denote an interval of accuracy that a person skilled in the art will understand to still ensure the technical effect of the feature in question. The term typically indicates a deviation from the indicated numerical value of ±10 %, preferably ±8 %, more preferably ±5 %, even more preferably ±2 %. It is to be understood that the term “comprising" and "encompassing" is not limiting. For the purposes of the present invention the term "consisting of‘ is considered to be a preferred embodiment of the term "comprising of‘. If hereinafter a group is defined to comprise at least a certain number of embodiments, this is meant to also encompass a group which preferably consists of these embodiments only. Furthermore, the terms “first", “second", “third" or “(a)", “(b)“, “(c)“, “(d)“ etc. and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein. In case the terms "first", "second", "third" or "(a)", "(b)", “(c)“, "(d)", "i", “K“ etc. relate to steps of a method or use or assay there is no time or time interval coherence between the steps, i.e. the steps may be carried out simultaneously or there may be time intervals of seconds, minutes, hours, days, weeks, months or even years between such steps, unless otherwise indicated in the application as set forth herein above or below. It is to be understood that this invention is not limited to the particular methodology, protocols, reagents etc. described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention that will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0043] As used herein the term “does not comprise", “does not contain", or “free of’ means in the context that the composition of the present invention is free of a specific compound or group of compounds, which may be combined under a collective term, that the composition does not comprise said compound or group of compounds in an amount of more than 0.8 % by weight, based on the total weight of the composition. Furthermore, it is preferred that the composition according to the present invention does not comprise said compounds or group of compounds in an amount of more than 0.5 % by weight, preferably the composition does not comprise said compounds or group of compounds at all.

[0044] When referring to compositions and the weight percent of the therein comprised ingredients it is to be understood that according to the present invention the overall amount of ingredients does not exceed 100% (± 1 % due to rounding). Detailed Description

[0045] In various aspects, the present invention provides a multi column modular chromatography system, which is suitable for being run in continuous mode, preferably in periodic counter-current continuous mode (PCC). The chromatography system is designed to be able to combine existing single-column chromatography units, such as are AKTA start, AKTA go, AKTA pure, AKTA avant, AKTA pilot, or AKTA ready available by Cytiva, resulting in a multi column modular chromatography system. This allows a modular design. Furthermore, it allows easy exchange not only of the used columns, but of the whole chromatography units in the system. Finally, the system is easily extendible in view of chromatography units, e.g., chromatography columns.

[0046] The most general embodiment of the present invention is depicted in Figure 1. Hence, the present invention provides a chromatography system comprising:

[0047] ■ a mobile phase origin (A),

[0048] ■ an eluent phase origin (B),

[0049] ■ a sample origin (C),

[0050] ■ a waste sink (D),

[0051] ■ a target sink (E),

[0052] ■ a central control unit (F), and

[0053] ■ at least two chromatography units (Gx), wherein each chromatography unit (Gx) comprises:

[0054] - an inlet valve (ax),

[0055] - a pump (bx),

[0056] - a chromatography column having an inlet and an outlet (cx),

[0057] - an outlet valve (dx), and

[0058] - a client control unit (fx), wherein the inlet valve (ax) is in fluid connection with the pump (bx) via an inlet conduit, wherein the pump (bx) is in fluid connection with the inlet of the chromatography column (cx) via a column conduit, wherein the outlet of the chromatography column (cx) is in fluid connection with the outlet (dx) of the chromatography unit (Gx) via an outlet conduit, wherein the mobile phase origin (A) and the eluent phase origin (B) are in fluid connection with each inlet valve (ax) of each of the at least two chromatography units (Gx), preferably via separate flow paths, wherein the sample origin (C) is in fluid connection with the mobile phase origin (A), each inlet valve (ax) of each of the at least two chromatography units (Gx), preferably via a separate flow path, each inlet conduit of each of the at least two chromatography units (Gx), preferably via a sample-inlet joint (six), each column conduit of each of the at least two chromatography units (Gx), preferably via a sample-column joint (sjx), or mixtures thereof, wherein each outlet of each of the at least two chromatography units (Gx) is in fluid connection with the waste sink (D) via a waste conduit, wherein each outlet of each of the at least two chromatography units (Gx) is in fluid connection with the target sink (E) via a target conduit, and wherein each client control unit (fx) of each of the at least two chromatography units (Gx) is in communication with the central control unit (F), wherein the outlet valve (dx) is in fluid connection with the inlet valve (ax+i) of a subsequent chromatography unit (Gx+i) of the at least two chromatography units (Gx), preferably via a separate flow path, the inlet conduit of the subsequent chromatography unit (Gx+i), preferably via the sample-inlet joint (six+i) of the subsequent chromatography unit (Gx+i) , the column conduit of the subsequent chromatography unit (Gx+i), preferably via the sample-column joint (sjx+i) of the subsequent chromatography unit (Gx+i), or mixtures thereof, wherein, if the chromatography unit is the last chromatography unit (Gx), the subsequent chromatography unit is the first chromatography unit (Gi).

[0059] Preferably, the at least two chromatography units (Gx) are chromatography modules, such as AKTA start, AKTA go, AKTA pure, AKTA avant, AKTA pilot, or AKTA ready. Preferably, the chromatography columns (cx) of the at least two chromatography units (Gx) include a chromatography matrix, which is able to absorb either the impurities to be separated from the target species or the target species itself. This has the advantage that the column can be loaded with target species and eluted later to yield a cleaned target species. Hence, preferably, the chromatography matrix is suitable for affinity chromatography.

[0060] As set out above, the sample origin (C) is in fluid connection with the mobile phase origin (A), each inlet valve (ax) of each of the at least two chromatography units (Gx), preferably via a separate flow path, each inlet conduit of each of the at least two chromatography units (Gx), preferably via a sample-inlet joint (six), each column conduit of each of the at least two chromatography units (Gx), preferably via a sample-column joint (sjx), or mixtures thereof. Hence, the sample origin (C) could be added to the system via the mobile phase origin (A). Hence, in such an embodiment, the sample would be added directly to the mobile phase origin (A). This embodiment has the disadvantage that the sample will be distributed over a large volume of mobile phase, therefore resulting in broadened chromatogram curves and reduced separation efficiency. Hence, preferably, the sample origin (C) is in fluid connection with each inlet valve (ax) of each of the at least two chromatography units (Gx), preferably via a separate flow path, each inlet conduit of each of the at least two chromatography units (Gx), preferably via a sample-inlet joint (six), each column conduit of each of the at least two chromatography units (Gx), preferably via a sample-column joint (sjx), or mixtures thereof. Such embodiments ensure that the sample is added to small volumes only and a broadening of the chromatogram curve is reduced. Particularly, the fluid connection between the sample origin (C) and the inlet valve (ax) has the advantage that the control of the fluid connections occurs in just one central control unit, i.e., the inlet valve (ax). Furthermore, the fluid connection between the sample origin (C) and each column conduit of each of the at least two chromatography units (Gx), preferably via a samplecolumn joint (sjx) , has the advantage that the dead volume of the system is minimized, as the sample-column joint (sjx) is usually located as close to the inlet of the chromatography column (cx) as possible. This reduces the dilution and, hence, improves separation efficiency.

[0061] In a preferred embodiment of the invention, the waste conduit of the chromatography system fluidly connects the outlet (dx) with the waste sink (D). Usually, in a PCC chromatography process, a certain amount of dilute, elute, and or washing fluid will be present having no or just low amounts of target species therein. These liquids are usually collected in the waste sink (D) to be cleaned and reused after the process has finished. Preferably, they are at least partially cleaned and separated during the process and reintroduced as (A) or (B).

[0062] Likewise, preferably, the target conduit of the chromatography system fluidly connects the outlet (dx) with the target sink (E).

[0063] Preferably, the mobile phase origin (A) is a mobile phase reservoir, preferably a mobile phase vessel. Generally, any type of reservoir or vessel can be used. Preferably, the reservoir or vessel of the mobile phase origin (A) is made from glass or a polymer, preferably polyethylene, polypropylene or polyethylene terephthalate.

[0064] Preferably, the eluent phase origin (B) is an eluent phase reservoir, preferably an eluent phase vessel. Generally, any type of reservoir or vessel can be used. Preferably, the reservoir or vessel of the eluent phase origin (B) is made from glass or a polymer, preferably polyethylene, polypropylene or polyethylene terephthalate. Also preferably, the sample origin (C) is a sample reservoir, preferably a sample vessel. Generally, any type of reservoir or vessel can be used. Preferably, the reservoir or vessel of the sample origin (C) is made from glass or a polymer, preferably polyethylene, polypropylene or polyethylene terephthalate.

[0065] Preferably, the waste sink (D) is a waste reservoir, preferably a waste vessel. Generally, any type of reservoir or vessel can be used. Preferably, the reservoir or vessel of the waste sink (D) is made from glass or a polymer, preferably polyethylene, polypropylene or polyethylene terephthalate.

[0066] Also preferably, the target sink (E) is a waste reservoir, preferably a waste vessel. Generally, any type of reservoir or vessel can be used. Preferably, the reservoir or vessel of the target sink (E) is made from glass or a polymer, preferably polyethylene, polypropylene or polyethylene terephthalate.

[0067] Generally, the reservoirs and vessels of the chromatography system should have an appropriate size, which is oriented at the volumes of the column and the separation efficiency of the column. Such a respective size reduces the risk of an underrun in the system.

[0068] The central control unit (F) can be any device for receiving, sending, and storing data as well as calculating using said data. Hence, preferably, the central control unit (F) is a computer, preferably a computer connected to a network. However, the central control unit (F) could also be any other electronic device, which is able to carry out the operations as described herein. Hence, the operations of the central control unit (F) could be either implemented by hardware, such as in electronic circuits, or could be implemented by software and configured to run on a computer. In one preferred embodiment of the present invention, the central control unit (F) is a dedicated computer, i.e., server, which is connected via network to the at least two chromatography units (Gx). In an alternative preferred embodiment of the present invention, the central control unit (F) could be provided by at least one of the at least two chromatography units (Gx). This embodiment has the advantage that the chromatography system does not necessarily need a dedicated computer.

[0069] Generally, each inlet (ax) of each of the at least two chromatography units (Gx) is configured to allow distinct or combined, preferably distinct, fluid connections between the mobile phase origin (A), the eluent phase origin (B), and, and / or or the target origin (C). More preferably, each inlet (ax) of each of the at least two chromatography units (Gx) is configured to allow distinct or combined, preferably distinct, fluid connections between the mobile phase origin (A), the eluent phase origin (B), the target origin (C), and / or the fluid connection of the outlet valve (dx.i) of a preceding chromatography unit with the inlet valve (ax).Thus, the inlet (ax) preferably is configured to allow for separate fluid connections between the inlet conduit and one of the mobile phase origin (A), the eluent phase origin (B), the target origin (C), or the fluid connection of the outlet valve (dx-i) of a preceding chromatography unit with the inlet valve (ax). Thus, each inlet (ax) of each of the at least two chromatography units (Gx) preferably is a valve having at least 4 ports, preferably an electrically actuated valve having at least 4 ports. More preferably, each inlet (ax) of each of the at least two chromatography units (Gx) is a valve having at least 5 ports, preferably an electrically actuated valve having at least 5 ports.

[0070] Generally, each outlet (dx) of each of the at least two chromatography units (Gx) is configured to allow distinct or combined, preferably distinct, fluid connections between the waste sink (D), the target sink (E), and / or the fluid connection of the outlet valve (dx) with the outlet valve (ax+i) of a subsequent chromatography unit (Gx+i). Thus, each outlet (dx) of each of the at least two chromatography units (Gx) preferably is a valve having at least 3 ports, preferably an electrically actuated valve having at least 3 ports.

[0071] Preferably, in the chromatography system according to the present invention the at least two chromatography units (Gx) comprise at least three chromatography units (Gx). This enables the usage of post load washing steps.

[0072] More preferably, in the chromatography system according to the present invention the at least three chromatography units (Gx) comprise at least four chromatography units (Gx).

[0073] In a preferred embodiment of the chromatography system according to the present invention the mobile phase origin (A) and the eluent phase origin (B) are each provided by an active phase origin (A1) and an inversely active phase origin (B1), wherein the active phase origin (A1) and the inversely active phase origin (B1) are in fluid connection with each inlet valve (ax) of each of the at least two chromatography units (Gx), wherein the sample origin (C) is in fluid connection with each inlet valve (ax) of each of the at least two chromatography units (Gx), each inlet conduit of each of the at least two chromatography units (Gx), preferably via a sample-inlet joint (six), each column conduit of each of the at least two chromatography units (Gx), preferably via a samplecolumn joint (sjx), or mixtures thereof, and wherein the inlet valve (ax) is configured to provide mixtures of fluids provided by the active phase origin (A1), the inversely active phase origin (B1), and / or the sample origin (C). The active phase origin (A1) usually activates the interaction between the compound to be separated and the chromatography matrix. Thereby it depends whether the chromatography system is run in a bind / elute mode or a flow-through mode. In case of a bind / elute mode the active phase origin (A1) will activate the interaction between the target species and the chromatography matrix, whereas the inversely active origin (B1) will deactivate such binding. Likewise, in flow-through mode, the active phase origin (A1) will activate the interaction between the impurity to be removed and the chromatography matrix, whereas the inversely active origin (B1) will deactivate such binding. Hence, the ratio of these two origins can control the binding behaviour of the target species and / or the impurity in the column. Thus, in such a preferred embodiment, the inlet valve (ax) usually is configured to allow for a mixing of the active phase origin (A1) and the inversely active phase origin (B1). The advantage of such a preferred embodiment is that the loading step, the eluting step, and optionally the post load washing step can be carried out with the same dilute components, but only with different ratios of (A1) and (B1).

[0074] Thus, preferably, the inlet valve (ax) is a mixing valve.

[0075] Also preferably, the chromatography system according to the present invention comprises a mixer (mx) in the inlet conduit. This ensures thorough mixing of the phases in the conduit.

[0076] Preferably, the active phase origin (A1) is an active phase reservoir, preferably an active phase vessel. Generally, any type of reservoir or vessel can be used. Preferably, the reservoir or vessel of the active phase origin (A1) is made from glass or a polymer, preferably polyethylene, polypropylene or polyethylene terephthalate.

[0077] Also preferably, the inversely active phase origin (B1) is an inversely active phase reservoir, preferably an inversely active phase vessel. Generally, any type of reservoir or vessel can be used. Preferably, the reservoir or vessel of the inversely active phase origin (B1) is made from glass or a polymer, preferably polyethylene, polypropylene or polyethylene terephthalate.

[0078] Preferably, in the chromatography system according to the preferred embodiment of the present invention the sample column joint is an injection valve (sjx). Preferably the injection valve (sjx) is a 6-port rotary valve, preferably a 6-port electrically actuated valve.

[0079] Preferably, in the chromatography system according to the present invention in each of the at least two chromatography units (Gx) the column conduit comprises a washing valve (swx), wherein each washing valve (swx) of each of the at least two chromatography units (Gx) is in fluid connection with the waste sink (D) via a washing conduit. Preferably, the washing valve (swx) is a 3-port valve, preferably a 3-port electrically actuated valve.

[0080] Preferably, in the chromatography system according to the present invention each outlet conduit of each of the at least two chromatography units (Gx) comprises at least one detector (gx), wherein the detector is selected from the list consisting of an ultraviolet light absorption (UV) detector, a visible light absorption (VIS) detector, a photo diode array (PDA) detector, a refractive-index detector, an evaporative light scattering detector, a multi-angle light scattering detector, a mass spectrometer, a conductivity detector, a fluorescence detector, a chemiluminescence detector, an optical rotation detector, and an electrochemical detector. Preferably, the detector (gx) is an ultraviolet light absorption (UV) detector or a visible light absorption (VIS) detector. More preferably, each outlet conduit of each of the at least two chromatography units (Gx) comprises at least two detectors (gx). Most preferably, the at least two detectors (gx) are an ultraviolet light absorption (UV) detector and a conductivity detector. The detector has the advantage that the breakthrough of target species and / or impurities in a column can be precisely and reliably detected.

[0081] Preferably, the detector (gx) can be a part of an on-line analysis, an in-line analysis, or at-line analysis.

[0082] In a preferred embodiment of the invention, the client control unit of each of the at least two chromatography units (Gx) of the chromatography system is configured to control the inlet valve (ax), the outlet valve (dx), and the pump (bx), more preferably is configured to control the inlet valve (ax), the outlet valve (dx), the pump (bx), the at least one detector (gx), and the injection valve (sjx), more preferably is configured to control the inlet valve (ax), the outlet valve (dx), the pump (bx), the washing valve (swx), the at least one detector (gx), and the injection valve (sjx). Thereby, the term controlling in particular comprises the action of actuating the valves, more preferably opening and closing fluid connections with the valves.

[0083] Furthermore, in a preferred embodiment of the chromatography system of the present invention, the central control unit (F) is configured to control on each of the at least two chromatography units (Gx) via the client control unit (fx) the inlet valve (ax), the outlet valve (dx), and the pump (bx), preferably is configured to control the inlet valve (ax), the outlet valve (dx), the pump (bx), the at least one detector (gx), and the injection valve (sjx), more preferably is configured to control the inlet valve (ax), the outlet valve (dx), the pump (bx), the washing valve (swx), the at least one detector (gx), and the injection valve (sjx). Thereby, controlling the valve in particular comprises the action of actuating the valve, more preferably opening and closing fluid connections with the valve. Furthermore, controlling the detector comprises retrieving the measurement values from the detector. Controlling the pump comprises starting and stopping the pump and adjusting the flow rate with the pump.

[0084] Preferably, in the chromatography system according to the present invention, the central control unit (F) is configured to achieve that the chromatography system can carry out a periodic counter-current chromatography (PCC). The periodic countercurrent chromatography can be carried out in flow-through mode or in bind / elute mode, preferably in bind-elute mode. Furthermore, the periodic counter-current chromatography can be carried out with or without a post load washing step included. If the central control unit (F) is configured to carry out a post load washing step, the at least two chromatography units (Gx) comprise at least three chromatography units.

[0085] Figure 1 shows the most general embodiment of the present invention including at least two chromatography units (Gx). The central control unit (F) of the chromatography system of the embodiment of Figure 1 can be configured to run the chromatography system in bind / elute mode or in flow-through mode.

[0086] If central control unit (F) of the chromatography system of the embodiment of Figure 1 is configured to run the chromatography system in bind / elute mode, it is configured to achieve the following steps (cf. also Figure 7):

[0087] In a starting step ai, sii , and / or sji are actuated to achieve a fluid connection of the sample origin (C) with the inlet conduit, the column conduit and / or the column of the first chromatography unit (Gi). Alternatively, a fluid connection to the mobile phase origin (A) can be achieved, if the sample origin (C) is connected to the mobile phase origin (A) or if ai is a mixing valve allowing for a fluid connection of both, the sample origin (C) and the mobile phase origin (A). Furthermore, di is actuated to achieve a fluid connection between di and a2, si2, and / or sj2, and a2, si2, and / or sj2 are actuated to achieve a fluid connection of di with the inlet conduit, the column conduit and / or the column of the second chromatography unit (G2). At the same time, the outlet valve d2 is actuated to achieve a fluid connection to the waste sink (D). Then, the pumps bi and b2 are started and the detector gi is monitored for a breakthrough of the target species through the column (ci) of the first chromatography unit (Gi).

[0088] If the breakthrough is reached, ai, sii, and / or sji are actuated in a first column elution step to close the fluid connection to the sample origin (C) and to achieve a fluid connection of the elute phase origin (B) with the inlet conduit, the column conduit and / or the column of the first chromatography unit (Gi). At the same time, the outlet valve di is actuated to close the fluid connection to a2, si2, and / or sj2, and to achieve a fluid connection to the target sink (E). Furthermore, a2, si2, and / or sj2are actuated to achieve a fluid connection of the sample origin (C) with the inlet conduit, the column conduit and / or the column of the second chromatography unit (G2). Alternatively, a fluid connection to the mobile phase origin (A) can be achieved, if the sample origin (C) is connected to the mobile phase origin (A) or if a2is a mixing valve allowing for a fluid connection of both, the sample origin (C) and the mobile phase origin (A). Then the detector gi is monitored for an end of the elution of the column (ci) of the first chromatography unit (Gi).

[0089] If the end of the elution and optionally cleaning and recalibration of the first column (ci) is reached, ai, sii, and / or sji are actuated in a second column loading step to achieve a fluid connection of the outlet (d2) of the second chromatography unit (G2) with the inlet conduit, the column conduit and / or the column of the first chromatography unit (Gi). Furthermore, d2is actuated to achieve a fluid connection to ai, sii , and / or sji of the first chromatography unit (Gi). At the same time, the outlet valve di is actuated to close the fluid connection to the target sink (E), and to achieve a fluid connection to the waste sink (D). Then, the detector g2is monitored for a breakthrough of the target species through the column (c2) of the second chromatography unit (G2).

[0090] If the breakthrough is reached, a2, si2, and / or sj2are actuated in a second column elution step to close the fluid connection to the sample origin (C) and to achieve a fluid connection of the elute phase origin (B) with the inlet conduit, the column conduit and / or the column of the first chromatography unit (Gi). At the same time, the outlet valve d2is actuated to close the fluid connection to ai, sii, and / or sji, and to achieve a fluid connection to the target sink (E). Furthermore, ai, sii , and / or sji are actuated to achieve a fluid connection of the sample origin (C) with the inlet conduit, the column conduit and / or the column of the first chromatography unit (Gi). Alternatively, a fluid connection to the mobile phase origin (A) can be achieved, if the sample origin (C) is connected to the mobile phase origin (A) or if ai is a mixing valve allowing for a fluid connection of both, the sample origin (C) and the mobile phase origin (A). Then the detector g2is monitored for an end of the elution of the column (c2) of the first chromatography unit (G2).

[0091] If the end of the elution and optionally cleaning and recalibration of the second column (c2) is reached, a2, si2, and / or sj2are actuated in a first column loading step to achieve a fluid connection of the outlet (di) of the first chromatography unit (Gi) with the inlet conduit, the column conduit and / or the column of the second chromatography unit (G2). Furthermore, di is actuated to achieve a fluid connection to a2, si2, and / or sj2of the second chromatography unit (G2). At the same time, the outlet valve d2is actuated to close the fluid connection to the target sink (E), and to achieve a fluid connection to the waste sink (D). Then, the detector gi is monitored for a breakthrough of the target species through the column (ci) of the first chromatography unit (G1).

[0092] If the breakthrough is reached, the central control unit (F) is configured to either invoke the first column elution step again followed by the second column loading step and the second column elution step, thereby achieving a continuous chromatography.

[0093] If the process should be stopped, the central control unit (F) is configured to invoke either the first or the second column loading step without redirection of the respective outlet to the other column, but rather directly to the waste sink (D) and subsequent first or second column elution step.

[0094] In case of the embodiment according to Figure 2, in which the mobile phase origin (A) and the elute phase origin (B) are formed by certain ratios of the active phase origin (A1) and the inversely active phase origin (B1), the central control unit is configured to achieve the same steps as for the embodiment of Figure 1 with the exception that ax, six, and / or sjxare not actuated to switch between (A1) and (B1), but rather to achieve a certain ratio needed for the loading step and the elution step. Hence, ax, six, and / or sjxare actuated to achieve a ratio of (A1) and (B1) which allows for an adsorption of the target species to the chromatography matrix in the respective loading step and to achieve a ratio of (A1) and (B1) which allows for a desorption of the target species from the chromatography matrix in the respective elution step.

[0095] If central control unit (F) of the chromatography system of the embodiment of Figure 1 is configured to run the chromatography system in flow-through mode, it is configured to achieve the following steps (cf. also Figure 8):

[0096] In a starting step ai, sii , and / or sji are actuated to achieve a fluid connection of the sample origin (C) with the inlet conduit, the column conduit and / or the column of the first chromatography unit (G1). Alternatively, a fluid connection to the mobile phase origin (A) can be achieved, if the sample origin (C) is connected to the mobile phase origin (A) or if ai is a mixing valve allowing for a fluid connection of both, the sample origin (C) and the mobile phase origin (A). Furthermore, di is actuated to achieve a fluid connection between di and a2, si2, and / or sj2, and a2, si2, and / or sj2are actuated to achieve a fluid connection of di with the inlet conduit, the column conduit and / or the column of the second chromatography unit (G2). At the same time, the outlet valve d2is actuated to achieve a fluid connection to the target sink (E). Then, the pumps bi and b2are started and the detector gi is monitored for a breakthrough of the impurity through the column (ci) of the first chromatography unit (Gi). If the breakthrough is reached, ai, sii , and / or sji are actuated in a first column washing step to close the fluid connection to the sample origin (C) and to achieve a fluid connection of the elute phase origin (B) with the inlet conduit, the column conduit and / or the column of the first chromatography unit (Gi). At the same time, the outlet valve di is actuated to close the fluid connection to a2, si2, and / or sj2, and to achieve a fluid connection to the waste sink (D). Furthermore, a2, si2, and / or sj2are actuated to achieve a fluid connection of the sample origin (C) with the inlet conduit, the column conduit and / or the column of the second chromatography unit (G2). Alternatively, a fluid connection to the mobile phase origin (A) can be achieved, if the sample origin (C) is connected to the mobile phase origin (A) or if a2is a mixing valve allowing for a fluid connection of both, the sample origin (C) and the mobile phase origin (A). Then the detector gi is monitored for an end of the washing of the column (ci) of the first chromatography unit (Gi).

[0097] If the end of the washing and optionally recalibration of the first column (ci) is reached, ai, sii , and / or sji are actuated in a second column separation step to achieve a fluid connection of the outlet (d2) of the second chromatography unit (G2) with the inlet conduit, the column conduit and / or the column of the first chromatography unit (Gi). Furthermore, d2is actuated to achieve a fluid connection to ai, sii , and / or sji of the first chromatography unit (Gi). At the same time, the outlet valve di is actuated to close the fluid connection to the wate sink (D), and to achieve a fluid connection to the sample sink (E). Then, the detector g2is monitored for a breakthrough of the impurity through the column (c2) of the second chromatography unit (G2).

[0098] If the breakthrough is reached, a2, si2, and / or sj2are actuated in a second column washing step to close the fluid connection to the sample origin (C) and to achieve a fluid connection of the elute phase origin (B) with the inlet conduit, the column conduit and / or the column of the first chromatography unit (Gi). At the same time, the outlet valve d2is actuated to close the fluid connection to ai, sii , and / or sji , and to achieve a fluid connection to the waste sink (D). Furthermore, ai, sii, and / or sji are actuated to achieve a fluid connection of the sample origin (C) with the inlet conduit, the column conduit and / or the column of the first chromatography unit (Gi). Alternatively, a fluid connection to the mobile phase origin (A) can be achieved, if the sample origin (C) is connected to the mobile phase origin (A) or if ai is a mixing valve allowing for a fluid connection of both, the sample origin (C) and the mobile phase origin (A). Then the detector g2is monitored for an end of the elution of the column (c2) of the first chromatography unit (G2). If the end of the washing and optionally recalibration of the second column (c2) is reached, a2, si2, and / or sj2are actuated in a first column loading step to achieve a fluid connection of the outlet (di) of the first chromatography unit (Gi) with the inlet conduit, the column conduit and / or the column of the second chromatography unit (G2). Furthermore, di is actuated to achieve a fluid connection to a2, si2, and / or sj2of the second chromatography unit (G2). At the same time, the outlet valve d2is actuated to close the fluid connection to the waste sink (D), and to achieve a fluid connection to the sample sink (E). Then, the detector gi is monitored for a breakthrough of the impurity through the column (ci) of the first chromatography unit (Gi).

[0099] If the breakthrough is reached, the central control unit (F) is configured to either invoke the first column washing step again followed by the second column separation step and the second column washing step, thereby achieving a continuous chromatography.

[0100] If the process should be stopped, the central control unit (F) is configured to invoke either the first or the second column separation step without redirection of the respective outlet to the other column, but rather directly to the sample sink (E) and subsequent first or second column washing step.

[0101] In case of the embodiment according to Figure 2, in which the mobile phase origin (A) and the elute phase origin (B) are formed by certain ratios of the active phase origin (A1) and the inversely active phase origin (B1), the central control unit is configured to achieve the same steps as for the embodiment of Figure 1 with the exception that ax, six, and / or sjxare not actuated to switch between (A1) and (B1), but rather to achieve a certain ratio needed for the separation step and the washing step. Hence, ax, six, and / or sjxare actuated to achieve a ratio of (A1) and (B1) which allows for an adsorption of the impurity to the chromatography matrix in the respective separation step and to achieve a ratio of (A1) and (B1) which allows for a desorption of the impurity from the chromatography matrix in the respective washing step.

[0102] Figures 3-6 show embodiments of the present invention including at least three chromatography units (Gx). This allows for the implementation of a post load washing step. The advantage of such a step is that after loading of a column at least a part of the target species is present in the mobile phase on the column, whereas this part is not adsorbed to the chromatography matrix. As also impurities are present in the mobile phase, this mobile phase has to be sent to waste before elution to ensure the purity of the product. However, in case of three chromatography column present in the system, the washing solution can be sent through the third, non-loaded column, on which the target species still present in the mobile phase can adsorb. This significantly enhances the separation efficiency and yield of the chromatography system. To enable post load washing, another interconnection of the columns is necessary, as the washing solution in the post washing step is sent to the subsequent-to-subsequent chromatography unit.

[0103] Hence, preferably, in the chromatography system according to the present invention the outlet valve (dx) is in fluid connection with the inlet valve (ax+2) of a subsequent-to- subsequent chromatography unit (Gx+2) of the at least two chromatography units (Gx), preferably via a separate flow path, the inlet conduit of the subsequent-to-subsequent chromatography unit (Gx+2), preferably via the sample-inlet joint (six+2) of the subsequent-to-subsequent chromatography unit (Gx+2), the column conduit of the subsequent-to-subsequent chromatography unit (Gx+2), preferably via the samplecolumn joint (sjx+2) of the subsequent chromatography unit (Gx+2), or mixtures thereof, wherein, if the chromatography unit is the last chromatography unit (Gx), the subsequent-to-subsequent chromatography unit is the second chromatography unit (G2) and, if the chromatography unit is the second last chromatography unit (Gx-i), the subsequent-to-subsequent chromatography unit is the first chromatography unit (G1). As stated above, this feature enables the system to send the mobile phase to the subsequent-to-subsequent chromatography unit (Gx+2), being the requirement for post load washing.

[0104] If the central control unit (F) of the chromatography system of the embodiment of Figure 5 is configured to run the chromatography system in bind / elute mode including post load washing, it is configured to achieve the following steps (cf. also Figure 9):

[0105] In a starting step ai, sii , and / or sji are actuated to achieve a fluid connection of the sample origin (C) with the inlet conduit, the column conduit and / or the column of the first chromatography unit (G1). Alternatively, a fluid connection to the mobile phase origin (A) can be achieved, if the sample origin (C) is connected to the mobile phase origin (A) or if ai is a mixing valve allowing for a fluid connection of both, the sample origin (C) and the mobile phase origin (A). Furthermore, di is actuated to achieve a fluid connection between di and a2, si2, and / or sj2, and a2, si2, and / or sj2 are actuated to achieve a fluid connection of di with the inlet conduit, the column conduit and / or the column of the second chromatography unit (G2). At the same time, the outlet valve d2 is actuated to achieve a fluid connection to the waste sink (D). Then, the pumps bi and b2 are started and the detector gi is monitored for a breakthrough of the target species through the column (ci) of the first chromatography unit (G1).

[0106] If the breakthrough is reached, ai, sii , and / or sji are actuated in a first column washing step to close the fluid connection to the sample origin (C) and to achieve a fluid connection of the mobile phase origin (A) with the inlet conduit, the column conduit and / or the column of the first chromatography unit (G1). At the same time, the outlet valve di is actuated to close the fluid connection to a2, si2, and / or sj2, and to achieve a fluid connection to a3, si3, and / or sj3. Furthermore, a3, si3, and / or sj3are actuated to achieve a fluid connection of the outlet di with the inlet conduit, the column conduit and / or the column of the third chromatography unit (G3). Moreover, a2, si2, and / or sj2are actuated to achieve close the fluid connection to di and to achieve a fluid connection of the sample origin (C) with the inlet conduit, the column conduit and / or the column of the second chromatography unit (G2). Alternatively, a fluid connection to the mobile phase origin (A) can be achieved, if the sample origin (C) is connected to the mobile phase origin (A) or if a2is a mixing valve allowing for a fluid connection of both, the sample origin (C) and the mobile phase origin (A). Furthermore, d3is actuated to achieve a fluid connection to the waste sink (D). Then the detector gi is monitored for an end of the washing of the column (ci) of the first chromatography unit (Gi).

[0107] If the end of the washing has been detected, ai, sii , and / or sji are actuated in a first column elution step to close the fluid connection to the mobile origin (A) and to achieve a fluid connection of the elute phase origin (B) with the inlet conduit, the column conduit and / or the column of the first chromatography unit (Gi). At the same time, the outlet valve di is actuated to close the fluid connection to a3, si3, and / or sj3, and to achieve a fluid connection to the target sink (E). Moreover, the outlet valve d2is actuated to close the fluid connection to the waste sink (D) and to achieve a fluid connection to a3, si3, and / or sj3. Furthermore, a3, si3, and / or sj3are actuated to achieve a fluid connection of the outlet d2with the inlet conduit, the column conduit and / or the column of the third chromatography unit (G3). Then the detector gi is monitored for an end of the elution of the column (ci) of the first chromatography unit (Gi).

[0108] If the breakthrough is reached, a2, si2, and / or sj2are actuated in a second column washing step to close the fluid connection to the sample origin (C) and to achieve a fluid connection of the mobile phase origin (A) with the inlet conduit, the column conduit and / or the column of the second chromatography unit (G2). At the same time, the outlet valve d2is actuated to close the fluid connection to a3, si3, and / or sj3, and to achieve a fluid connection to ai, sii, and / or sji. Furthermore, ai, sii, and / or sji are actuated to achieve a fluid connection of the outlet d2with the inlet conduit, the column conduit and / or the column of the first chromatography unit (Gi). Moreover, a3, si3, and / or sj3are actuated to achieve close the fluid connection to d2and to achieve a fluid connection of the sample origin (C) with the inlet conduit, the column conduit and / or the column of the third chromatography unit (G3). Alternatively, a fluid connection to the mobile phase origin (A) can be achieved, if the sample origin (C) is connected to the mobile phase origin (A) or if a3is a mixing valve allowing for a fluid connection of both, the sample origin (C) and the mobile phase origin (A). Furthermore, di is actuated to achieve a fluid connection to the waste sink (D). Then the detector g2is monitored for an end of the washing of the column (c2) of the second chromatography unit (G2).

[0109] If the end of the washing has been detected, a2, si2, and / or sj2are actuated in a second column elution step to close the fluid connection to the mobile origin (A) and to achieve a fluid connection of the elute phase origin (B) with the inlet conduit, the column conduit and / or the column of the second chromatography unit (G2). At the same time, the outlet valve d2is actuated to close the fluid connection to ai, sii , and / or sji , and to achieve a fluid connection to the target sink (E). Moreover, the outlet valve d2is actuated to close the fluid connection to the waste sink (D) and to achieve a fluid connection to ai, sii , and / or sji . Furthermore, ai, sii , and / or sji are actuated to achieve a fluid connection of the outlet d2with the inlet conduit, the column conduit and / or the column of the first chromatography unit (Gi). Then the detector gi is monitored for an end of the elution of the column (ci) of the first chromatography unit (Gi).

[0110] If the breakthrough is reached, a2, sis, and / or sj2are actuated in a third column washing step to close the fluid connection to the sample origin (C) and to achieve a fluid connection of the mobile phase origin (A) with the inlet conduit, the column conduit and / or the column of the third chromatography unit (G3). At the same time, the outlet valve da is actuated to close the fluid connection to ai, sii , and / or sji , and to achieve a fluid connection to a2, si2, and / or sj2. Furthermore, a2, si2, and / or sj2are actuated to achieve a fluid connection of the outlet d3with the inlet conduit, the column conduit and / or the column of the second chromatography unit (G2). Moreover, ai, sii , and / or sji are actuated to close the fluid connection to d3and to achieve a fluid connection of the sample origin (C) with the inlet conduit, the column conduit and / or the column of the first chromatography unit (Gi). Alternatively, a fluid connection to the mobile phase origin (A) can be achieved, if the sample origin (C) is connected to the mobile phase origin (A) or if ai is a mixing valve allowing for a fluid connection of both, the sample origin (C) and the mobile phase origin (A). Furthermore, d2is actuated to achieve a fluid connection to the waste sink (D). Then the detector gs is monitored for an end of the washing of the column (C3) of the second chromatography unit (G3).

[0111] If the end of the washing has been detected, as, sis, and / or sjs are actuated in a third column elution step to close the fluid connection to the mobile origin (A) and to achieve a fluid connection of the elute phase origin (B) with the inlet conduit, the column conduit and / or the column of the third chromatography unit (G3). At the same time, the outlet valve ds is actuated to close the fluid connection to a2, si2, and / or sj2, and to achieve a fluid connection to the target sink (E). Moreover, the outlet valve di is actuated to close the fluid connection to the waste sink (D) and to achieve a fluid connection to a2, si2, and / or sj2. Furthermore, a2, si2, and / or sj2are actuated to achieve a fluid connection of the outlet di with the inlet conduit, the column conduit and / or the column of the second chromatography unit (G2). Then the detector g3is monitored for an end of the elution of the column (c3) of the first chromatography unit (G3).

[0112] If the end of the elution is reached, the central control unit (F) is configured to either invoke the first column washing step again followed by the second column separation step and the second column washing step, thereby achieving a continuous chromatography.

[0113] If the process should be stopped, the central control unit (F) is configured to invoke either the first, the second, or the third column washing step without redirection of the respective outlet to the other column, but rather directly to the waste sink (D).

[0114] In case of the embodiment according to Figure 6, in which the mobile phase origin (A) and the elute phase origin (B) are formed by certain ratios of the active phase origin (A1) and the inversely active phase origin (B1), the central control unit is configured to achieve the same steps as for the embodiment of Figure 1 with the exception that ax, six, and / or sjxare not actuated to switch between (A1) and (B1), but rather to achieve a certain ratio needed for the separation step and the washing step. Hence, ax, six, and / or sjxare actuated to achieve a ratio of (A1) and (B1) which allows for an adsorption of the impurity to the chromatography matrix in the respective separation step and to achieve a ratio of (A1) and (B1) which allows for a desorption of the impurity from the chromatography matrix in the respective washing step.

[0115] As illustrated in the Figures, two or more self-contained chromatography units Gx may be provided in a parallel configuration, each controllable via a respective client control unit (fx). This allows for easier system capacity expansion as further chromatography modules may be added as required to increase capacity (e.g. in a “plug-and-play” type configuration). Also, replacement of any such chromatography units is facilitated as they may be easily swapped out without interrupting operation of the other chromatography units. The chromatography units are preferably provided as identical, or substantially identical, units.

[0116] A further benefit of various embodiments of the present invention is that, compared to regular PCC chromatography, a simpler system is provided which (as with PCC) is also not limited to operations using only two chromatography columns. For example, a reduced number of valves and other complicated expensive pieces of equipment (additional UV monitors and pumps etc.) is needed. Furthermore, simplified reliable control strategies are also enabled, rather than the more sophisticated control strategies that may be involved for conventional PCC and SMB set-ups. Additionally, various embodiments of the present in invention can also be run non-stop, e.g. automatically. In one test run, such a system (in this case using columns that were overloaded using a Delta-UV methodology) was run continuously for 48 hours without encountering any issues. The chromatography units may also be provided as single use (Sil) units. This allows embodiments of the present invention to provide a “PCC like” chromatography system with single-use technology. Currently, there are no known single use PCC or SMB systems, due to the high cost of the existing technologies used.

[0117] Various other advantages of the present invention will also be apparent to those skilled in the art.

Claims

Claims:

1. A chromatography system comprising:■ a mobile phase origin (A),■ an eluent phase origin (B),■ a sample origin (C),■ a waste sink (D),■ a target sink (E),■ a central control unit (F), and■ at least two chromatography units (Gx), wherein each chromatography unit (Gx) comprises:- an inlet valve (ax),- a pump (bx),- a chromatography column having an inlet and an outlet (cx),- an outlet valve (dx), and- a client control unit (fx), wherein the inlet valve (ax) is in fluid connection with the pump (bx) via an inlet conduit, wherein the pump (bx) is in fluid connection with the inlet of the chromatography column (cx) via a column conduit, wherein the outlet of the chromatography column (cx) is in fluid connection with the outlet (dx) of the chromatography unit (Gx) via an outlet conduit, wherein the mobile phase origin (A) and the eluent phase origin (B) are in fluid connection with each inlet valve (ax) of each of the at least two chromatography units (Gx), preferably via separate flow paths, wherein the sample origin (C) is in fluid connection with the mobile phase origin (A), each inlet valve (ax) of each of the at least two chromatography units (Gx), preferably via a separate flow path, each inlet conduit of each of the at least two chromatography units (Gx), preferably via a sample-inlet joint (six), each column conduit of each of the at least two chromatography units (Gx), preferably via a sample-column joint (sjx), or mixtures thereof, wherein each outlet of each of the at least two chromatography units (Gx) is in fluid connection with the waste sink (D) via a waste conduit, wherein each outlet of each of the at least two chromatography units (Gx) is in fluid connection with the target sink (E) via a target conduit, andwherein each client control unit (fx) of each of the at least two chromatography units (Gx) is in communication with the central control unit (F), wherein the outlet valve (dx) is in fluid connection with the inlet valve (ax+i) of a subsequent chromatography unit (Gx+i) of the at least two chromatography units (Gx), preferably via a separate flow path, the inlet conduit of the subsequent chromatography unit (Gx+i), preferably via the sample-inlet joint (six+i) of the subsequent chromatography unit (Gx+i), the column conduit of the subsequent chromatography unit (Gx+i), preferably via the sample-column joint (sjx+i) of the subsequent chromatography unit (Gx+i), or mixtures thereof, wherein, if the chromatography unit is the last chromatography unit (Gx), the subsequent chromatography unit is the first chromatography unit (Gi).

2. The chromatography system according to claim 1 , wherein the outlet valve (dx) is in fluid connection with the inlet valve (ax+2) of a subsequent-to-subsequent chromatography unit (Gx+2) of the at least two chromatography units (Gx), preferably via a separate flow path, the inlet conduit of the subsequent-to- subsequent chromatography unit (Gx+2), preferably via the sample-inlet joint (six+2) of the subsequent-to-subsequent chromatography unit (Gx+2), the column conduit of the subsequent-to-subsequent chromatography unit (Gx+2), preferably via the sample-column joint (sjx+2) of the subsequent chromatography unit (Gx+2), or mixtures thereof, wherein, if the chromatography unit is the last chromatography unit (Gx), the subsequent-to-subsequent chromatography unit is the second chromatography unit (G2) and, if the chromatography unit is the second last chromatography unit (Gx-i), the subsequent-to-subsequent chromatography unit is the first chromatography unit (G1).

3. The chromatography system according to claims 1 or 2, wherein the waste conduit fluidly connects the outlet (dx) with the waste sink (D).

4. The chromatography system according to any of the preceding claims, wherein the target conduit fluidly connects the outlet (dx) with the target sink (E).

5. The chromatography system according to any of the preceding claims, wherein each inlet of each of the at least two chromatography units (Gx) is a valve having at least 4 ports, preferably an electrically actuated valve having at least 4 ports.

6. The chromatography system according to any of the preceding claims, wherein each outlet of each of the at least two chromatography units (Gx) is a valve having at least 3 ports, preferably an electrically actuated valve having at least 3 ports.

7. The chromatography system according to any of the preceding claims, wherein the mobile phase origin (A) and the eluent phase origin (B) are each provided by an active phase origin (A1) and an inversely active phase origin (B1), wherein the active phase origin (A1) and the inversely active phase origin (B1) are in fluid connection with each inlet valve (ax) of each of the at least two chromatography units (Gx), wherein the sample origin (C) is in fluid connection with each inlet valve (ax) of each of the at least two chromatography units (Gx), each inlet conduit of each of the at least two chromatography units (Gx), preferably via a sample-inlet joint (six), each column conduit of each of the at least two chromatography units (Gx), preferably via a sample-column joint (sjx), or mixtures thereof, and wherein the inlet valve (ax) is configured to provide mixtures of fluids provided by the active phase origin (A1), the inversely active phase origin (B1), and / or the sample origin (C), preferably wherein the inlet valve (ax) is a mixing valve.

8. The chromatography system according to claim 7, wherein the inlet conduit comprises a mixer (mx).

9. The chromatography system according to any of the preceding claims, wherein each outlet conduit of each of the at least two chromatography units (Gx) comprises at least one detector (gx), wherein the detector is selected from the list consisting of a Ultraviolet light absorption (UV) detector, a visible light absorption (VIS) detector, a photo diode array (PDA) detector, a refractive-index detector, an evaporative light scattering detector, a multi-angle light scattering detector, a mass spectrometer, a conductivity detector, a fluorescence detector, a chemiluminescence detector, an optical rotation detector, and an electrochemical detector.

10. The chromatography system according to any of the preceding claims, wherein the client control unit is configured to control the inlet valve (ax), the outlet valve (dx), and the pump (bx), preferably is configured to control the inlet valve (ax), the outlet valve (dx), the pump (bx), the at least one detector (gx), and the injection valve (sjx), more preferably is configured to control the inlet valve (ax), the outlet valve (dx), the pump (bx), the washing valve (swx), the at least one detector (gx), and the injection valve (sjx).

11. The chromatography system according to claim 10, wherein the central control unit (F) is configured to control on each of the at least two chromatography units (Gx) via the client control unit (fx) the inlet valve (ax), the outlet valve (dx), and thepump (bx), preferably is configured to control the inlet valve (ax), the outlet valve (dx), the pump (bx), the at least one detector (gx), and the injection valve (sjx), more preferably is configured to control the inlet valve (ax), the outlet valve (dx), the pump (bx), the washing valve (swx), the at least one detector (gx), and the injection valve (sjx).

12. The chromatography system according to claim 11 , wherein the central control unit (F) is configured to actuate via client control unit (fx) the inlet valve (ax), the outlet valve (dx), and the pump (bx), preferably is configured to control the inlet valve (ax), the outlet valve (dx), the pump (bx), the at least one detector (gx), and the injection valve (sjx), more preferably is configured to control the inlet valve (ax), the outlet valve (dx), the pump (bx), the washing valve (swx), the at least one detector (gx), and the injection valve (sjx), to achieve that the chromatography system can carry out a periodic counter-current chromatography (PCC).

13. The chromatography system according to claims 11 or 12, wherein the central control unit (F) is configured to carry out the periodic counter-current chromatography in flow-through mode or in bind-elute mode, preferably in bind- elute mode.

14. The chromatography system according to any of the preceding claims, wherein the at least two chromatography units (Gx) comprise at least three chromatography units (Gx).

15. The chromatography system according to claim 14, wherein the central control unit (F) is configured to carry out at least one post-load washing step.

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