Protein purification device and method for producing purified protein

Abstract

Provided is a technology for more efficiently producing purified protein from a cell culture liquid. A device disclosed herein is for purifying protein from a cell culture liquid. The device comprises a hydrocyclone, a first reservoir tank for retaining a first liquid, a first pump, and a second pump. The first pump is provided at a site where the flow rate of liquid discharged from the hydrocyclone can be adjusted. The second pump is provided at a site where the flow rate of the first liquid discharged from the first reservoir tank can be adjusted.

Description

Protein purification device and method for producing purified protein

[0001] The present disclosure relates to a protein purification apparatus and a method for producing purified proteins.

[0002] International Publication No. 2019 / 189064 discloses an apparatus for continuously purifying proteins from cell culture fluid using a module combining an in-line mixer and a cyclone. This apparatus comprises a first module and a second module. In the first module, a piping connecting a culture vessel or cell separation device to the first module and a piping connecting a first storage tank to the first module are connected to the inlet of the in-line mixer via a branch valve. The outlet of the in-line mixer is connected to the inlet of the hydrocyclone. A piping connecting a first reaction module to the second module is connected to the lower outlet of the hydrocyclone. A piping is connected to the upper outlet of the hydrocyclone. In the second module, a piping connecting the first module to the second module and a piping connecting a second storage tank to the second module are connected to the inlet of the in-line mixer via a branch valve. The outlet of the in-line mixer is connected to the inlet of the hydrocyclone. A pipe connecting the second module to the other modules is connected to the outlet at the bottom of the hydrocyclone. A pipe is connected to the outlet at the top of the hydrocyclone. The publication states that by using a device with this configuration, it is possible to continuously recover and purify a target protein from a suspended sample containing a small amount of cells, cell debris, or host microorganisms in a short period of time.

[0003] Japanese Patent Application Publication No. 2000-509325 discloses a method for separating an emulsion containing a water-miscible phase, a water-immiscible phase, and fine solid particles into the water-miscible phase and the water-immiscible phase, comprising steps (a) to (d). Step (a) is a step of introducing the emulsion into a first hydrocyclone, thereby separating the emulsion into an overflow emulsion and an underflow emulsion. Step (b) is a step of inverting the phase of the overflow emulsion. Step (c) is a step of introducing the overflow emulsion into one or more subsequent hydrocyclones arranged in series. Step (d) is a step of recovering the water-miscible phase and the water-immiscible phase and recovering the solid particles in the discontinuous phase of the overflow emulsion of step (c). The publication states that by using a method configured as described above, it is possible to recover high-purity oil in high yield from an oil / water / biocatalyst emulsion in a water-continuous system, such as that which may result from a biological desulfurization process.

[0004] International Publication No. 2019 / 189064 Japanese Patent Application Publication No. 2000-509325

[0005] The present inventors would like to produce purified proteins more efficiently using cell culture media.

[0006] According to the technology disclosed herein, an apparatus for purifying a protein from a cell culture medium is disclosed. The apparatus includes a hydrocyclone, a first reservoir tank for storing a first liquid, a first pump, and a second pump. The first pump is provided at a location where the flow rate of the liquid exiting the hydrocyclone can be adjusted. The second pump is provided at a location where the flow rate of the first liquid exiting the first reservoir tank can be adjusted. By using an apparatus configured in this manner, purified proteins can be produced more efficiently using cell culture medium.

[0007] The apparatus may further include an in-line mixer, a first liquid transfer line, and a second liquid transfer line. The in-line mixer may be located downstream of the hydrocyclone. The first liquid transfer line may connect the hydrocyclone and the in-line mixer. The second liquid transfer line may extend from the first storage tank and merge with the first liquid transfer line. The first pump may be provided on the first liquid transfer line closer to the hydrocyclone than the junction with the second liquid transfer line. The second pump may be provided on the second liquid transfer line closer to the first storage tank than the junction with the first liquid transfer line.

[0008] The hydrocyclone may be configured to separate a liquid fed to the hydrocyclone into liquid A and liquid B. The hydrocyclone may have a first outlet for discharging liquid A and a second outlet for discharging liquid B. The first outlet may be connected to a first liquid feed line. The first pump may be provided between a junction of the first outlet and the second liquid feed line.

[0009] The device may be used to purify antibodies produced from cultured cells.

[0010] The protein to be purified may be an antibody. An antibody-binding protein that is a ligand may be used to purify the antibody.

[0011] The technology disclosed herein provides a method for producing a purified protein. This method includes purifying a protein from a cell culture medium using the above-described device. By using this production method, purified proteins can be produced more efficiently using cell culture medium.

[0012] Fig. 1 is a schematic diagram of the device 100. Fig. 2 is a schematic diagram of the device 1. Fig. 3 is an enlarged schematic diagram of the device 1. Fig. 4 is an enlarged schematic diagram of the device 1.

[0013] Preferred embodiments of the technology disclosed herein are described below. Matters necessary for implementing the technology disclosed herein, other than those specifically mentioned in this specification, can be understood based on the technical content taught by this specification and the common general technical knowledge of a person skilled in the art. The technology disclosed herein can be implemented based on the content disclosed in this specification and the common general technical knowledge of a person skilled in the art. In this specification and claims, the terms A to B (A and B are arbitrary numerical values) refer to values ​​greater than A and less than B, and also encompass values ​​greater than A and less than B.

[0014] The technology disclosed herein provides an apparatus for purifying a protein from a cell culture medium. As used herein, "cells" refer to cells that produce the protein to be purified and that can be cultured outside the body (in vitro). "Cells" include, for example, cell lines and cell masses, tissues, or organs extracted from a living organism. "Cell lines" refer to clones of primary cells that can be stably cultured in vitro for many generations. Examples of cells include microbial cells such as Escherichia coli; yeast; insect cells such as S2 cells, Sf9 cells, and Sf21 cells; and mammalian cells such as COS cells, NS0 cells, Sp2 / 0 cells, CHO cells, HEK cells, HeLa cells, and hybridomas. Mammalian cells are preferred, and NS0 cells, Sp2 / 0 cells, CHO cells, HEK cells, hybridomas, and the like are particularly preferred. Cells may also be genetically modified to produce the protein to be purified. As used herein, the term "culture medium" refers to a culture medium in which the above-mentioned cells are cultured and in which the protein to be purified is produced and secreted.

[0015] Proteins to be purified by the technology disclosed herein are, for example, proteins that can be used as pharmaceuticals. Examples of proteins include antibodies; hematopoietic proteins such as erythropoietin; cytokines such as interferon α, interferon β, interferon γ, G-CSF, and GM-CSF; enzymes such as t-PA; and hormones such as insulin, human growth hormone, and estrogen. Among these, the protein is preferably an antibody. The antibody is preferably a monoclonal antibody, and may be, for example, a mouse antibody or a chimeric antibody, but is preferably a humanized antibody or a fully humanized antibody. The antigen recognized by the antibody is not particularly limited and can be appropriately selected depending on the purpose.

[0016] Fig. 1 is a schematic diagram of an apparatus 100. As shown in Fig. 1, the apparatus 100 disclosed herein includes a hydrocyclone 110, a first storage tank 120 that stores a first liquid, a first pump 130, and a second pump 140. The first pump 130 is provided at a location where the flow rate of the liquid discharged from the hydrocyclone 110 can be adjusted. The second pump 140 is provided at a location where the flow rate of the first liquid discharged from the first storage tank 120 can be adjusted. Although not shown in Fig. 1, the apparatus 100 may also include valves, pressure gauges, flow meters, and the like as needed.

[0017] The hydrocyclone 110 is configured to separate, for example, a liquid sent to the hydrocyclone 110 into liquid A and liquid B. As shown in FIG. 1 , the hydrocyclone 110 is funnel-shaped and has a first outlet 111 at its lower end, a second outlet 112 at its upper end, and an inlet 113 on its upper side. The first outlet 111 is, for example, a portion through which liquid A is discharged from the hydrocyclone 110. In the embodiment shown in FIG. 1 , the first outlet 111 is connected to a first liquid supply line 161. The second outlet 112 is a portion through which liquid B is discharged from the hydrocyclone 110. The second outlet 112 is connected to, for example, a drain line outside the apparatus 100 or another liquid supply line. The inlet 113 is a portion through which liquid sent from upstream is introduced into the hydrocyclone 110. In this embodiment, the inlet 113 is connected to a pipe 171. In this embodiment, a supply source (e.g., a reservoir tank for various liquids, a cell culture tank, etc.) that supplies liquid to the hydrocyclone 110 is disposed upstream of the hydrocyclone 110. The piping 171 may directly or indirectly connect the supply source to the hydrocyclone 110. For example, an inline mixer, a hydrocyclone, etc. may be disposed between the supply source and the hydrocyclone 110, and pumps, valves, various sensors, etc. may also be disposed as necessary. In this specification, the term "hydrocyclone" refers to a separator that does not have a driving unit and that can separate, classify, concentrate, etc., liquid introduced into the separator while stationary.

[0018] The first reservoir 120 is a portion for storing the first liquid. As shown in Fig. 1, the first reservoir 120 has a connection port 121. The connection port 121 is a supply port for the first liquid from the first reservoir 120 to the second liquid transfer line 162, and is connected to the second liquid transfer line 162. The first liquid may be, for example, a buffer solution such as a washing liquid, an elution liquid, or an equilibration liquid.

[0019] The liquid sent to the hydrocyclone 110 may contain solids (e.g., a carrier, as described below). When the hydrocyclone 110 separates liquid A containing solids from the liquid, if an ejector is provided in the piping downstream of the hydrocyclone 110 (in the embodiment shown in FIG. 1 , the first liquid feed line 161 through which liquid A flows), the piping is prone to clogging and retention due to solids, making flow rate control difficult. The present inventors have investigated a configuration that can more easily control the flow rate of the liquid in the piping downstream of the hydrocyclone 110 by suppressing clogging and retention due to solids, and that can efficiently merge the liquid with liquid A from the first storage tank 120. After extensive investigation, the present inventors have arrived at a configuration in which a first pump 130 and a second pump 140 are provided in specific locations of the apparatus 100.

[0020] The first pump 130 is provided at a position where the flow rate of the liquid discharged from the hydrocyclone 110 can be adjusted. In the embodiment shown in Fig. 1, the first pump 130 is provided on the first liquid feed line 161. In this example, the first pump 130 is provided on the first liquid feed line 161, closer to the hydrocyclone 110 than the junction with the second liquid feed line 162 (connection port 161A in Fig. 1). The first pump 130 is provided between the first outlet 111 of the hydrocyclone 110 and the junction.

[0021] The second pump 140 is provided at a position where the flow rate of the first liquid discharged from the first storage tank 120 can be adjusted. In the embodiment shown in Fig. 1, the second pump 140 is provided on the second liquid feed line 162. Here, the second pump 140 is provided on the second liquid feed line 162, closer to the first storage tank 120 than the confluence portion (connection port 161A in Fig. 1). The second pump 140 is provided between the connection port 121 of the first storage tank 120 and the confluence portion.

[0022] In the embodiment shown in FIG. 1 , the apparatus 100 further includes an in-line mixer 150. The in-line mixer 150 is, for example, a mixer that mixes the liquid discharged from the hydrocyclone 110 with the first liquid discharged from the first storage tank 120. As shown in FIG. 1 , the in-line mixer 150 is disposed downstream of the hydrocyclone 110. The in-line mixer 150 is provided in the first liquid feed line 161, downstream of the junction with the second liquid feed line 162 (connection port 161A in FIG. 1 ). The in-line mixer 150 here has a pipe 151, an inlet 152, and an outlet 153. The pipe 151 is tubular (here, cylindrical), is the main body of the in-line mixer 150, and is a flow path for the liquid. The inlet 152 is, for example, the portion where the liquid discharged from the hydrocyclone 110 flows into the pipe 151. 1 , the inlet 152 is provided at one end of the pipeline 151. The inlet 152 is connected to a first liquid feed line 161. The outlet 153 is, for example, a portion where a liquid is discharged from the in-line mixer 150. In the embodiment shown in FIG. 1 , the outlet 153 is provided at the other end of the pipeline 151. In this example, the outlet 153 is connected to a pipe 172.

[0023] For example, another in-line mixer, another hydrocyclone, or the like may be arranged downstream of in-line mixer 150, and a pump may also be arranged as needed. In this specification, the term "in-line mixer" refers to a static mixer that does not have a driving unit and mixes at least two types of liquids by using energy generated by the flow velocity of the liquids flowing inside the device.

[0024] In the embodiment shown in FIG. 1 , the apparatus 100 includes a first liquid feed line 161 and a second liquid feed line 162. Here, the first liquid feed line 161 and the second liquid feed line 162 are pipes. In this embodiment, the first liquid feed line 161 connects the hydrocyclone 110 and the in-line mixer 150. One end of the first liquid feed line 161 is connected to the first outlet 111 of the hydrocyclone 110. The other end of the first liquid feed line 161 is connected to the inlet 152 of the in-line mixer 150. The first liquid feed line 161 is provided with a connection port 161A for connecting to the second liquid feed line 162. Here, the connection port 161A is the junction of the first liquid feed line 161 and the second liquid feed line 162. In this embodiment, the second liquid supply line 162 extends from the first storage tank 120 and merges with the first liquid supply line 161. One end of the second liquid supply line 162 is connected to the connection port 121 of the first storage tank 120. The other end of the second liquid supply line 162 is connected to the connection port 161A of the first liquid supply line 161.

[0025] The apparatus 100 is configured to operate, for example, as follows. First, when a switch of a supply source disposed upstream of the hydrocyclone 110 is turned on, liquid is supplied from the supply source. The liquid flows downstream from the supply source, passes through the pipe 171, and is introduced into the hydrocyclone 110 from the inlet 113. The liquid is then separated into liquid A and liquid B by the hydrocyclone 110. Here, liquid A flows into the first liquid feed line 161 through the first outlet 111. Liquid B flows into another liquid feed line different from the first liquid feed line 161 through the second outlet 112. Although not particularly limited, the other liquid feed line may be, for example, a drain line, a liquid feed line leading to a protein purification process, or the like. As liquid A flows downstream through the first liquid feed line 161 (toward the junction with the second liquid feed line 162 in the embodiment shown in FIG. 1 ), its flow rate is adjusted by the operation of the first pump 130. Liquid A flows through the first liquid feed line 161 under the action of the first pump 130 and reaches the connection port 161A.

[0026] Meanwhile, the first liquid flows downstream from the first storage tank 120. The first liquid passes through the connection port 121 of the first storage tank 120 and flows into the second liquid feed line 162. As the first liquid flows downstream through the second liquid feed line 162 (towards the junction with the first liquid feed line 161 in the embodiment shown in FIG. 1 ), the flow rate of the first liquid is adjusted by the operation of the second pump 140. The first liquid, having flowed through the second liquid feed line 162 while being subjected to the operation of the second pump 140, reaches the connection port 161A, which is the junction with the first liquid feed line 161.

[0027] Liquid A and the first liquid join at connection port 161A and continue to flow downstream. In the embodiment shown in FIG. 1 , liquid A and the first liquid join at connection port 161A and flow toward in-line mixer 150. Liquid A and the first liquid then flow through inlet 152 into pipe 151 of in-line mixer 150. Liquid A and the first liquid flow downstream while being mixed within pipe 151. The mixture of liquid A and the first liquid is discharged outside in-line mixer 150 through outlet 153 and flows into pipe 172. The mixture can then be subjected to processing such as joining with and mixing with a pump, a hydrocyclone, or other liquids provided downstream of pipe 172.

[0028] As described above, the apparatus 100 is an apparatus for purifying a protein from a cell culture medium. The apparatus 100 includes a hydrocyclone 110, a first reservoir 120 for storing a first liquid, a first pump 130, and a second pump 140. The first pump 130 is provided at a position where the flow rate of the liquid exiting the hydrocyclone 110 can be adjusted. The second pump 140 is provided at a position where the flow rate of the first liquid exiting the first reservoir 120 can be adjusted.

[0029] In the apparatus 100, the first pump 130 is provided at a location where the flow rate of the liquid discharged from the hydrocyclone 110 can be adjusted, and the second pump 140 is provided at a location where the flow rate of the first liquid discharged from the first storage tank 120 can be adjusted. In other words, in the apparatus 100, the flow rate of the liquid discharged from the hydrocyclone 110 can be controlled, for example, by appropriately changing the output, etc., of the first pump 130. This makes it possible to suppress clogging and retention of solids in the piping downstream of the hydrocyclone 110, even when the liquid discharged from the hydrocyclone 110 contains solids, making it easier to control the flow rate. The flow rate of the liquid discharged from the first storage tank 120 can be controlled by appropriately changing the output, etc., of the second pump 140. Therefore, by providing the first pump 130 and the second pump 140, liquid can be supplied more stably from each of the hydrocyclone 110 and the first storage tank 120. This allows the liquid coming out of the hydrocyclone 110 to be mixed better with the first liquid coming out of the first storage tank 120, and ultimately allows for more efficient production of purified protein from the cell culture liquid.

[0030] The apparatus 1 may further include an in-line mixer 150, a first liquid feed line 161, and a second liquid feed line 162. The in-line mixer 150 may be located downstream of the hydrocyclone 110. The first liquid feed line 161 may connect the hydrocyclone 110 and the in-line mixer 150. The second liquid feed line 162 may extend from the first storage tank 120 and merge with the first liquid feed line 161. The first pump 130 may be provided on the first liquid feed line 161 closer to the hydrocyclone 110 than the merger with the second liquid feed line 162 (connection port 161A in FIG. 1 ). The second pump 140 may be provided on the second liquid feed line 162 closer to the first storage tank 120 than the merger with the first liquid feed line 161. According to this configuration, the first pump 130 is provided in the first liquid feed line 161 closer to the hydrocyclone 110 than the joining point with the second liquid feed line 162, and the second pump 140 is provided in the second liquid feed line 162 closer to the first storage tank 120 than the joining point with the first liquid feed line 161. In other words, the liquid discharged from the hydrocyclone 110 reaches the joining point after its volume has been adjusted by the first pump 130. The first liquid discharged from the first storage tank 120 reaches the joining point after its volume has been adjusted by the second pump 140. Therefore, both liquids are joined together with their volumes adjusted and sent to the in-line mixer 150. This further improves the mixing efficiency of the liquid discharged from the hydrocyclone 110 and the first liquid discharged from the first storage tank 120.

[0031] The hydrocyclone 110 may be configured to separate the liquid sent to the hydrocyclone 110 into liquid A and liquid B. The hydrocyclone 110 may have a first outlet 111 from which liquid A is discharged and a second outlet 112 from which liquid B is discharged. The first outlet 111 may be connected to a first liquid feed line 161. The first pump 130 may be provided between the first outlet 111 and the above-mentioned joining portion. With this configuration, the efficiency of mixing of the liquid discharged from the hydrocyclone 110 and the first liquid discharged from the first storage tank 120 can be further improved.

[0032] The device 100 may be used to purify antibodies produced from cultured cells, and such a configuration allows for more efficient purification of antibodies from cell culture media.

[0033] An embodiment of the apparatus 100 will be described below with reference to FIGS. 2 to 4. FIG. 2 is a schematic diagram of the apparatus 1. FIGS. 3 and 4 are enlarged schematic diagrams of the apparatus 1. FIG. 2 schematically illustrates the overall configuration of the apparatus 1. FIG. 3 schematically illustrates the configuration of a reaction module 1A, a first cleaning module 1B, and a portion of an elution module 1C. FIG. 4 schematically illustrates the configuration of the remaining portion of the elution module 1C, a second cleaning module 1D, and an equilibration module 1E. The apparatus 1 is an apparatus for purifying proteins from a cell culture solution and includes the configuration of the apparatus 100 described above. As shown in FIG. 2, the apparatus 1 includes a reaction module 1A, a first cleaning module 1B, an elution module 1C, a second cleaning module 1D, and an equilibration module 1E. The apparatus 1 is connected to, for example, a computer (not shown) that controls the operation of the apparatus 1. In this embodiment, the liquid flows from upstream to downstream.

[0034] Reaction module 1A is, for example, a module in which a cell culture solution and a first slurry react with each other. In this embodiment, in reaction module 1A, the cell culture solution reacts with a first slurry containing a carrier equipped with a ligand that binds to a protein derived from the cells, thereby producing a complex in which the protein and the ligand are bound. The cell culture solution is as described above.

[0035] The first slurry contains a carrier equipped with a ligand that binds to a protein derived from cells. The cells and proteins are as described above. The carrier equipped with the ligand may be, for example, dispersible in a liquid. The carrier equipped with the ligand is preferably one in which the ligand and the carrier are not easily separated when the device 100 is used, and more preferably, the two do not separate. Any carrier used for this type of application can be used without particular limitation. Resin beads, for example, are preferably used as the carrier. The resin may be, for example, a resin made of a natural polymer compound or a resin made of a synthetic polymer compound. Examples of suitable resins include resins made of natural polymer compounds such as agarose, dextran, cellulose, chitin, chitosan, and mannan; and resins made of synthetic polymer compounds such as polystyrene resin and methacrylate resin. The carrier may be beads containing an inorganic material, such as beads made of glass, silica, or zirconia; beads made of stainless steel; or beads made of a tungsten alloy. The carrier may be a substrate formed by mixing the inorganic material described above with a natural or synthetic polymer compound.

[0036] The ligand is capable of forming a complex with the protein to be purified. In forming the complex, the ligand and the protein can bind reversibly. The type of ligand is not particularly limited and can be appropriately selected depending on the type of protein to be purified. The ligand can be, for example, an antibody-binding protein such as protein A, protein G, or protein L; an antibody that recognizes the protein to be purified (including a primary antibody and a secondary antibody); a protein tag such as a histidine tag (His tag), a glutathione-S-transferase tag (GST tag), or a FLAG (registered trademark) tag; a receptor for the protein to be purified; or the like.

[0037] The first slurry may contain a buffer solution in addition to a carrier having a ligand that binds to a cell-derived protein. A buffer solution that does not denature the ligand and the protein to be purified may be used. The buffer solution is preferably a buffer solution having a pH of 5 to 8, more preferably a buffer solution having a pH of 6.5 to 8.0. Examples of the buffer solution include Tris-HCl buffer, acetate buffer, citrate buffer, and phosphate buffer. If necessary, 0.1 M to 1 M sodium chloride, potassium chloride, or the like may be added to the buffer solution.

[0038] As shown in Figures 2 and 3, the reaction module 1A includes a cell culture medium supply source 11, a first slurry storage tank 12, an in-line mixer 13, a hydrocyclone 21, pumps 14A and 14B, valves 15A to 15E, 27A, and 27C, pressure gauges 16A, 16B, and 26A, and flow meters 17A, 17B, and 28A. In this embodiment, the reaction module 1A includes a culture medium supply line 10A from the supply source 11, a first slurry supply line 10B from the first slurry storage tank, and a drain line 20C. The culture medium supply line 10A and the first slurry supply line 10B join at a joining point 10C. A pipe 18C is disposed at the joining point 10C. An in-line mixer 13 is connected to the pipe 18C. In the embodiment shown in FIGS. 2 and 3, four in-line mixers 13 connected in series are connected to the pipe 18C, and a hydrocyclone 21 is further connected downstream thereof.

[0039] As shown in Figure 3, culture medium supply line 10A includes, from upstream to downstream, supply source 11, valve 15A, valve 15B, pump 14A, pressure gauge 16A, flow meter 17A, valve 15C, in-line mixer 13, hydrocyclone 21, pressure gauge 26A, and valve 27A. In culture medium supply line 10A, supply source 11, valve 15A, pump 14A, flow meter 17A, and valve 15C are interconnected in this order by piping 18A. Valve 15B is provided in branch pipe 18A1 branching from piping 18A at portion 10D between valve 15A and pump 14A. Pressure gauge 16A is positioned between pump 14A and flow meter 17A so as to measure the pressure within piping 18A.

[0040] As shown in Figure 3, the pipe 18C is connected to the inlet 213 of the hydrocyclone 21. A valve 27A is connected to the first outlet 211 of the hydrocyclone 21 via a pipe 29A. A pressure gauge 26A is disposed between the hydrocyclone 21 and the valve 27A so as to be able to measure the pressure inside the pipe 29A.

[0041] 3, the first slurry supply line 10B includes, from the upstream side, the first slurry storage tank 12, valves 15D and 15E, a pump 14B, a pressure gauge 16B, and a flow meter 17B. In the first slurry supply line 10B, the first slurry storage tank 12, valve 15D, pump 14B, and flow meter 17B are interconnected in this order by piping 18B. The valve 15E is provided in a branch pipe 18B1 branching off from the piping 18B at a position 10E between the valve 15D and the pump 14B. The pressure gauge 16B is positioned between the pump 14B and the flow meter 17B so as to measure the pressure in the piping 18B.

[0042] 2 and 3, the drain line 20C includes, from the upstream side, the hydrocyclone 21, the flow meter 28A, and the valve 27C. In the embodiment shown in Fig. 3, a pipe 29C is connected to the second outlet 212 of the hydrocyclone 21. In the drain line 20C, the hydrocyclone 21, the flow meter 28A, and the valve 27C are interconnected in this order from the upstream side by the pipe 29C. In this embodiment, the pipe 29C is connected to a drain path (not shown).

[0043] The first cleaning module 1B is, for example, connected to the reaction module 1A and is a module that cleans the composite using a first cleaning liquid. The first cleaning liquid may be, for example, the dispersion medium of the first slurry. As shown in FIGS. 2 and 3 , the first cleaning module 1B includes a first cleaning liquid storage tank 22, a first pump 23, a second pump 24, an in-line mixer 25, pressure gauges 26B, 26C, and 36A, valves 27B, 27D to 27F, 37A, and 37L, flow meters 28B and 38A, and a hydrocyclone 31A. The first cleaning module 1B includes a composite supply line 20A connected to the reaction module 1A, a cleaning liquid supply line 20B extending from the first cleaning liquid storage tank 22, and a drain line 30C. The composite supply line 20A and the cleaning liquid supply line 20B join at a joining point 20D.

[0044] 3, the composite supply line 20A includes, from the upstream side, a first pump 23, a pressure gauge 26B, a valve 27B, an in-line mixer 25, a hydrocyclone 31A, a pressure gauge 36A, and a valve 37A. In the composite supply line 20A, from the upstream side, the first pump 23, the valve 27B, and the in-line mixer 25 are interconnected in this order by a pipe 29A. The pressure gauge 26B is disposed between the first pump 23 and the valve 27B so as to be able to measure the pressure inside the pipe 29A. The in-line mixer 25 is connected to a pipe 29D on the downstream side.

[0045] As shown in Figure 3, pipe 29D is connected to an inlet 313A of hydrocyclone 31A. A valve 37A is connected to a first outlet 311A ​​of hydrocyclone 31A via pipe 39AX. A pressure gauge 36A is disposed between hydrocyclone 31A and valve 37A so as to measure the pressure in pipe 39AX.

[0046] 3, the cleaning liquid supply line 20B includes, from the upstream side, the first cleaning liquid storage tank 22, valve 27D, valve 27E, second pump 24, pressure gauge 26C, valve 27F, and flow meter 28B. In the cleaning liquid supply line 20B, from the upstream side, the first cleaning liquid storage tank 22, valve 27D, second pump 24, valve 27F, and flow meter 28B are interconnected in this order by piping 29B. Valve 27E is provided in a branch pipe 29B1 branching from piping 29B at a position 20E between valve 27D and second pump 24. The pressure gauge 26C is positioned between the second pump 24 and valve 27F so as to measure the pressure in piping 29B.

[0047] 3, the drain line 30C includes, from upstream, a hydrocyclone 31A, a flow meter 38A, and a valve 37L. In this embodiment, a pipe 39C is connected to the second outlet 312A of the hydrocyclone 31A. In the drain line 30C, from upstream, the hydrocyclone 31A, the flow meter 38A, and the valve 37L are interconnected by the pipe 39C in this order. In this embodiment, the pipe 39C is connected to a drain path (not shown).

[0048] The elution module 1C is connected to the first washing module 1B and uses an elution solution to elute the protein from the complex that has passed through the first washing module 1B. Any elution solution can be used without particular limitations, as long as it can, for example, release the bond between the ligand and the protein and elute the protein from the complex. The elution solution is preferably an elution solution of pH 5 or less, more preferably a buffer solution of pH 2 to pH 5, and even more preferably an elution solution of pH 2.0 to pH 2.5. The elution solution may be, for example, an acetate buffer, a citrate buffer, a phosphate buffer, a dilute solution of phosphoric acid or hydrochloric acid, or the like. If necessary, 0.1 M to 1 M (preferably 0.1 M to 0.5 M, more preferably 0.1 M to 0.3 M) of sodium chloride, potassium chloride, or the like may be added to the buffer.

[0049] 2 to 4, the elution module 1C includes hydrocyclones 31B, 31C, and 41A, an eluate reservoir 32, first pumps 33A to 33C, a second pump 34, in-line mixers 35A to 35C, pressure gauges 36B to 36I and 46A, valves 37B to 37K, 37M, 37N, 47A, and 47I, and flow meters 38B to 38F and 48A. As shown in FIG. 2, the elution module 1C includes a complex supply line 30A connected to the first cleaning module 1B and serving as a complex flow path, an eluate supply line 30B extending from the eluate reservoir 32, and a recovery line 30F for recovering the eluted protein. The complex supply line 30A and the eluate supply line 30B are joined at respective confluences 30D1 to 30D3.

[0050] 2, the composite supply line 30A includes a first block 30A1, a second block 30A2, and a third block 30A3, which are connected in series in this order from the upstream side.

[0051] As shown in FIG. 3 , the first block 30A1 includes a first pump 33A, a pressure gauge 36B, a valve 37B, an in-line mixer 35A, a hydrocyclone 31B, a pressure gauge 36C, and a valve 37C. In the configuration shown in FIG. 3 , in the first block 30A1, the first pump 33A, the valve 37B, and the in-line mixer 35A are interconnected in this order from the upstream side by a pipe 39AX. The pressure gauge 36B is positioned between the first pump 33A and the valve 37B so as to measure the pressure in the pipe 39AX. In this embodiment, the in-line mixer 35A and the hydrocyclone 31B are interconnected in this order by a pipe 39D1. In this embodiment, the pipe 39D1 is connected to the inlet 313B of the hydrocyclone 31B. The hydrocyclone 31B and the valve 37C are connected to each other by a pipe 39AY. The pipe 39AY is connected to a first outlet 311B of the hydrocyclone 31B. A pressure gauge 36C is disposed between the hydrocyclone 31B and the valve 37C so as to measure the pressure in the pipe 39AY.

[0052] As shown in FIG. 3 , the second block 30A2 includes a first pump 33B, a pressure gauge 36D, a valve 37D, an in-line mixer 35B, a hydrocyclone 31C, a pressure gauge 36E, and a valve 37E. In the configuration shown in FIG. 3 , the second block 30A2 includes, from upstream, the first pump 33B, the valve 37D, and the in-line mixer 35B, which are interconnected in this order by a pipe 39AY. The pressure gauge 36D is positioned between the first pump 33B and the valve 37D so as to measure the pressure within the pipe 39AY. In this embodiment, the in-line mixer 35B and the hydrocyclone 31C are interconnected in this order by a pipe 39D2. In this embodiment, the pipe 39D2 is connected to the inlet 313C of the hydrocyclone 31C. The hydrocyclone 31C and the valve 37E are interconnected by a pipe 39AZ. The pipe 39AZ is connected to a first outlet 311C of the hydrocyclone 31C. A pressure gauge 36E is disposed between the hydrocyclone 31C and the valve 37E so as to measure the pressure in the pipe 39AZ.

[0053] As shown in FIG. 3 , the third block 30A3 includes a first pump 33C, a pressure gauge 36F, a valve 37F, an in-line mixer 35C, a hydrocyclone 41A, a pressure gauge 46A, and a valve 47A. In the configuration shown in FIGS. 3 and 4 , in the third block 30A3, the first pump 33C, the valve 37F, and the in-line mixer 35C are interconnected in this order via a pipe 39AZ. The pressure gauge 36F is positioned between the first pump 33C and the valve 37F so as to measure the pressure within the pipe 39AZ. In this embodiment, the in-line mixer 35C and the hydrocyclone 41A are interconnected in this order via a pipe 39D3. In this embodiment, the pipe 39D3 is connected to an inlet 413A of the hydrocyclone 41A. The hydrocyclone 41A and the valve 47A are connected to each other by a pipe 49AX. The pipe 49AX is connected to a first outlet 411A of the hydrocyclone 41A. A pressure gauge 46A is disposed between the hydrocyclone 41A and the valve 47A so as to be able to measure the pressure in the pipe 49AX.

[0054] 2 and 3, the eluate supply line 30B includes an eluate reservoir 32, a valve 37G, a valve 37H, a second pump 34, a first line 30B1, a second line 30B2, and a third line 30B3. In this embodiment, the eluate reservoir 32, the valve 37G, and the second pump 34 are connected to each other in this order via a pipe 39B, and the first line 30B1, the second line 30B2, and the third line 30B3 are disposed downstream of the second pump 34. The valve 37H is provided in a branch pipe 39B1 branching off from the pipe 39B at a position 30E between the valve 37G and the second pump 34.

[0055] As shown in FIG. 3 , first line 30B1 connects branch point 39P on pipe 39B to confluence 30D1. Here, first line 30B1 includes pressure gauge 36G, valve 37I, and flow meter 38D. In the configuration shown in FIG. 3 , in first line 30B1, valve 37I and flow meter 38D are interconnected in this order from the branch point 39P side by pipe 39BX. Pressure gauge 36G is positioned between branch point 39P and valve 37I so as to measure the pressure within pipe 39BX. Pipe 39BX extends from branch point 39P as a base end and confluences with pipe 39AX at confluence 30D1.

[0056] As shown in FIG. 3 , second line 30B2 connects branch point 39Q on pipe 39B to confluence 30D2. Here, second line 30B2 includes pressure gauge 36H, valve 37J, and flow meter 38E. In the configuration shown in FIG. 3 , second line 30B2 is interconnected by pipe 39BY, in this order, with valve 37J and flow meter 38E from the branch point 39Q side. Pressure gauge 36H is positioned between branch point 39Q and valve 37J so as to measure the pressure within pipe 39BY. Pipe 39BY extends from branch point 39Q as its base end and confluences with pipe 39AY at confluence 30D2.

[0057] As shown in FIG. 3 , third line 30B3 connects branch point 39R on pipe 39B to confluence 30D3. Here, third line 30B3 includes pressure gauge 36I, valve 37K, and flow meter 38F. In the configuration shown in FIG. 3 , in third line 30B3, valve 37K and flow meter 38F are interconnected in this order from the branch point 39R side by pipe 39BZ. Pressure gauge 36I is positioned between branch point 39R and valve 37K so as to measure the pressure within pipe 39BZ. Pipe 39BZ extends from branch point 39R as its base end and confluences with pipe 39AZ at confluence 30D3.

[0058] As shown in FIG. 3, the return line 30F includes a first return line 30F1, a second return line 30F2, and a third return line 30F3.

[0059] As shown in FIG. 3 , the first recovery line 30F1 connects the second outlet 312B of the hydrocyclone 31B to a branch point 39X on the pipe 39E (the main pipe of the recovery line 30F). Here, the first recovery line 30F1 includes a flow meter 38B and a valve 37M. In the configuration shown in FIG. 3 , the first recovery line 30F1 is interconnected in this order by a pipe 39E1, starting from the second outlet 312B. The pipe 39E1 merges with the pipe 39E at a branch point 39X. As shown in FIG. 3 , the second recovery line 30F2 connects the second outlet 312C of the hydrocyclone 31C to a branch point 39Y on the pipe 39E. Here, the second recovery line 30F2 includes a flow meter 38C and a valve 37N. In the configuration shown in FIG. 3, in the second return line 30F2, from the second outlet 312C side, the flow meter 38C and the valve 37N are interconnected in this order by piping 39E2. Pipe 39E2 merges with piping 39E at branch point 39Y. As shown in FIG. 4, the third return line 30F3 connects the second outlet 412A of the hydrocyclone 41A to branch point 39Z on piping 39E. Here, the third return line 30F3 includes a flow meter 48A and a valve 47I. In the configuration shown in FIG. 4, in the third return line 30F3, from the second outlet 412A side, the flow meter 48A and the valve 47I are interconnected in this order by piping 49C. Pipe 49C merges with piping 39E at branch point 39Z.

[0060] The second washing module 1D is, for example, connected to the elution module 1C and is a module that washes the ligand and carrier after the protein has been eluted with a second washing solution. As the second washing solution, for example, any of the various buffer solutions described as the first washing solution can be used without any particular limitation. The second washing solution may be the same as the first washing solution or may be different from the first washing solution.

[0061] 2 and 4, the second cleaning module 1D includes hydrocyclones 41B and 51A, a second cleaning liquid storage tank 42, first pumps 43A and 43B, a second pump 44, in-line mixers 45A and 45B, pressure gauges 46B to 46F and 56A, valves 47B to 47H, 47J, 57A and 57L, and flow meters 48B to 48D and 58A. As shown in FIG. 2, the second cleaning module 1D includes a carrier supply line 40A that serves as a flow path for carriers (carriers having a ligand), a cleaning liquid supply line 40B extending from the second cleaning liquid storage tank 42, and a drain line 40C. The carrier supply line 40A and the cleaning liquid supply line 40B join at joining portions 40D1 and 40D2, respectively.

[0062] In the configurations shown in FIGS. 2 and 4, the carrier supply line 40A includes a first block 40A1 and a second block 40A2. The first block 40A1 and the second block 40A2 are connected in series in this order from the upstream side. The first block 40A1 includes a first pump 43A, a pressure gauge 46B, a valve 47B, an in-line mixer 45A, a hydrocyclone 41B, a pressure gauge 46C, and a valve 47C. In the configuration shown in FIG. 4, in the first block 40A1, the first pump 43A, the valve 47B, and the in-line mixer 45A are interconnected in this order from the upstream side by a pipe 49AX. The pressure gauge 46B is disposed between the first pump 43A and the valve 47B so as to measure the pressure in the pipe 49AX. In this embodiment, the in-line mixer 45A and the hydrocyclone 41B are connected to each other in this order by a pipe 49D1. In this embodiment, the pipe 49D1 is connected to the inlet 413B of the hydrocyclone 41B. The hydrocyclone 41B and the valve 47C are connected to each other by a pipe 49AY. The pipe 49AY is connected to the first outlet 411B of the hydrocyclone 41B. The pressure gauge 46C is disposed between the hydrocyclone 41B and the valve 47C so as to measure the pressure in the pipe 49AY.

[0063] As shown in FIG. 4 , the second block 40A2 includes a first pump 43B, a pressure gauge 46D, a valve 47D, an in-line mixer 45B, a hydrocyclone 51A, a pressure gauge 56A, and a valve 57A. In the configuration shown in FIG. 4 , in the second block 40A2, the first pump 43B, the valve 47D, and the in-line mixer 45B are interconnected in this order from the upstream side by a pipe 49AY. The pressure gauge 46D is positioned between the first pump 43B and the valve 47D so as to measure the pressure in the pipe 49AY. In this embodiment, the in-line mixer 45B and the hydrocyclone 51A are interconnected in this order by a pipe 49D2. In this embodiment, the pipe 49D2 is connected to the inlet 513A of the hydrocyclone 51A. The hydrocyclone 51A and the valve 57A are connected to each other by a pipe 59AX. The pipe 59AX is connected to a first outlet 511A of the hydrocyclone 51A. A pressure gauge 56A is disposed between the hydrocyclone 51A and the valve 57A so as to measure the pressure in the pipe 59AX.

[0064] 2 and 4, the cleaning liquid supply line 40B includes a second cleaning liquid reservoir 42, a valve 47G, a valve 47H, a second pump 44, a first line 40B1, and a second line 40B2. In this embodiment, the cleaning liquid supply line 40B includes, from the upstream side, the second cleaning liquid reservoir 42, the valve 47G, and the second pump 44, which are interconnected in this order by a pipe 49B, and the first line 40B1 and the second line 40B2 are disposed downstream of the second pump 44. The valve 47H is provided in a branch pipe 49B1 branching off from the pipe 49B at a position 40F between the valve 47G and the second pump 44.

[0065] As shown in FIG. 4 , first line 40B1 connects branch point 49P on pipe 49B to confluence 40D1. Here, first line 40B1 includes pressure gauge 46E, valve 47E, and flow meter 48C. In the configuration shown in FIG. 4 , in first line 40B1, valve 47E and flow meter 48C are interconnected in this order from the branch point 49P side by pipe 49BX. Pressure gauge 46E is positioned between branch point 49P and valve 47E so as to measure the pressure within pipe 49BX. Pipe 49BX extends from branch point 49P as its base end and confluences with pipe 49AX at confluence 40D1.

[0066] As shown in FIG. 4 , second line 40B2 connects branch point 49Q on pipe 49B to confluence 40D2. Here, second line 40B2 includes pressure gauge 46F, valve 47F, and flow meter 48D. In the configuration shown in FIG. 4 , second line 40B2 is interconnected in this order from branch point 49Q by pipe 49BY, with valve 47F and flow meter 48D. Pressure gauge 46F is positioned between branch point 49Q and valve 47F so as to measure the pressure within pipe 49BY. Pipe 49BY extends from branch point 49Q as its base end and merges with pipe 49AY at confluence 40D2.

[0067] In this embodiment, the drain line 40C includes a first drain line 40C1 and a second drain line 40C2. As shown in FIG. 4 , the first drain line 40C1 includes, from upstream, a hydrocyclone 41B, a flow meter 48B, and a valve 47J. In this embodiment, a pipe 49E is connected to the second outlet 412B of the hydrocyclone 41B. In the first drain line 40C1, the hydrocyclone 41B, the flow meter 48B, and the valve 47J are interconnected in this order from upstream by the pipe 49E. In this embodiment, the pipe 49E is connected to a drain path (not shown). In the embodiment shown in FIG. 4 , the second drain line 40C2 includes a hydrocyclone 51A, a flow meter 58A, and a valve 57L. Here, a pipe 59CX is connected to the second outlet 512A of the hydrocyclone 51A. 4, in the second drainage line 40C2, the hydrocyclone 51A, the flow meter 58A, and the valve 57L are connected to one another in this order from the upstream side by a pipe 59CX. In this embodiment, the pipe 59CX is connected to a drainage path (not shown).

[0068] The equilibration module 1E is, for example, connected to the second cleaning module 1D, and is a module that equilibrates, using an equilibration liquid, the ligands (ligands provided on the carriers) after being washed in the second cleaning module 1D. Considering that the carriers with the ligands equilibrated in this module will be returned to the first slurry storage tank 12, it is preferable that the equilibration liquid be the same as the dispersion medium contained in the first slurry.

[0069] 2 and 4, the equilibration module 1E includes hydrocyclones 51B and 51C, an equilibration liquid reservoir 52, first pumps 53A and 53B, a second pump 54, in-line mixers 55A and 55B, pressure gauges 56B to 56E, 56G, and 56H, valves 57B to 57E, 57H to 57K, 57M, and 57N, and flow meters 58B to 58E. As shown in FIG. 2, the equilibration module 1E includes a carrier supply line 50A serving as a flow path for carriers (carriers containing ligands), an equilibration liquid supply line 50B extending from the equilibration liquid reservoir 52, and a drain line 50C. The carrier supply line 50A and the equilibration liquid supply line 50B join at confluences 50D1 and 50D2, respectively.

[0070] In the configurations shown in FIGS. 2 and 4, the carrier supply line 50A includes a first block 50A1 and a second block 50A2. The first block 50A1 and the second block 50A2 are connected in series from the upstream side. As shown in FIG. 4, the first block 50A1 includes a first pump 53A, a pressure gauge 56B, a valve 57B, an in-line mixer 55A, a hydrocyclone 51B, a pressure gauge 56C, and a valve 57C. In the configuration shown in FIG. 4, in the first block 50A1, the first pump 53A, the valve 57B, and the in-line mixer 55A are interconnected in this order from the upstream side by a pipe 59AX. The pressure gauge 56B is disposed between the first pump 53A and the valve 57B so as to measure the pressure in the pipe 59AX. In this embodiment, the in-line mixer 55A and the hydrocyclone 51B are connected to each other in this order by a pipe 59D1. In this embodiment, the pipe 59D1 is connected to the inlet 513B of the hydrocyclone 51B. The hydrocyclone 51B and the valve 57C are connected to each other by a pipe 59AY. The pipe 59AY is connected to the first outlet 511B of the hydrocyclone 51B. The pressure gauge 56C is disposed between the hydrocyclone 51B and the valve 57C so as to measure the pressure in the pipe 59AY.

[0071] As shown in FIG. 4 , the second block 50A2 includes a first pump 53B, a pressure gauge 56D, a valve 57D, an in-line mixer 55B, a hydrocyclone 51C, a pressure gauge 56E, and a valve 57E. In the configuration shown in FIG. 4 , in the second block 50A2, the first pump 53B, the valve 57D, and the in-line mixer 55B are interconnected in this order from the upstream side by a pipe 59AY. The pressure gauge 56D is positioned between the first pump 53B and the valve 57D so as to measure the pressure in the pipe 59AY. In this embodiment, the in-line mixer 55B and the hydrocyclone 51C are interconnected in this order by a pipe 59D2. In this embodiment, the pipe 59D2 is connected to the inlet 513C of the hydrocyclone 51C. The hydrocyclone 51C and the valve 57E are interconnected by a pipe 59AZ. The pipe 59AZ is connected to a first outlet 511C of the hydrocyclone 51C. A pressure gauge 56E is disposed between the hydrocyclone 51C and the valve 57E so as to measure the pressure in the pipe 59AZ.

[0072] 4, the equilibration solution supply line 50B includes an equilibration solution reservoir 52, a valve 57H, a valve 57I, a second pump 54, a first line 50B1, and a second line 50B2. In this embodiment, the equilibration solution reservoir 52, the valve 57H, and the second pump 54 are connected to each other in this order via a pipe 59B, with the first line 50B1 and the second line 50B2 located downstream of the second pump 54. The valve 57I is provided in a branch pipe 59B1 branching off from the pipe 59B at a position 50F between the valve 57H and the second pump 54.

[0073] As shown in FIG. 4 , first line 50B1 connects branch point 59P on pipe 59B to confluence 50D1. Here, first line 50B1 includes pressure gauge 56G, valve 57J, and flow meter 58D. In the configuration shown in FIG. 4 , in first line 50B1, valve 57J and flow meter 58D are interconnected in this order from the branch point 59P side by pipe 59BX. Pressure gauge 56G is positioned between branch point 59P and valve 57J so as to measure the pressure within pipe 59BX. Pipe 59BX extends from branch point 59P as a base end and confluences with pipe 59AX at confluence 50D1.

[0074] As shown in FIG. 4 , the second line 50B2 connects the branch point 59Q on the pipe 59B to the junction 50D2. Here, the second line 50B2 includes a pressure gauge 56H, a valve 57K, and a flow meter 58E. In the configuration shown in FIG. 4 , the valve 57K and the flow meter 58E are interconnected in this order from the branch point 59Q side of the second line 50B2 by the pipe 59BY. The pressure gauge 56H is positioned between the branch point 59Q and the valve 57K so as to measure the pressure within the pipe 59BY. The pipe 59BY extends from the branch point 59Q as its base end and merges with the pipe 59AY at the junction 50D2.

[0075] As shown in FIG. 2 , the drain line 50C includes a first drain line 50C1 and a second drain line 50C2. The first drain line 50C1 includes a hydrocyclone 51B, a flow meter 58B, and a valve 57M. Here, a pipe 59CY is connected to the second outlet 512B of the hydrocyclone 51B. As shown in FIG. 4 , the hydrocyclone 51B, the flow meter 58B, and the valve 57M are interconnected in this order from the upstream side by the pipe 59CY. In this embodiment, the pipe 59CY is connected to a drain path (not shown). The second drain line 50C2 includes a hydrocyclone 51C, a flow meter 58C, and a valve 57N. Here, a pipe 59CZ is connected to the second outlet 512C of the hydrocyclone 51C. 4, in the second drainage line 50C2, the hydrocyclone 51C, the flow meter 58C, and the valve 57N are connected to each other in this order from the upstream side by a pipe 59CZ. In this embodiment, the pipe 59CZ is connected to a drainage path (not shown).

[0076] In this embodiment, a return line 50E is connected to the equilibration module 1E. Here, the return line 50E is a line that returns the equilibrated carrier to the first slurry storage tank 12. In the configuration shown in FIG. 4, a pipe 59AQ (the main pipe of the return line 50E in this embodiment) is connected downstream of the valve 57E of the equilibration module 1E. As shown in FIG. 4, the return line 50E has a pump 535, a pressure gauge 56F, a valve 57F, and a valve 57G. In the configurations shown in FIGS. 2 and 4, in the return line 50E, the pump 535, the valve 57F, and the valve 57G are connected to each other in this order from the upstream side by the pipe 59AQ. The pressure gauge 56F is disposed between the pump 535 and the valve 57F so as to measure the pressure in the pipe 59AQ.

[0077] Next, the operation of the apparatus 1 will be described with reference to FIGS. 2 to 4. However, the following description is merely an example of the operation of the apparatus 1 and is not intended to limit the technology disclosed herein. When a switch of a computer controlling the operation of the apparatus 1 is turned on and then a switch of the apparatus 1 is turned on, the apparatus 1 becomes operational. First, in the reaction module 1A, the first slurry is sent from the first slurry storage tank 12 to the first slurry supply line 10B. At this time, the valve 15D is preferably in an open state and the pump 14B is in an operable state. The valve 15E is preferably in a closed state. The first slurry passes from the first slurry storage tank 12 through the pipe 18B and reaches the confluence 10C while the liquid volume in the pipe 18B is adjusted by the pump 14B. The output of the pump 14B can be appropriately set, for example, based on the measured values ​​of a pressure gauge 16B and a flow meter 17B provided in the first slurry supply line 10B.

[0078] As described above, while the first slurry is being delivered, cell culture solution is delivered from supply source 11 to culture solution supply line 10A. At this time, valves 15A and 15C are preferably open, and pump 14A is preferably operable. Valve 15B is preferably closed. The culture solution flows from supply source 11 through pipe 18A and reaches confluence 10C while the amount of liquid in pipe 18A is adjusted by pump 14A. The output of pump 14A can be appropriately set, for example, based on the measured values ​​of pressure gauge 16A and flow meter 17A provided in culture solution supply line 10A.

[0079] The culture solution and first slurry that meet at meeting point 10C pass through pipe 18C and are mixed by four in-line mixers 13 connected in series. During this process, proteins in the culture solution bind to ligands in the first slurry, and a protein-ligand complex is produced in the mixture of the culture solution and the first slurry (hereinafter also referred to as the "first mixture"). The first mixture then passes through in-line mixer 13 and is sent to hydrocyclone 21.

[0080] The first mixture enters the hydrocyclone 21 through the inlet 213 from the pipe 18C. The hydrocyclone 21 separates the first mixture into component A and component B. Component A includes, for example, both carriers carrying the complex and carriers carrying unreacted ligands. Component B is, for example, the remaining component. Component A is sent from the first outlet 211 of the hydrocyclone 21 to the pipe 29A, passes through the valve 27A, and enters the first cleaning module 1B. At this time, it is preferable that the valves 27A and 27B are open and the first pump 23 is operable. Component A passes from the first outlet 211 through the pipe 29A and reaches the confluence 20D while the amount of liquid in the pipe 29A is adjusted by the first pump 23. The output of the first pump 23 can be appropriately set, for example, based on the measured values ​​of the pressure gauges 26A and 26B.

[0081] Component B is discharged to the drainage path via drain line 20C. In this case, component B is discharged to the drainage path from second outlet 212 of hydrocyclone 21 through pipe 29C. At this time, valve 27C is preferably in an open state.

[0082] At this time, the first cleaning liquid is sent from the first cleaning liquid storage tank 22 to the pipe 29B in the cleaning liquid supply line 20B. At this time, it is preferable that the valve 27D is open and the second pump 24 is operable. It is preferable that the valve 27E is closed. The first cleaning liquid flows from the first cleaning liquid storage tank 22 through the pipe 29B, and reaches the junction 20D while the amount of liquid in the pipe 29B is adjusted by the second pump 24, and then joins the composite supply line 20A. The output of the second pump 24 can be set appropriately based on, for example, the measured values ​​of the pressure gauge 26C and the flow meter 28B.

[0083] Component A and the first washing solution that have joined at confluence 20D pass through pipe 29A and are mixed by in-line mixer 25. At this time, for example, by washing the complex with the first washing solution, components (e.g., proteins derived from the culture medium) that are nonspecifically attached to the carrier, complex, etc. are removed and become suspended in the solution. Then, the mixture of component A and the first washing solution (hereinafter also simply referred to as the "second mixture") passes through in-line mixer 25 and is sent to hydrocyclone 31A.

[0084] The second mixture then passes from pipe 29D through inlet 313A and enters hydrocyclone 31A. Hydrocyclone 31A separates the second mixture into component C and component D. Component C includes, for example, a carrier bearing a complex and a carrier bearing unreacted ligand. Component C is sent from first outlet 311A ​​of hydrocyclone 31A to pipe 39AX, passes through valve 37A, and enters first block 30A1 of complex supply line 30A in elution module 1C. At this time, valves 37A and 37B are preferably open, and first pump 33A is preferably operable. Component C passes from first outlet 311A ​​through pipe 39AX and reaches confluence 30D1 while the amount of liquid in pipe 39AX is adjusted by first pump 33A. The output of the first pump 33A can be set appropriately based on, for example, the measured values ​​of the pressure gauges 36A and 36B. The component D is discharged to the drainage path via the drainage line 30C. In this case, the component D is discharged from the second outlet 312A of the hydrocyclone 31A through the pipe 39C to the drainage path. At this time, it is preferable that the valve 37L is in an open state.

[0085] Meanwhile, in the eluate supply line 30B, the eluate is sent from the eluate reservoir 32 to the pipe 39B. At this time, the valve 37G is preferably in an open state, and the second pump 34 is preferably in an operable state. The valve 37H is preferably in a closed state. Furthermore, the valve 37I in the pipe 39BX is preferably in an open state. The eluate flows from the eluate reservoir 32 through the pipes 39B and 39BX, and while the liquid volume in the pipes 39B and 39BX is being adjusted by the second pump 34, the eluate reaches the junction 30D1 and joins the first block 30A1 of the composite supply line 30A. The output of the second pump 34 can be set appropriately based on, for example, the measurement values ​​of a pressure gauge 36G and a flow meter 38D provided on the pipe 39BX, the measurement values ​​of a pressure gauge 36H and a flow meter 38E provided on the pipe 39BY described later, and the measurement values ​​of a pressure gauge 36I and a flow meter 38F provided on the pipe 39BZ described later.

[0086] Component C and the eluate that have joined at the joining point 30D1 pass through pipe 39AX and are mixed by in-line mixer 35A. At this time, for example, the protein is eluted from the complex into the liquid by the eluate. The mixture of component C and the eluate (hereinafter also simply referred to as the "third mixture") then passes through in-line mixer 35A and is sent to hydrocyclone 31B.

[0087] Next, the third mixture enters the hydrocyclone 31B through the inlet 313B from the pipe 39D1. The hydrocyclone 31B separates the third mixture into component E and component F. Component E may include carriers bearing ligands from which proteins have been eluted in the first block 30A1, and carriers bearing complexes from which proteins have not yet been eluted in the first block 30A1. Component F is the remaining component and may include the protein eluted in the first block 30A1. Component E is sent from the first outlet 311B of the hydrocyclone 31B to the pipe 39AY, passes through the valve 37C, and enters the second block 30A2. At this time, it is preferable that the valves 37C and 37D are open, and the first pump 33B is operable. Component E flows from first outlet 311B through pipe 39AY and reaches confluence 30D2 while the amount of liquid in pipe 39AY is adjusted by first pump 33B. Note that the output of first pump 33B can be set appropriately based on the measured values ​​of pressure gauges 36C and 36D, for example.

[0088] Component F is sent to recovery line 30F via first recovery line 30F1. Component F passes from second outlet 312B of hydrocyclone 31B through pipe 39E1 and is sent to pipe 39E from branch point 39X. At this time, valve 37M should be open. Component F then joins with components H and J (described below) in pipe 39E and is sent to the protein purification process.

[0089] Meanwhile, in the eluate supply line 30B, the valve 37G is open, the second pump 34 is operable, and the eluate is being sent from the eluate reservoir tank 32 to the pipe 39B, and the valve 37J in the pipe 39BY is opened, thereby opening the second line 30B2. The eluate flows from the eluate reservoir tank 32 through the pipes 39B and 39BY, and reaches the junction 30D2 while the liquid volume in the pipes 39B and 39BY is adjusted by the second pump 34, and then merges with the second block 30A2 of the composite supply line 30A.

[0090] Component E and the eluate that join at confluence 30D2 pass through pipe 39AY and are mixed by in-line mixer 35B. At this time, for example, the protein is eluted from the complex into the liquid by the eluate. Then, the mixture of component E and the eluate (hereinafter simply referred to as the "fourth mixture") passes through in-line mixer 35B and is sent to hydrocyclone 31C.

[0091] Next, the fourth mixture passes from pipe 39D2 through inlet 313C and enters hydrocyclone 31C. Hydrocyclone 31C separates the fourth mixture into component G and component H. Component G may include carriers bearing ligands from which proteins have been eluted in first block 30A1 and second block 30A2, and carriers bearing complexes from which proteins have not been eluted in second block 30A2. Component H is the remaining component and may include proteins eluted in second block 30A2. Component G is sent from first outlet 311C of hydrocyclone 31C to pipe 39AZ, passes through valve 37E, and enters third block 30A3. At this time, valves 37E and 37F are preferably open, and first pump 33C is preferably operable. Component G flows from first outlet 311C through pipe 39AZ and reaches confluence 30D3 while the amount of liquid in pipe 39AZ is adjusted by first pump 33C. Note that the output of first pump 33C can be set appropriately based on the measured values ​​of pressure gauges 36E and 36F, for example.

[0092] Component H is sent to recovery line 30F via second recovery line 30F2. Component H is sent from second outlet 312C of hydrocyclone 31C through pipe 39E2 and from branch point 39Y to pipe 39E. At this time, valve 37N should be open. Thereafter, component H is joined with component F and component J (described below) in pipe 39E and sent to the protein purification process.

[0093] Meanwhile, in the eluate supply line 30B, as described above, the eluate is being sent from the eluate reservoir tank 32 to the pipe 39B, and the valve 37K in the pipe 39BZ is opened to open the third line 30B3. The eluate flows from the eluate reservoir tank 32 through the pipes 39B and 39BZ, and while the liquid amount in the pipes 39B and 39BZ is adjusted by the second pump 34, the eluate reaches the junction 30D3 and joins the third block 30A3 of the composite supply line 30A.

[0094] Component G and the eluate that join at confluence 30D3 pass through pipe 39AZ and are mixed by in-line mixer 35C. At this time, for example, the protein is eluted from the complex into the liquid by the eluate. The mixture of component G and the eluate (hereinafter also simply referred to as the "fifth mixture") then passes through in-line mixer 35C and is sent to hydrocyclone 41A.

[0095] The fifth mixture enters the hydrocyclone 41A through the inlet 413A from the pipe 39D3. The hydrocyclone 41A separates the fifth mixture into component I and component J. Component I may include carriers with ligands remaining after proteins have been eluted by the first block 30A1, the second block 30A2, and the third block 30A3. Component J is the remaining component and may include proteins eluted in the third block 30A3. Component I is sent from the first outlet 411A of the hydrocyclone 41A to the pipe 49AX, passes through the valve 47A, and enters the first block 40A1 of the carrier supply line 40A in the second cleaning module 1D. At this time, it is preferable that the valves 47A and 47B are open, and the first pump 43A is operable. Component I flows from first outlet 411A through pipe 49AX and reaches confluence 40D1 while the amount of liquid in pipe 49AX is adjusted by first pump 43A. Note that the output of first pump 43A can be set appropriately based on the measured values ​​of pressure gauges 46A and 46B, for example.

[0096] Component J is sent to recovery line 30F via third recovery line 30F3. Component J passes from second outlet 412A of hydrocyclone 41A through pipe 49C and is sent to pipe 39E from branch point 39Z. At this time, valve 47I should be open. Component J then joins with components F and H at pipe 39E and is sent to the protein purification process.

[0097] Meanwhile, in the cleaning liquid supply line 40B, the second cleaning liquid is sent from the second cleaning liquid storage tank 42 to the pipe 49B. At this time, the valve 47G is preferably open and the second pump 44 is preferably operable. The valve 47H is preferably closed. Furthermore, the valve 47E in the pipe 49BX is preferably open. The second cleaning liquid flows from the second cleaning liquid storage tank 42 through the pipes 49B and 49BX, and reaches the junction 40D1 to join the first block 40A1 of the carrier supply line 40A while the liquid volume in the pipes 49B and 49BX is adjusted by the second pump 44. The output of the second pump 44 can be appropriately set, for example, based on measurements from a pressure gauge 46E and a flow meter 48C provided in the pipe 49BX, and measurements from a pressure gauge 46F and a flow meter 48D provided in the pipe 49BY (described later).

[0098] The component I and the second cleaning liquid that have joined at the joining portion 40D1 pass through the pipe 49AX and are mixed by the in-line mixer 45A. At this time, for example, the support is washed with the second cleaning liquid. Then, the mixed liquid of the component I and the second cleaning liquid (hereinafter also simply referred to as the "sixth mixed liquid") passes through the in-line mixer 45A and is sent to the hydrocyclone 41B.

[0099] Next, the sixth mixed liquid enters the hydrocyclone 41B. The hydrocyclone 41B separates the sixth mixed liquid into component K and component L. Component K may include the carrier after cleaning in the first block 40A1. Component L is the remaining component. Component K is sent from the first outlet 411B of the hydrocyclone 41B to the pipe 49AY, passes through the valve 47C, and enters the second block 40A2. At this time, it is preferable that valves 47C and 47D are open and the first pump 43B is operable. Component K passes from the first outlet 411B through the pipe 49AY and reaches the confluence 40D2 while the amount of liquid in the pipe 49AY is adjusted by the first pump 43B. The output of the first pump 43B can be appropriately set, for example, based on the measurements of the pressure gauges 46C and 46D.

[0100] The component L is discharged to the drainage path via the first drainage line 40C1. In this case, the component L is discharged from the second outlet 412B of the hydrocyclone 41B through the pipe 49E to the drainage path. At this time, it is preferable that the valve 47J is in an open state.

[0101] Meanwhile, in the cleaning liquid supply line 40B, the valve 47G is open and the second pump 44 is operable to send the second cleaning liquid from the second cleaning liquid storage tank 42 to the pipe 49B, and the valve 47F in the pipe 49BY is opened to open the second line 40B2. The second cleaning liquid flows from the second cleaning liquid storage tank 42 through the pipes 49B and 49BY, and reaches the junction 40D2 while the liquid volume in the pipes 49B and 49BY is adjusted by the second pump 44, and then merges with the second block 40A2 of the carrier supply line 40A.

[0102] The component K and the second cleaning liquid that have joined at the joining point 40D2 pass through the pipe 49AY and are mixed by the in-line mixer 45B. At this time, for example, the carrier is washed with the second cleaning liquid. Then, the mixture of the component K and the second cleaning liquid (hereinafter also simply referred to as the "seventh mixed liquid") passes through the in-line mixer 45B and is sent to the hydrocyclone 51A.

[0103] The seventh mixed liquid enters the hydrocyclone 51A from the pipe 49D2 through the inlet 513A. The hydrocyclone 51A separates the seventh mixed liquid into component M and component N. Component M may include the carrier after cleaning in the second block 40A2. Component N is the remaining component. Component M is sent from the first outlet 511A of the hydrocyclone 51A to the pipe 59AX, passes through the valve 57A, and enters the first block 50A1 of the carrier supply line 50A of the balancing module 1E. At this time, it is preferable that the valves 57A and 57B are open and the first pump 53A is operable. Component M passes from the first outlet 511A through the pipe 59AX and reaches the confluence 50D1 while the amount of liquid in the pipe 59AX is adjusted by the first pump 53A. The output of the first pump 53A can be set appropriately based on the measured values ​​of the pressure gauges 56A and 56B, for example.

[0104] The component N is discharged to the drainage path via the second drainage line 40C2. In this case, the component N is discharged from the second outlet 512A of the hydrocyclone 51A through the pipe 59CX to the drainage path. At this time, it is preferable that the valve 57L is in an open state.

[0105] Meanwhile, in the equilibration liquid supply line 50B, the equilibration liquid is sent from the equilibration liquid reservoir 52 to the pipe 59B. At this time, the valve 57H is preferably open and the second pump 54 is preferably operable. The valve 57I is preferably closed. Furthermore, the valve 57J in the pipe 59BX is preferably open. The equilibration liquid flows from the equilibration liquid reservoir 52 through the pipes 59B and 59BX, and reaches the junction 50D1 to join the first block 50A1 of the carrier supply line 50A while the liquid volume in the pipes 59B and 59BX is adjusted by the second pump 54. The output of the second pump 54 can be appropriately set, for example, based on measurements from a pressure gauge 56G and a flow meter 58D provided in the pipe 59BX, and measurements from a pressure gauge 56H and a flow meter 58E provided in the pipe 59BY (described later).

[0106] The component M and the equilibration liquid that join at the joining point 50D1 pass through pipe 59AX and are mixed by in-line mixer 55A. At this time, the carrier is equilibrated by the equilibration liquid, for example. The mixture of component M and the equilibration liquid (hereinafter also simply referred to as the "eighth mixture") then passes through in-line mixer 55A and is sent to hydrocyclone 51B.

[0107] Next, the eighth mixed liquid enters the hydrocyclone 51B through the inlet 513B from the pipe 59D1. The eighth mixed liquid is separated into component O and component P by the hydrocyclone 51B. Component O may include carriers with ligands after equilibration by the first block 50A1. Component P is the remaining component. Component O is sent from the first outlet 511B of the hydrocyclone 51B to the pipe 59AY, passes through the valve 57C, and enters the second block 50A2 of the carrier supply line 50A. At this time, valves 57C and 57D may be open, and the first pump 53B may be operable. Component O passes from the first outlet 511B through the pipe 59AY and reaches the confluence 50D2 while the amount of liquid in the pipe 59AY is adjusted by the first pump 53B. The output of the first pump 53B can be set appropriately based on the measured values ​​of the pressure gauges 56C and 56D, for example.

[0108] The component P is discharged to the drainage path via the first drainage line 50C1. In this case, the component P is discharged from the second outlet 512B of the hydrocyclone 51B through the pipe 59CY to the drainage path. At this time, it is preferable that the valve 57M is in an open state.

[0109] Meanwhile, in the equilibration liquid supply line 50B, the valve 57H is open and the second pump 54 is operable to send the equilibration liquid from the equilibration liquid reservoir 52 to the pipe 59B, and the valve 57K in the pipe 59BY is opened to open the second line 50B2. The equilibration liquid flows from the equilibration liquid reservoir 52 through the pipes 59B and 59BY, and reaches the junction 50D2 to join the second block 50A2 of the carrier supply line 50A, while the amount of the equilibration liquid in the pipes 59B and 59BY is adjusted by the second pump 54.

[0110] The component O and the equilibration liquid that join at the joining point 50D2 pass through the pipe 59AY and are mixed by the in-line mixer 55B. At this time, for example, the carrier that has been treated with the equilibration liquid in the first block 50A1 is further treated with the equilibration liquid. The mixture of the component O and the equilibration liquid (hereinafter simply referred to as the "ninth mixture") then passes through the in-line mixer 55B and is sent to the hydrocyclone 51C.

[0111] The ninth mixture then enters the hydrocyclone 51C through the inlet 513C from the pipe 59D2. The hydrocyclone 51C separates the ninth mixture into component Q and component R. Component Q may include carriers with ligands after equilibration in the second block 50A2. Component R is the remaining component. Component Q is sent from the first outlet 511C of the hydrocyclone 51C to the pipe 59AZ, passes through the valve 57E, and enters the return line 50E. At this time, valves 57E and 57F may be open, and the pump 535 may be operable. Component Q passes from the first outlet 511C through the pipe 59AZ and enters the return line 50E while the pump 535 adjusts the amount of liquid in the pipe 59AQ. The component Q that has entered the return line 50E is finally sent to the first slurry storage tank 12 of the reaction module 1A. The output of the pump 535 can be appropriately set, for example, based on the measured values ​​of the pressure gauges 56E and 56F.

[0112] The component R is discharged to the drainage path via the second drainage line 50C2. In this case, the component R is discharged from the second outlet 512C of the hydrocyclone 51C through the pipe 59CZ to the drainage path. At this time, it is preferable that the valve 57N is in an open state.

[0113] As described above, the configuration of the device 100 shown in Fig. 1 can be included in the device 1 shown in Figs. 2 to 4. The device 100 may have the configuration of the hydrocyclone 110, first reservoir 120, first pump 130, and second pump 140 shown in Fig. 1 (hereinafter, also simply referred to as the "configuration shown in Fig. 1"). The hydrocyclones 21, 31A to 31C, 41A, 41B, 51A, and 51B of the device 1 shown in Figs. 2 to 4 can correspond to the hydrocyclone 110 in Fig. 1. The first cleaning solution reservoir 22, the eluate reservoir 32, the second cleaning solution reservoir 42, and the equilibration solution reservoir 52 of the device 1 can correspond to the first reservoir 120 in Fig. 1. The first pumps 23, 33A to 33C, 43A, 43B, 53A, and 53B of the apparatus 1 may correspond to the first pump 130 in Fig. 1. The second pumps 24, 34, 44, and 54 of the apparatus 1 may correspond to the second pump 140 in Fig. 1. The apparatus 1 having the configuration shown in Fig. 1 can more efficiently produce a purified protein from a cell culture solution.

[0114] In this embodiment, in the apparatus 1, a carrier dispersible in a liquid is used as a carrier having a ligand for the protein to be purified to capture the protein. In this regard, in the apparatus 1, first pumps 23, 33A-33C, 43A, 43B, 53A, and 53B are disposed downstream of the hydrocyclones 21, 31A-31C, 41A, 41B, 51A, and 51B. The first pumps 23, 33A-33C, 43A, 43B, 53A, and 53B suppress clogging and retention of the carrier-containing liquid in the piping that flows out of the hydrocyclones 21, 31A-31C, 41A, 41B, 51A, and 51B located upstream, thereby making it easier to control the flow rate of the liquid. This allows the functions of each module to be better realized.

[0115] 1 is preferably provided in elution module 1C, although this is not a limitation. By providing elution module 1C with the configuration shown in Fig. 1, the liquids discharged from hydrocyclones 31B, 31C, and 41A can be mixed more effectively with the eluate discharged from eluate reservoir 32, which allows the protein to be eluted from the complex more efficiently, and ultimately allows the purified protein to be produced more efficiently.

[0116] The protein to be purified may be an antibody. The ligand may be an antibody-binding protein. This configuration allows for more efficient purification of antibodies from cell culture media.

[0117] The technology disclosed herein provides a method for producing a purified protein, which includes purifying a protein from a cell culture medium using the apparatus 1 or the apparatus 100. By using such a production method, a purified protein can be produced from a cell culture medium more efficiently.

[0118] As described above, the apparatus 1 is only required to have the configuration shown in Fig. 1, and there are no particular limitations on the number of hydrocyclones, in-line mixers, first pumps, second pumps, etc. in each module. These numbers can be set appropriately depending on the scale of the apparatus, the amount of raw material that can be processed in one operation of the apparatus, etc.

[0119] The technology disclosed herein may include aspects described in the following items: -Item 1- An apparatus for purifying a protein from a cell culture medium, comprising: a hydrocyclone; a first reservoir tank for storing a first liquid; a first pump; and a second pump, wherein the first pump is provided at a position where it is possible to adjust the flow rate of the liquid exiting the hydrocyclone, and the second pump is provided at a position where it is possible to adjust the flow rate of the first liquid exiting the first reservoir tank. -Item 2- The apparatus according to Item 1, further comprising: an in-line mixer located downstream of the hydrocyclone; a first liquid feed line connecting the hydrocyclone and the in-line mixer; and a second liquid feed line extending from the first storage tank and joining the first liquid feed line, wherein the first pump is provided on the first liquid feed line closer to the hydrocyclone than the joining point with the second liquid feed line, and the second pump is provided on the second liquid feed line closer to the first storage tank than the joining point with the first liquid feed line. -Item 3- The hydrocyclone is configured to separate a liquid fed to the hydrocyclone into liquid A and liquid B, and has a first outlet for discharging liquid A and a second outlet for discharging liquid B, the first outlet being connected to the first liquid feed line, and the first pump being provided between a junction of the first outlet and the second liquid feed line. -Item 4- The apparatus according to any one of items 1 to 3, which is used to purify an antibody produced from cultured cells. -Item 5- The apparatus according to any one of items 1 to 4, wherein the protein is an antibody, and an antibody-binding protein that is a ligand is used to purify the antibody. -Item 6- A method for producing a purified protein, which comprises purifying a protein from a cell culture medium using the apparatus according to any one of items 1 to 5.

[0120] Although specific examples of the present invention have been described above in detail, these are merely examples and do not limit the scope of the claims. The technology described in the claims includes various modifications and alterations of the specific examples exemplified above.

[0121] 100 Apparatus 110 Hydrocyclone 120 First reservoir 130 First pump 140 Second pump 150 In-line mixer 1 Apparatus 1A Reaction module 1B First washing module 1C Elution module 1D Second washing module 1E Equilibration module

Claims

1. An apparatus for purifying a protein from a cell culture solution, comprising: a hydrocyclone; a first storage tank for storing a first liquid; a first pump; and a second pump, wherein the first pump is provided at a position where the flow rate of the liquid exiting the hydrocyclone can be adjusted; and the second pump is provided at a position where the flow rate of the first liquid exiting the first storage tank can be adjusted.

2. The apparatus according to claim 1, further comprising: an in-line mixer downstream of the hydrocyclone; a first liquid supply line connecting the hydrocyclone and the in-line mixer; and a second liquid supply line extending from the first storage tank and merging with the first liquid supply line, wherein the first pump is provided on the first liquid supply line closer to the hydrocyclone than the junction with the second liquid supply line, and the second pump is provided on the second liquid supply line closer to the first storage tank than the junction with the first liquid supply line.

3. The apparatus described in claim 2, wherein the hydrocyclone is configured to separate liquid sent to the hydrocyclone into liquid A and liquid B, and has a first outlet for discharging liquid A and a second outlet for discharging liquid B, the first outlet being connected to the first liquid supply line, and the first pump being provided between a junction of the first outlet and the second liquid supply line.

4. The device according to any one of claims 1 to 3, which is used to purify antibodies produced from cultured cells.

5. The device of claim 4, wherein the protein is an antibody and an antibody-binding protein that is a ligand is used to purify the antibody.

6. A method for producing a purified protein, comprising purifying a protein from a cell culture medium using the device according to any one of claims 1 to 3.

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