Protein purification device

Abstract

Provided is a technology for improving the efficiency of separating a complex of a protein to be purified and a carrier in a hydrocyclone of a device including an in-line mixer and the hydrocyclone. A protein purification device disclosed herein comprises a first supply unit, a second supply unit, an in-line mixer, a hydrocyclone, and a dilution line. The first supply unit supplies a first slurry containing a carrier including a ligand that binds to a protein. The second supply unit supplies a second slurry which is a cell culture solution containing a protein. The in-line mixer mixes the first slurry and the second slurry. The hydrocyclone separates, downstream of the in-line mixer, a complex of the protein and the carrier from a mixed slurry of the first slurry and the second slurry. The dilution line is provided between the in-line mixer and the hydrocyclone and introduces a diluted liquid into the mixed slurry.

Description

Protein purification device

[0001] This disclosure relates to a protein purification apparatus. This application claims priority under Japanese Patent Application No. 2024-188847, filed on 28 October 2024, the entire contents of which are incorporated herein by reference.

[0002] International Publication No. 2019 / 189064 discloses an apparatus for the continuous purification of proteins from cell culture media using a module combining an inline mixer and a cyclone. This apparatus consists of a first module and a second module. In the first module, the inlet of the inline mixer is connected via a branch valve to piping connecting the culture tank or cell separation device to the first module and to piping connecting the first storage tank to the first module. The outlet of the inline mixer is connected to the inlet of the hydrocyclone. The lower outlet of the hydrocyclone is connected to piping connecting the first reaction module and the second module. Piping is connected to the upper outlet of the hydrocyclone. In the second module, the inlet of the inline mixer is connected via a branch valve to piping connecting the first module and the second module and to piping connecting the second storage tank and the second module. The outlet of the inline 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 also connected to the outlet at the top of the hydrocyclone. The publication states that by using an apparatus with this configuration, even a suspension containing a small amount of cells, cell fragments, or host microorganisms can be continuously recovered and purified in a short time.

[0003] International Publication No. 2019 / 189064

[0004] Incidentally, as described in Patent Document 1, there is an apparatus including an in-line mixer and a hydrocyclone connected to the in-line mixer downstream thereof. For example, in Patent Document 1, a step of mixing a cell culture solution containing a specific protein and a slurry containing a resin using such an apparatus to bind the protein in the cell culture solution to the resin in the slurry to produce a complex, and a step of separating the complex from the mixed slurry continuously fed from the in-line mixer in the hydrocyclone are described as being carried out. Here, the present inventor has focused on separating a complex of a protein to be purified and a carrier having a ligand that binds to the protein using a hydrocyclone.

[0005] When producing a complex of a protein to be purified and a carrier using an apparatus having the above-described configuration, for example, as the volume ratio of the carrier in the slurry containing the carrier increases, the adsorption rate of the protein to the carrier improves, and the production efficiency of the complex improves. However, as the volume ratio of the complex in the slurry supplied to the hydrocyclone increases, for example, the separation efficiency of the complex from the hydrocyclone tends to decrease. On the other hand, in order to increase the separation efficiency of the complex from the hydrocyclone, for example, if an attempt is made to reduce the concentration of the complex in the mixed slurry with the cell culture solution by lowering the volume ratio of the carrier in the slurry containing the carrier, it may be necessary to lengthen the mixing time of the slurry containing the carrier and the cell culture solution, use a larger in-line mixer, and the like.

[0006] In view of such circumstances, the present inventor desires to increase the efficiency of separating a complex of a protein to be purified and a carrier in the hydrocyclone included in an apparatus including an in-line mixer and a hydrocyclone connected to the in-line mixer downstream thereof.

[0007] The technology disclosed herein provides a device for purifying proteins from cell culture medium (protein purification device). The protein purification device comprises a first feed unit, a second feed unit, an in-line mixer, a hydrocyclone, and a dilution line. The first feed unit supplies a first slurry. The first slurry contains a carrier equipped with a ligand that binds to a protein. The second feed unit supplies a second slurry. The second slurry is a cell culture medium containing a protein. The in-line mixer mixes the first slurry supplied from the first feed unit and the second slurry supplied from the second feed unit. The hydrocyclone is connected to the in-line mixer downstream of it. The hydrocyclone separates the protein-carrier complex from the mixed slurry of the first and second slurries obtained by the in-line mixer. The dilution line introduces a diluent into the mixed slurry between the in-line mixer and the hydrocyclone. This configuration can improve the efficiency of separating the complex of the protein to be purified and the support in a hydrocyclone.

[0008] Figure 1 is a schematic diagram of apparatus 1. Figure 2 is an enlarged schematic diagram of the adsorption section 10. Figure 3 is a schematic diagram of the apparatus used in test 2. Figure 4 is a schematic diagram of the apparatus used in test 2.

[0009] Preferred embodiments of the technology disclosed herein are described below. Matters other than those specifically mentioned herein that are necessary for carrying out the technology disclosed herein can be understood based on the technical content taught herein and the common technical knowledge of those skilled in the art. The technology disclosed herein can be carried out based on the content disclosed herein and the common technical knowledge of the art. The A to B (where A and B are arbitrary numbers) in this specification and the claims mean A or greater and B or less, and also include the case where A is greater than and B is less than or equal to.

[0010] The technology disclosed herein provides a protein purification apparatus. The protein purification apparatus is, for example, an apparatus for purifying proteins from a cell culture medium. In this specification, “cells” means cells that produce the protein to be purified and that can be cultured in vitro. “Cells” includes, for example, cell lines and cell aggregates, tissues, or organs excised from living organisms. “Cell line” means a clone 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, 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 preferably used as cells, and among them, NS0 cells, Sp2 / 0 cells, CHO cells, HEK cells, and hybridomas are preferably used. The cells may be genetically modified cells capable of producing the protein to be purified. In this specification, "culture medium" refers to the culture medium in which the above-mentioned cells are cultured and the protein to be purified is produced and secreted.

[0011] The proteins to be purified using 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, antibodies are preferable. The antibodies are preferably monoclonal antibodies, and may be, for example, mouse antibodies or chimeric antibodies, but humanized antibodies or fully humanized antibodies are preferred. The antigens recognized by the antibodies are not particularly limited and can be appropriately set depending on the purpose.

[0012] Figure 1 is a schematic diagram of apparatus 1. Apparatus 1 shown in Figure 1 is an example of the protein purification apparatus disclosed herein. As shown in Figure 1, apparatus 1 has an adsorption unit 10, a first washing unit 20, an elution unit 30, a second washing unit 40, and an equilibration unit 50. Each functional block will be described below.

[0013] The adsorption unit 10 is a functional block that, for example, adsorbs a protein to be purified from a cell culture medium onto a carrier. Figure 2 is an enlarged schematic diagram of the adsorption unit 10. In Figure 2, the adsorption unit 10 of the apparatus 1 shown in Figure 1 is shown in an enlarged view. As shown in Figures 1 and 2, the adsorption unit 10 includes a first supply unit 110, a second supply unit 120, an inline mixer 130, a dilution line 10A, a backflow prevention unit 10B, a hydrocyclone 140, a first pump 170, and a second pump 180. The adsorption unit 10 may also be equipped with pumps, valves, pressure gauges, flow meters, etc., as needed, although these are not shown.

[0014] The first supply unit 110 is, for example, a part that supplies the first slurry. In this embodiment, the first supply unit 110 is a storage tank for storing the first slurry. As shown in Figure 2, the first supply unit 110 has a connection port 111. The connection port 111 is a supply port for the first slurry from the first supply unit 110 to the first liquid delivery line 151 and is connected to the first liquid delivery line 151. In this embodiment, a first pump 170 is provided upstream of the confluence point 152X on the first liquid delivery line 151 to facilitate the delivery of liquid from the first supply unit 110.

[0015] The first slurry here contains a carrier equipped with a ligand that binds to a protein. The protein is the protein to be purified as described above. The carrier equipped with the ligand is preferably, for example, dispersible in liquid. When using the apparatus 1, it is preferable that the ligand and the carrier do not separate easily, and more preferably that they do not separate at all. Any carrier used for this type of application can be used without particular limitations. For example, resin beads can be 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. The resin may be, for example, a resin made of a natural polymer compound such as agarose, dextran, cellulose, chitin, chitosan, mannan; or a resin made of a synthetic polymer compound such as polystyrene resin or methacrylate resin. The carrier may be beads containing an inorganic material, such as beads made of glass, silica, zirconia; beads made of stainless steel; or beads made of tungsten alloy. The carrier may be composed of a substrate formed by mixing the above-mentioned inorganic material with a natural polymer compound or a synthetic polymer compound.

[0016] A ligand is a substance that can form a complex with the protein to be purified. In complex formation, 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. Ligands may include, for example, antibody-binding proteins such as protein A, protein G, and protein L; antibodies that recognize the protein to be purified (including primary and secondary antibodies); protein tags such as histidine tags (His tags), glutathione-S-transferase tags (GST tags), and FLAG® tags; receptors for the protein to be purified; and so on.

[0017] The first slurry may contain a carrier equipped with a ligand that binds to a cell-derived protein, as well as a buffer. The buffer may be one that does not denature the ligand or the protein to be purified. The buffer is preferably pH 5 to pH 8, and more preferably pH 6.5 to 0H8.0. Examples of buffers include Tris-HCl buffer, acetate buffer, citrate buffer, and phosphate buffer. If necessary, 0.1 M to 1 M sodium chloride, potassium chloride, etc., may be added to the buffer.

[0018] The second supply unit 120 is, for example, a part that supplies the second slurry. In this embodiment, the second supply unit 120 is a storage tank for storing the second slurry. As shown in Figure 2, the second supply unit 120 has a connection port 121. The connection port 121 is a supply port for the second slurry from the second supply unit 120 to the second liquid delivery line 152, and is connected to the second liquid delivery line 152. In this embodiment, a second pump 180 is provided upstream of the confluence point 152X on the second liquid delivery line 152 to facilitate the delivery of liquid from the second supply unit 120. The second slurry in this case is a cell culture medium containing protein. The protein and cell culture medium are as described above.

[0019] The inline mixer 130 is a mixer that mixes, for example, a first slurry supplied from a first supply unit 110 and a second slurry supplied from a second supply unit 120. As shown in Figure 2, the inline mixer 130 is located downstream of the first supply unit 110 and the second supply unit 120. The inline mixer 130 is located downstream of the confluence point 152X with the first supply line 151 in the second liquid delivery line 152. The inline mixer 130 here has a pipe 131, an inlet 132, and an outlet 133. The pipe 131 is cylindrical (in this case cylindrical) and is the main body of the inline mixer 130 and the flow path for the mixed slurry of the first slurry and the second slurry. The inlet 132 is, for example, the point where the first slurry and the second slurry, which merge at the confluence point 152X, flow into the pipeline 131. In the configuration shown in Figure 2, the inlet 132 is located at one end of the pipeline 131. The inlet 132 is connected to the second liquid supply line 152. The outlet 133 is, for example, the point where the mixed slurry is discharged from the inline mixer 130. In the configuration shown in Figure 2, the outlet 133 is located at the other end of the pipeline 131. Here, the outlet 133 is connected to the piping 161.

[0020] In this specification, "inline mixer" refers to a stationary mixer without a drive unit that mixes at least two types of liquids using the energy generated by the flow velocity of the liquids flowing inside the device.

[0021] The mixed slurry contains, for example, a carrier and a protein. In this embodiment, the volume percentage of the carrier in the mixed slurry is set so that the volume percentage of the carrier in the slurry X flowing downstream of the confluence section 161X, which will be described later, is the desired volume percentage. The mixed slurry is preferably prepared to contain approximately 5% to 40% by volume of carrier, with the entire mixed slurry being 100% by volume. From the viewpoint of miniaturizing the apparatus 1 and from the viewpoint of improving the adsorption efficiency of the protein to the carrier and shortening the protein purification time by the apparatus 1, the mixed slurry is prepared to contain preferably 7.5% or more by volume, more preferably 10% or more by volume of carrier, with the entire mixed slurry being 100% by volume. On the other hand, from the viewpoint of suppressing clogging in the hydrocyclone 140 and from the viewpoint of efficiently diluting the mixed slurry by the dilution line 190, which will be described later, the mixed slurry is prepared to contain preferably 35% or less by volume, more preferably 30% or less by volume, and even more preferably 25% or less by volume of carrier, with the entire mixed slurry being 100% by volume. While not particularly limited, the mixed slurry is preferably prepared such that, for example, the entire mixed slurry, as 100% by volume, contains 5 mg to 20 mg of protein per 1 ml of carrier.

[0022] While not particularly limited, the length of the inline mixer 130 is preferably set so that the first slurry and the second slurry can be mixed in approximately 0.6 to 60 seconds. From the viewpoint of improving the adsorption of the protein to the carrier, the length of the inline mixer 130 is set so that the first slurry and the second slurry can be mixed in, for example, 0.8 seconds or more, preferably 1 second or more, more preferably 2 seconds or more, even more preferably 3 seconds or more, and particularly preferably 5 seconds or more. On the other hand, from the viewpoint of saving space in the apparatus 1 and shortening the protein purification time by the apparatus 1, the length of the inline mixer 130 is preferably set so that the first slurry and the second slurry can be mixed in a time of 55 seconds or less, more preferably 50 seconds or less. The length of the inline mixer 130 is preferably set so that the first slurry and the second slurry can be mixed in a time of 3 to 50 seconds, more preferably 4 to 45 seconds, and particularly preferably 10 to 40 seconds. As described above, by setting the length of the inline mixer 130 to achieve the aforementioned mixing time between the first slurry and the second slurry, approximately 70% or more of the protein to be purified contained in the second slurry can be adsorbed onto the carrier.

[0023] In this specification, "length of inline mixer 130" refers to the length of the conduit 131 of the inline mixer 130. In the configuration shown in Figure 2, one inline mixer 130 is shown, but the device 1 may be equipped with multiple inline mixers 130. In this case, "length of inline mixer 130" refers to the sum of the lengths of the conduits 131 of each inline mixer 130.

[0024] The dilution line 10A is, for example, a line that introduces a diluent into the mixed slurry between the inline mixer 130 and the hydrocyclone 140. In the configuration shown in Figure 2, the dilution line 10A is located on the piping 161. Here, the dilution line 10A is connected to the piping 161 at the confluence point 161X.

[0025] As shown in Figure 2, the dilution line 10A includes a diluent supply unit 10A1 and a pump 10A2. The diluent supply unit 10A1 is, for example, a part that supplies the diluent. In this embodiment, the diluent supply unit 10A1 is a storage tank for storing the diluent. As shown in Figure 2, the diluent supply unit 10A1 has a connection port 10A11. The connection port 10A11 is a supply port for the diluent from the diluent supply unit 10A1 to the piping 10A3 and is connected to the piping 161. The pump 10A2 is, for example, a pump that adjusts the flow rate of the diluent supplied from the diluent supply unit 10A1. In the configuration shown in Figure 2, the pump 10A2 is located on the piping 10A3. The pump 10A2 is located between the connection port 10A11 and the confluence point 161X. As the diluent, for example, the same buffer solution contained in the first slurry is used.

[0026] The dilution line 10A is preferably configured to introduce a diluent into the mixed slurry such that, for example, the volume ratio of the carrier in slurry X is approximately 1 / 8 to 2 / 3 of the volume ratio of the carrier in the mixed slurry. Here, slurry X refers to the slurry containing the diluent and the mixed slurry that has passed through the point where the diluent and the mixed slurry merge (here, the merging point 161X). From the viewpoint of suppressing clogging by the carrier in the hydrocyclone 140, the dilution line 10A is configured to introduce a diluent into the mixed slurry such that the volume ratio of the carrier in slurry X is preferably 1 / 2 or less, more preferably 1 / 3 or less of the volume ratio of the carrier in the mixed slurry. On the other hand, from the viewpoint of shortening the time required to separate the composite in the hydrocyclone 140, the dilution line 10A is configured to introduce a diluent into the mixed slurry such that the volume ratio of the carrier in slurry X is preferably 1 / 6 or more, more preferably 1 / 4 or more of the volume ratio of the carrier in the mixed slurry.

[0027] The backflow prevention unit 10B has the function of preventing the mixed slurry from entering the dilution line 10A from the point where the diluent and the mixed slurry merge (here, the merging point 161X). In this embodiment, the backflow prevention unit 10B is a check valve. The backflow prevention unit 10B is provided, for example, on the dilution line. As shown in Figures 1 and 2, the backflow prevention unit 10B is provided between the pump 10A2 and the merging point 161X. In this embodiment, the backflow prevention unit 10B is provided on the piping 10A3.

[0028] The hydrocyclone 140 is a separator that separates protein-carrier complexes from a mixed slurry of a first slurry and a second slurry obtained by the inline mixer 130, for example. In this embodiment, the hydrocyclone 140 separates the complexes from slurry X, which contains a diluent and a mixed slurry. In this embodiment, by passing through the hydrocyclone 140, slurry X is separated into liquid A and liquid B. Liquid A contains, for example, the complexes. Liquid A may also contain, for example, carriers that did not form complexes with proteins. Liquid B is, for example, the residue after removing the complexes and carriers that did not form complexes with proteins from slurry X. Therefore, the larger the amount of carriers contained in liquid A, the higher the separation efficiency of the complexes in the hydrocyclone 140. In this embodiment, in the configuration shown in Figure 2, the hydrocyclone 140 is downstream of the inline mixer 130 and connected to the inline mixer 130. As shown in Figure 2, the hydrocyclone 140 is funnel-shaped and has a first outlet 141 at its lower end, a second outlet 142 at its upper end, and an inlet 143 on its upper side. The first outlet 141 is, for example, the part from which liquid A is discharged from the hydrocyclone 140. In the configuration shown in Figure 2, the first outlet 141 is connected to piping 162. In this embodiment, the first outlet 141 is connected to the first cleaning unit 20 via piping 162. The second outlet 142 is the part from which liquid B is discharged from the hydrocyclone 140. The second outlet 142 is connected to, for example, a drain line outside the device 1 or another liquid supply line. The inlet 143 is the part from which liquid sent from upstream is introduced into the hydrocyclone 140. In this embodiment, the inlet 143 is connected to piping 161. In the configuration shown in Figure 2, an inline mixer 130 is positioned upstream of the hydrocyclone 140. The piping 161 may connect the inline mixer 130 and the hydrocyclone 140 directly or indirectly. In this specification, "hydrocyclone" refers to a separator without a drive unit that can separate, classify, concentrate, etc., a liquid introduced in a stationary state.

[0029] The first pump 170 is, for example, a pump that adjusts the flow rate of the first slurry supplied from the first supply unit 110. In the configuration shown in Figure 2, the first pump 170 is located on the first liquid delivery line 151. Here, in the first liquid delivery line 151, it is located on the side of the first supply unit 110 that is closer to the junction 152X with the second liquid delivery line 152. The first pump 170 is located between the connection port 111 and the junction 152X.

[0030] The second pump 180 is, for example, a pump that adjusts the flow rate of the second slurry supplied from the second supply unit 120. In the configuration shown in Figure 2, the second pump 180 is located on the second liquid delivery line 152. Here, in the second liquid delivery line 152, it is located on the side of the second supply unit 120 that is closer to the junction 152X with the first liquid delivery line 151. The second pump 180 is located between the connection port 121 and the junction 152X.

[0031] The first cleaning unit 20 is a functional block connected to, for example, the adsorption unit 10, and cleans the composite using the first cleaning solution. As shown in Figure 1, the first cleaning unit 20 includes a first cleaning solution supply unit 210, a pump 220, an inline mixer 230, and a hydrocyclone 240. In the configuration shown in Figure 1, the first cleaning unit 20 includes the inline mixer 230 and the hydrocyclone 240 from the adsorption unit 10 side. Here, the adsorption unit 10 and the inline mixer 230 are interconnected by piping 162. The inline mixer 230 and the hydrocyclone 240 are interconnected by piping 261. Piping 262 and piping 263 extend from the hydrocyclone 240. Piping 262 is connected to the elution unit 30. Piping 263 is connected to, for example, a drainage line (not shown).

[0032] In the configuration shown in Figure 1, a pipe 250 extends from the first cleaning fluid supply unit 210. Here, the pipe 250 merges with the pipe 162 at the junction 250X. In this embodiment, the pump 220 is provided on the pipe 250. As the first cleaning fluid, for example, the buffer solution used in the first slurry may be preferably used. The first cleaning unit 20 may also be equipped with a pump, valve, pressure gauge, flow meter, etc., which are not shown, as needed.

[0033] The elution unit 30 is a functional block connected to, for example, the first washing unit 20, which uses an eluent to elute proteins from the complex that has passed through the first washing unit 20. As shown in Figure 1, the elution unit 30 comprises an eluent supply unit 310, a pump 320, an in-line mixer 330, and a hydrocyclone 340. In the configuration shown in Figure 1, the elution unit 30 comprises the in-line mixer 330 and the hydrocyclone 340 from the first washing unit 20 side. Here, the first washing unit 20 and the in-line mixer 330 are interconnected by piping 262. The in-line mixer 330 and the hydrocyclone 340 are interconnected by piping 361. Piping 362 and piping 363 extend from the hydrocyclone 340. Piping 362 is connected to the second washing unit 40. Piping 363 is connected to, for example, a recovery line (not shown).

[0034] In the configuration shown in Figure 1, a pipe 350 extends from the eluent supply unit 310. Here, the pipe 350 merges with the pipe 262 at the confluence point 350X. In this embodiment, the pump 320 is provided on the pipe 350. The eluent is preferably an eluent with a pH of 5 or lower, more preferably a buffer solution with a pH of 2 to 5, and even more preferably an eluent with a pH of 2.5 to 3.5. The eluent may be, for example, an acetate buffer, a citrate buffer, a phosphate buffer, or a dilute solution of phosphoric acid or hydrochloric acid. 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, etc. may be added to the buffer. The eluent unit 30 may also be appropriately equipped with a pump, valve, pressure gauge, flow meter, etc., which are not shown, if necessary.

[0035] The second washing section 40 is a functional block connected to, for example, the elution section 30, and washes the carrier that has passed through the elution section 30 using the second washing liquid. As shown in Figure 1, the second washing section 40 includes a second washing liquid supply section 410, a pump 420, an inline mixer 430, and a hydrocyclone 440. In the configuration shown in Figure 1, the second washing section 40 includes the inline mixer 430 and the hydrocyclone 440 from the elution section 30 side. Here, the second washing section 40 and the inline mixer 430 are interconnected by piping 362. The inline mixer 430 and the hydrocyclone 440 are interconnected by piping 461. Piping 462 and piping 463 extend from the hydrocyclone 440. Piping 462 is connected to the equilibrium section 50. Piping 463 is connected to, for example, a drain line (not shown).

[0036] In the configuration shown in Figure 1, a pipe 450 extends from the second cleaning fluid supply unit 410. Here, the pipe 450 merges with the pipe 362 at the junction 450X. In this embodiment, the pump 420 is provided on the pipe 450. As the second cleaning fluid, for example, the buffer solution used in the first slurry may be preferably used. The second cleaning unit 40 may also be equipped with a pump, valve, pressure gauge, flow meter, etc., which are not shown, as needed.

[0037] The equilibration unit 50 is a functional block connected to, for example, the second washing unit 40, which equilibrates the ligands (ligands provided on the carrier) after they have been washed in the second washing unit 40 using an equilibration liquid. As shown in Figure 1, the equilibration unit 50 includes an equilibration liquid supply unit 510, a pump 520, an in-line mixer 530, and a hydrocyclone 540. In the configuration shown in Figure 1, the equilibration unit 50 includes the in-line mixer 530 and the hydrocyclone 540 from the second washing unit 40 side. Here, the equilibration unit 50 and the in-line mixer 530 are interconnected by piping 462. The in-line mixer 530 and the hydrocyclone 540 are interconnected by piping 561. Piping 562 and piping 563 extend from the hydrocyclone 540. In this embodiment, piping 562 is connected to the first supply unit 110 (not shown). The piping 563 is connected, for example, to a drainage line (not shown).

[0038] In the configuration shown in Figure 1, a pipe 550 extends from the equilibration liquid supply unit 510. Here, the pipe 550 merges with the pipe 462 at the junction 550X. In this embodiment, the pump 520 is provided on the pipe 550. As the equilibration liquid, for example, the buffer solution used in the first slurry can preferably be used. The equilibration unit 50 may also be equipped with a pump, valve, pressure gauge, flow meter, etc., as needed, although these are not shown.

[0039] Device 1 is configured to operate as follows, for example. First, when the switch of device 1 is turned on, a first slurry is supplied from the first supply unit 110. The first slurry enters the first liquid delivery line 151 through the connection port 111 and flows downstream, and its flow rate is adjusted by the operation of the first pump 170. The first slurry, having flowed through the first liquid delivery line 151 while being operated by the first pump 170, reaches the confluence point 152X. Meanwhile, a second slurry is supplied from the second supply unit 120. The second slurry enters the second liquid delivery line 152 through the connection port 121 and flows downstream, and its flow rate is adjusted by the operation of the second pump 180. The second slurry, having flowed through the second liquid delivery line 152 while being operated by the second pump 180, reaches the confluence point 152X.

[0040] The first slurry and the second slurry merge at the confluence point 152X and flow further downstream. In the configuration shown in Figure 2, the first slurry and the second slurry merge at the confluence point 152X and flow toward the inline mixer 130. The first slurry and the second slurry flow through the inlet 132 into the conduit 131 of the inline mixer 130. In the conduit 131, the first slurry and the second slurry are mixed and flow toward downstream. At this time, the mixed slurry of the first slurry and the second slurry achieves the volume ratio of the carrier and the protein concentration within the range described above. The mixing time between the first slurry and the second slurry is set to fall within the range described above.

[0041] Next, the mixed slurry flows out of the inline mixer 130 through the outlet 133 and into the piping 161. In this embodiment, a diluent is introduced from the dilution line 10A into the mixed slurry flowing through the piping 161. Here, the diluent is first supplied from the diluent supply unit 10A1. The diluent enters the piping 10A3 through the connection port 10A11 and flows downstream, its flow rate being adjusted by the operation of the pump 10A2. The diluent that has flowed through the piping 10A3 while being operated by the pump 10A2 passes through the backflow prevention unit 10B and reaches the confluence point 161X.

[0042] Next, the diluent flows from the confluence point 161X into the pipe 161 and merges with the mixed slurry flowing through the pipe 161. At this time, in the slurry X containing the diluent and the mixed slurry, the carrier and protein contained in the mixed slurry are diluted. This results in the above-mentioned volume ratio of carrier being achieved in slurry X. Subsequently, slurry X flows into a hydrocyclone 140 located downstream of pipe 161. In this embodiment, slurry X flows from pipe 161 through the inlet 143 into the hydrocyclone 140. Next, slurry X is separated into liquid A and liquid B by the hydrocyclone 140. Liquid B enters pipe 163 through, for example, the second outlet 142 and is sent to the drain line. Liquid A includes, for example, a carrier comprising a complex and a carrier comprising an unreacted ligand.

[0043] In this embodiment, liquid A enters the piping 162 through the first outlet 141 and is sent to the first washing section 20. Liquid A travels through the piping 162 to the confluence section 250X. Meanwhile, the first washing liquid is supplied from the first washing liquid supply section 210. The first washing liquid travels through the piping 250 to the confluence section 250X. In this embodiment, liquid A and the first washing liquid merge at the confluence section 250X. Liquid A and the first washing liquid, which merge at the confluence section 250X, travel through the piping 162 and are mixed by the inline mixer 230. At this time, for example, the complex is washed by the first washing liquid, which removes components (e.g., proteins derived from the culture medium) that are nonspecifically attached to the carrier, complex, etc., and they become suspended in the liquid. The mixture of liquid A and the first washing liquid (hereinafter also referred to as "mixture A") passes through the inline mixer 230 and is sent from the piping 261 to the hydrocyclone 240. Mixture A is separated into liquid C and liquid D by the hydrocyclone 240. Liquid D is, for example, wastewater. In this embodiment, liquid D enters the piping 263 from the hydrocyclone 240 and is sent to the wastewater line. Liquid C includes, for example, a carrier comprising a complex and a carrier comprising an unreacted ligand.

[0044] In this embodiment, the C liquid enters the pipe 262 from the hydrocyclone 240 and is sent to the elution section 30. The C liquid passes through the pipe 262 and reaches the confluence site 350X. On the other hand, the eluate is supplied from the eluate supply section 310. The eluate passes through the pipe 350 and reaches the confluence site 350X. In this embodiment, at the confluence site 350X, the C liquid and the eluate merge. The C liquid and the eluate merged at the confluence site 350X pass through the pipe 262 and are mixed by the in-line mixer 330. At this time, for example, the protein is eluted into the liquid by the eluate. Then, the mixed liquid of the C liquid and the eluate (hereinafter also referred to as "mixed liquid B") passes through the in-line mixer 330 and is sent from the pipe 361 to the hydrocyclone 340. The mixed liquid B is separated by the hydrocyclone 340 into an E liquid and an F liquid. The E liquid may contain the eluted protein in this embodiment. The E liquid enters the pipe 363 from the hydrocyclone 340 and is sent to the protein recovery process. The F liquid may contain the carrier after the protein is eluted in this embodiment.

[0045] In this embodiment, the F liquid enters the pipe 362 from the hydrocyclone 340 and is sent to the second washing section 40. The F liquid passes through the pipe 362 and reaches the confluence site 450X. On the other hand, the second washing liquid is supplied from the second washing liquid supply section 410. The second washing liquid passes through the pipe 450 and reaches the confluence site 450X. In this embodiment, at the confluence site 450X, the F liquid and the second washing liquid merge. The F liquid and the second washing liquid merged at the confluence site 450X pass through the pipe 362 and are mixed by the in-line mixer 430. At this time, for example, the carrier is washed by the second washing liquid. Then, the mixed liquid of the F liquid and the second washing liquid (hereinafter also referred to as "mixed liquid C") passes through the in-line mixer 430 and is sent to the hydrocyclone 440. The mixed liquid C is separated into a G liquid and an H liquid. The G liquid is the drainage liquid in this embodiment. The G liquid enters the pipe 463 from the hydrocyclone 440 and is sent to the drainage line. The H liquid may contain the carrier in this embodiment.

[0046] In this embodiment, liquid H enters the piping 462 from the hydrocyclone 440 and is sent to the equilibration unit 50. Liquid H passes through the piping 462 to the confluence point 550X. Meanwhile, equilibration liquid is supplied from the equilibration liquid supply unit 510. The equilibration liquid passes through the piping 550 to the confluence point 550X. In this embodiment, liquid H and equilibration liquid merge at the confluence point 550X. The liquid H and equilibration liquid that merge at the confluence point 550X pass through the piping 462 and are mixed by the inline mixer 530. At this time, for example, the ligand (ligand provided on the carrier) is equilibrated by the equilibration liquid. The mixture of liquid H and equilibration liquid (hereinafter also referred to as "mixture D") passes through the inline mixer 530 and is sent to the hydrocyclone 440. Mixture D is separated into liquid I and liquid J. Liquid I is the wastewater in this embodiment. Liquid I enters the piping 563 from the hydrocyclone 540 and is sent to the wastewater line. Liquid J may contain a carrier in this embodiment. Liquid J enters the piping 562 from the hydrocyclone 540 and is sent to the first supply unit 110.

[0047] The operation of apparatus 1 has been described above with reference to Figures 1 and 2. The configuration of apparatus 1 is not limited to that shown in Figures 1 and 2. For example, the number of inline mixers, hydrocyclones, pumps, etc. in each functional block are not limited. Such numbers can be set appropriately according to the scale of the apparatus, the amount of raw material that can be processed in a single operation of the apparatus, etc.

[0048] As described above, the apparatus 1 is an apparatus for purifying proteins from a cell culture solution. The apparatus 1 includes a first supply unit 110, a second supply unit 120, an in-line mixer 130, a hydrocyclone 140, and a dilution line 10A. The first supply unit 110 supplies a first slurry. The first slurry contains a carrier having a ligand that binds to a protein. The second supply unit 120 supplies a second slurry. The second slurry is a cell culture solution containing a protein. The in-line mixer 130 mixes the first slurry supplied from the first supply unit 110 and the second slurry supplied from the second supply unit 120. The hydrocyclone 140 is connected to the in-line mixer 130 downstream of the in-line mixer 130. The hydrocyclone 140 separates a complex of a protein and a carrier from a mixed slurry of the first slurry and the second slurry (here, slurry X containing a diluent and the mixed slurry) obtained by the in-line mixer 130. The dilution line 10A introduces a diluent into the mixed slurry between the in-line mixer 130 and the hydrocyclone 140.

[0049] By using the apparatus 1 including the first supply unit 110, the second supply unit 120, the in-line mixer 130, and the hydrocyclone 140, for example, the target protein can be separated and purified from a cell culture solution without using an affinity column or the like. Therefore, the apparatus itself can be made into a simpler and continuous configuration, and the time for purifying the protein can be shortened.

[0050] Apparatus 1 is equipped with a dilution line 10A between the inline mixer 130 and the hydrocyclone 140. This allows the mixed slurry introduced into the hydrocyclone 140 to be diluted. In other words, by providing the dilution line 10A, the volume proportion of the carrier (here, including the complex and the carrier not used in the formation of the complex) in the mixed slurry can be reduced. This increases the separation efficiency of the complex in the hydrocyclone 140. Furthermore, because the volume proportion of the carrier in the mixed slurry is reduced when introduced into the hydrocyclone 140, the occurrence of carrier clogging in the hydrocyclone 140 can be suppressed. In addition, in Apparatus 1, since the dilution line 10A is located downstream of the inline mixer 130, for example, it is not necessary to reduce the volume proportion of the carrier in the first slurry introduced into the inline mixer 130. That is, the volume proportion of the carrier in the first slurry can be increased, thereby increasing the adsorption efficiency of proteins from the second slurry in the inline mixer 130. This allows for a reduction in the time required for protein purification and enables the miniaturization of the apparatus 1.

[0051] The apparatus 1 may further include a backflow prevention unit 10B. The backflow prevention unit 10B may be provided on the dilution line 10A. The backflow prevention unit 10B may prevent the mixed slurry from entering the dilution line 10A from the point where the diluent and the mixed slurry merge (here, the merging point 161X). By including the backflow prevention unit 10B in the apparatus 1, for example, it is possible to prevent the mixed slurry from entering the dilution line 10A and causing a delay in the supply of the diluent by the dilution line 10A, and consequently, to prevent delays in protein purification by the apparatus 1.

[0052] While embodiments of the technology disclosed herein have been described above, the technology disclosed herein is not intended to be limited to the embodiments described herein. The technology disclosed herein may include modifications insofar as they can achieve the effects of the technology disclosed herein.

[0053] For example, in the above-described embodiment, the apparatus 1 was provided with a backflow prevention unit 10B on the dilution line 10A. However, the location where the backflow prevention unit 10B is provided is not limited to this, as long as the effects obtained by providing the backflow prevention unit 10B are realized. In other embodiments, the backflow prevention unit 10B may be provided, for example, between the inline mixer 131 and the point where the diluent and the mixed slurry merge (here, the merging point 161X).

[0054] Alternatively, from the viewpoint of further simplifying the configuration of device 1, device 1 does not necessarily have to include a backflow prevention unit 10B. Even if device 1 does not include a backflow prevention unit 10B, the effects of the technology disclosed herein will be realized.

[0055] Alternatively, the apparatus 1 may be equipped with another inline mixer (hereinafter simply referred to as "the other inline mixer") different from the inline mixer 130, either in place of the backflow prevention unit 10B or together with the backflow prevention unit 10B, on the hydrocyclone 140 side of the point where the diluent and the mixed slurry merge (here, the merging point 161X). The other inline mixer, in this case, is an inline mixer that mixes the diluent and the mixed slurry. By equipping the apparatus 1 with the other inline mixer, for example, the diluent and the mixed slurry can be mixed more effectively, and the dispersibility of the carrier in the slurry X can be improved. Therefore, the separation efficiency of the complex in the hydrocyclone 140 can be further improved. In addition to this, by mixing the diluent and the mixed slurry, components that are nonspecifically attached to the carrier, complex, etc. (for example, proteins derived from the culture medium) can be removed. Therefore, by equipping the apparatus 1 with the other inline mixer, a washing effect between the carrier and the complex can also be achieved in the adsorption unit 10. Furthermore, if the device 1 is equipped with both a backflow prevention unit 10B and other inline mixers, it is preferable to install the other inline mixers on the hydrocyclone 140 side.

[0056] Alternatively, the first washing section 20 in the apparatus 1 may be omitted. In the apparatus 1, the mixed slurry is diluted by the provision of a dilution line 10A. This makes it possible to remove components (for example, proteins derived from the culture medium) that are nonspecifically attached to the carrier, complex, etc. Therefore, even if the apparatus 1 does not have the first washing section 20, washing of the carrier and complex can be appropriately achieved. In this configuration, the hydrocyclone 140 may be directly connected to the elution section 30. This makes the configuration of the apparatus 1 simpler, and thus the apparatus 1 can be made smaller. If the apparatus 1 does not have the first washing section 20, it is preferable that the apparatus 1 be provided with another in-line mixer from the viewpoint of better washing of the carrier and complex.

[0057] The following describes some test examples conducted by the inventor with respect to the technology disclosed herein, but it is not intended to limit the technology disclosed herein to these test examples.

[0058] <Test 1> In Test 1, human IgG antibody and silica beads (manufactured by Osaka Soda Co., Ltd.) were mixed using a stirring blade. The antibody used in this test was prepared as an antibody sample solution with a concentration of 20 mg / mL. The silica beads used in this test were silica beads with protein A immobilized as a ligand, and had an average particle size of 50 μm.

[0059] In Experiment 1, five types of slurries were prepared, each containing 50 ml of a carrier at volume percentages of 5%, 10%, 20%, 25%, and 50%, respectively, relative to 100% by volume of the entire first slurry. Five types of slurries were also prepared, each containing 5 mg of antibody per 1 ml of carrier in each first slurry, each in 50 ml quantities. Next, each 50 ml of the first slurry and each 50 ml of the second slurry were stirred and mixed to prepare five types of mixed slurries. Each of the five mixed slurries contained 2.5%, 5%, 10%, 12.5%, or 25% by volume of carrier relative to 100% by volume of the entire mixed slurry. All five mixed slurries contained 5 mg of antibody per 1 ml of carrier. A stirring blade (Tornado SMT-101 from AS ONE Corporation) was used to agitate the first slurry and the second slurry. The rotation speed of the stirring blade was 200 rpm.

[0060] Next, the adsorption rate of antibodies to silica beads after a predetermined stirring time (hereinafter also simply referred to as "adsorption rate") was measured. A portion of the mixed slurry was taken at the start of stirring, and after 6 seconds, 13 seconds, 19 seconds, 26 seconds, 32 seconds, 60 seconds, and 120 seconds from the start of stirring. The antibodies adsorbed to the silica beads were eluted with acetate buffer, and the amount of antibody adsorbed to the carrier was quantified by measuring the absorbance of the eluate at a wavelength of 280 nm. The results are shown in Table 1.

[0061] Table 1 shows the adsorption rate (%) after various stirring times for each volume percentage of the carrier in a 100 ml mixed slurry. Based on the obtained data, the results were plotted with the adsorption rate (%) on the vertical axis and time (seconds) on the horizontal axis for each volume percentage of the carrier, and an approximation formula for the resulting curve was obtained. Then, using this approximation formula, the time (seconds) at which the adsorption rate (%) reached 70% was calculated for each volume percentage of the carrier. The results are shown in the corresponding column of Table 1.

[0062]

[0063] The results shown in Table 1 indicate that increasing the volume ratio of the carrier in the mixed slurry shortens the stirring time required to achieve an antibody adsorption rate of 70%. Furthermore, the inventors' studies have shown that the volume ratio of the carrier in the mixed slurry tested in Test 1 does not cause clogging of the carrier in the hydrocyclone. Thus, it is preferable to set the volume ratio of the carrier in the first slurry considering the adsorption efficiency between the protein and the carrier in the inline mixer, the risk of carrier clogging in the hydrocyclone, and other factors.

[0064] <Test 2> Figures 3 and 4 are schematic diagrams of the apparatus used in Test 2. Figure 3 shows the state in which a slurry containing a carrier is circulated in cyclone C before sample collection. Figure 4 shows the state in which samples are being collected from the lower outlet C2 and upper outlet C3 of cyclone C. In the state shown in Figure 3, the apparatus used in Test 2 (hereinafter also simply referred to as "apparatus") comprises a storage tank A, a pump B, and a hydrocyclone C. Storage tank A, pump B, and hydrocyclone C are connected by piping F. Pump B is installed between storage tank A and hydrocyclone C. Piping G is connected to the lower outlet C2 of hydrocyclone C. Piping H is connected to the upper outlet C3 of hydrocyclone C. The ends of piping G and piping H are inside storage tank A. In the configuration shown in Figure 4, the apparatus includes, in addition to the components described above, a first sampling tank D and a second sampling tank E. Here, the tip of pipe G is inside the first sampling tank D. The tip of pipe H is inside the second sampling tank E.

[0065] [Group 1] A slurry containing a carrier was stored in storage tank A. The volume percentage of the carrier in the slurry was 2.5% by volume when the total volume of the slurry was considered to be 100% by volume. Silica beads used in Test 1 were used as the carrier. First, with the apparatus in the state shown in Figure 3, the slurry containing the carrier was introduced from storage tank A to hydrocyclone C through piping F and inlet C1. The slurry containing the carrier was set to flow through piping F at a flow rate of 2 L / min by the operation of pump B. In hydrocyclone C, the slurry containing the carrier separated into a first liquid and a second liquid. The first liquid was discharged from the lower outlet C2 and entered storage tank A. The second liquid was discharged from the upper outlet C3 and entered storage tank A. The state shown in Figure 3 was maintained for a predetermined period of time to ensure that the slurry containing the carrier was stably introduced into hydrocyclone C and separated into a first liquid and a second liquid. Furthermore, storage tank A was kept agitated to ensure a uniform concentration of the slurry containing the carrier. Next, with the apparatus in the state shown in Figure 4, the tip of pipe G was inserted into the first sampling tank D to collect the first liquid, and the tip of pipe H was inserted into the second sampling tank E to collect the second liquid. The sampling time for each liquid was approximately 5 seconds.

[0066] The first liquid collected in the first sampling tank D and the second liquid collected in the second sampling tank E were dried, and the weight D (g) of the carrier collected in the first sampling tank D and the weight E (g) of the carrier collected in the second sampling tank E were measured. The separation rate (%) of the carrier from the lower outlet C2 of the hydrocyclone C was obtained using the following formula (P): Separation rate (%) = [(weight D) / (weight D + weight E)] (P) The results are shown in Table 2. In Group 1, the number of samples was 1 (n=1).

[0067] [Group 2] The volume percentage of the carrier in the slurry containing the carrier was set to 5% by volume when the total volume of the slurry is set to 100% by volume. In Group 2, the number of samples was 1 (n=1). Otherwise, the same apparatus and procedure as in Group 1 were used to obtain the separation rate (%) of the carrier from the lower outlet C2 of the hydrocyclone C. The results are shown in Table 2.

[0068] [Group 3] The volume percentage of the carrier in the slurry containing the carrier was set to 10% by volume when the total volume of the slurry is set to 100% by volume. In Group 3, the number of samples was 4 (n=4). Otherwise, the same apparatus and procedure as in Group 1 were used to obtain the separation rate (%) of the carrier from the lower outlet C2 of the hydrocyclone C. The results are shown in Table 2. Note that the separation rates shown in Table 2 are the average values ​​for the number of samples in Group 3.

[0069] [Group 4] The volume percentage of the carrier in the slurry containing the carrier was set to 12.5% ​​by volume when the entire slurry is considered to be 100% by volume. In Group 4, the number of samples was 1 (n=1). Otherwise, the same apparatus and procedure as in Group 1 were used to obtain the separation rate (%) of the carrier from the lower outlet C2 of the hydrocyclone C. The results are shown in Table 2.

[0070] [Group 5] The volume percentage of the carrier in the slurry containing the carrier was set to 15% by volume when the total volume of the slurry is set to 100% by volume. In Group 5, the number of samples was 2 (n=2). Otherwise, the same apparatus and procedure as in Group 1 were used to obtain the separation rate (%) of the carrier from the lower outlet C2 of the hydrocyclone C. The results are shown in Table 2. Note that the separation rates shown in Table 2 are the average values ​​for the number of samples in Group 5.

[0071] [Group 6] The volume percentage of the carrier in the slurry containing the carrier was set to 25% by volume when the total volume of the slurry is set to 100% by volume. In Group 6, the number of samples was 1 (n=1). Otherwise, the same apparatus and procedure as in Group 1 were used to obtain the separation rate (%) of the carrier from the lower outlet C2 of the hydrocyclone C. The results are shown in Table 2.

[0072]

[0073] In Test 2, the groups with a separation rate of 90% or higher, as shown in Table 2, were evaluated as having good separation efficiency. In Test 2, a higher separation rate is considered to indicate better carrier separation efficiency in Hydrocyclone C. From the results shown in Table 2, it was found that when the entire slurry containing the carrier was considered as 100% by volume, the carrier volume percentage was at least 2.5% to less than 25% by volume (slurries used in Groups 1 to 5) and was introduced into Hydrocyclone C, the carrier separation efficiency was good.

[0074] The technologies disclosed herein may include the inventions described in the following sections: Section 1: A protein purification apparatus for purifying a protein from a cell culture medium, comprising: a first supply unit for supplying a first slurry containing a carrier equipped with a ligand that binds to the protein; a second supply unit for supplying a second slurry which is a cell culture medium containing the protein; an in-line mixer for mixing the first slurry supplied from the first supply unit and the second slurry supplied from the second supply unit; a hydrocyclone connected to the in-line mixer downstream of the in-line mixer for separating the complex of the protein and the carrier from the mixed slurry of the first slurry and the second slurry obtained by the in-line mixer; and a dilution line between the in-line mixer and the hydrocyclone for introducing a diluent into the mixed slurry. Section 2: The protein purification apparatus according to Section 1, further comprising a backflow prevention unit on the dilution line to prevent the mixed slurry from entering the dilution line from a point where the diluent and the mixed slurry merge. Item 3: The protein purification apparatus according to item 1 or 2, further comprising another inline mixer, different from the inline mixer, for mixing the diluent and the mixed slurry, located on the hydrocyclone side of the point where the diluent and the mixed slurry merge. Item 4: The protein purification apparatus according to any one of items 1 to 3, further comprising an elution unit for eluting the protein from the complex that has passed through the hydrocyclone, wherein the hydrocyclone is directly connected to the elution unit.

[0075] 1 Apparatus 10 Adsorption section 110 First supply section 120 Second supply section 130 In-line mixer 140 Hydrocyclone 161X Confluence section 170 First pump 180 Second pump 10A Dilution line 10B Backflow prevention section 20 First washing section 30 Elution section 40 Second washing section 50 Equilibrium section

Claims

1. A protein purification apparatus comprising: a first supply unit for supplying a first slurry containing a carrier equipped with a ligand that binds to the protein; a second supply unit for supplying a second slurry which is a cell culture medium containing the protein; an inline mixer for mixing the first slurry supplied from the first supply unit and the second slurry supplied from the second supply unit; a hydrocyclone connected to the inline mixer downstream of the inline mixer for separating the complex of the protein and the carrier from the mixed slurry of the first slurry and the second slurry obtained by the inline mixer; and a dilution line between the inline mixer and the hydrocyclone for introducing a diluent into the mixed slurry.

2. The protein purification apparatus according to claim 1, further comprising a backflow prevention unit on the dilution line that prevents the mixed slurry from entering the dilution line from the point where the diluent and the mixed slurry merge.

3. The protein purification apparatus according to claim 1, further comprising another inline mixer, different from the inline mixer, located on the hydrocyclone side of the point where the diluent and the mixed slurry merge, for mixing the diluent and the mixed slurry.

4. The protein purification apparatus according to any one of claims 1 to 3, further comprising an elution unit for eluting the protein from the complex that has passed through the hydrocyclone, wherein the hydrocyclone is directly connected to the elution unit.

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