Protein adsorption method, protein purification method, and protein purification apparatus

Abstract

Provided is a technique for improving the efficiency of adsorption of a protein onto a carrier in an apparatus equipped with an in-line mixer and a hydrocyclone. An adsorption method disclosed herein is performed using an apparatus equipped with an in-line mixer and a hydrocyclone connected to the in-line mixer. The adsorption method is a method in which a protein is adsorbed onto a carrier provided with a ligand capable of binding to the protein. The adsorption method includes: mixing a first slurry containing a carrier with a second slurry containing a protein using an in-line mixer to prepare a mixed slurry; and adsorbing the protein onto the carrier in the mixed slurry to produce a complex between the protein and the carrier. The mixed slurry is prepared so as to contain the carrier in an amount of 10-25 vol% and the protein in an amount of 5-20 mg per 1 ml of the carrier when the whole amount of the mixed slurry is 100 vol%. The mixing time in the inline mixer is 0.6-60 seconds.

Description

Protein adsorption method, protein purification method, and protein purification device

[0001] The present disclosure relates to a protein adsorption method, a protein purification method, and a protein purification device. This application claims priority to Japanese Patent Application No. 2024-035889, filed on March 8, 2024, the entire contents of which are incorporated herein by reference.

[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] International Publication No. 2019 / 189064

[0004] As described in Patent Document 1, there is an apparatus including an in-line mixer and a hydrocyclone connected downstream to the in-line mixer. For example, Patent Document 1 describes that such an apparatus is used to perform the following steps: mixing a cell culture solution containing a specific protein with a slurry containing a resin to bind the protein in the cell culture solution to the resin in the slurry to form a complex; and separating the complex from the mixed slurry continuously delivered from the in-line mixer in the hydrocyclone. The present inventors hope to improve the adsorption efficiency of a protein and a carrier when a cell-derived protein is adsorbed onto a carrier equipped with a ligand that binds to the protein, using an in-line mixer included in an apparatus having the above-described configuration.

[0005] The adsorption method disclosed herein is carried out using an apparatus including an in-line mixer and a hydrocyclone connected to the in-line mixer downstream. The adsorption method involves adsorbing a cell-derived protein onto a carrier equipped with a ligand that binds to the protein. The adsorption method involves mixing a first slurry containing a carrier and a second slurry containing a protein in an in-line mixer, and adsorbing the protein onto the carrier in the mixed slurry of the first and second slurries to produce a protein-carrier complex. The mixed slurry is prepared to contain 10% to 25% by volume of the carrier, with the total mixed slurry being 100% by volume, and 5 mg to 20 mg of protein per ml of carrier. The mixing time using the in-line mixer is 0.6 to 60 seconds. This configuration allows the in-line mixer included in the apparatus having the above configuration to increase the adsorption efficiency of the protein and the carrier when adsorbing the cell-derived protein onto a carrier equipped with a ligand that binds to the protein.

[0006] The technology disclosed herein provides a method for purifying a protein from a cell culture solution containing the protein. This purification method includes preparing a complex using the above-described adsorption method and separating, in a hydrocyclone, the complex from a mixed slurry continuously delivered from an in-line mixer to the hydrocyclone. This configuration enables the in-line mixer included in the apparatus having the above-described configuration to increase the adsorption efficiency of the protein and the carrier when the cell-derived protein is adsorbed onto a carrier equipped with a ligand that binds to the protein.

[0007] According to the technology disclosed herein, an apparatus for purifying a protein from a cell culture solution is disclosed. The apparatus includes a first supply unit, a second supply unit, an in-line mixer, and a hydrocyclone. The first supply unit supplies a first slurry. The first slurry contains a carrier having a ligand that binds to the protein. The second supply unit supplies a second slurry. The second slurry is a cell culture solution containing the protein. The in-line mixer mixes the first slurry supplied from the first supply unit with the second slurry supplied from the second supply unit. The hydrocyclone is connected to the in-line mixer downstream of the in-line mixer. The hydrocyclone separates the protein-carrier complex from the mixed slurry of the first and second slurries obtained by the in-line mixer. The mixed slurry is prepared to contain 10% to 25% by volume of the carrier, with the total mixed slurry being 100% by volume, and 5 mg to 20 mg of protein per 1 ml of carrier. The length of the in-line mixer is set so that the first slurry and the second slurry can be mixed for a time of 0.6 seconds to 60 seconds. With this configuration, in the in-line mixer included in the apparatus having the above configuration, when a protein derived from a cell is adsorbed onto a carrier having a ligand that binds to the protein, the adsorption efficiency of the protein to the carrier can be increased.

[0008] Fig. 1 is a schematic diagram of the device 1. Fig. 2 is an enlarged schematic diagram of the suction unit 10.

[0009] 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.

[0010] <Protein Purification Apparatus> The technology disclosed herein provides a protein purification apparatus. The protein purification apparatus is, for example, an apparatus for purifying proteins from cell culture media. 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 multiple 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. The cells may be genetically modified cells so that they can 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 the protein to be purified is produced and secreted.

[0011] 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.

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

[0013] The adsorption unit 10 is a functional block that adsorbs, for example, a protein to be purified in a cell culture solution onto a carrier. Fig. 2 is an enlarged schematic diagram of the adsorption unit 10. Fig. 2 shows an enlarged view of the adsorption unit 10 of the apparatus 1 shown in Fig. 1. As shown in Figs. 1 and 2, the adsorption unit 10 includes a first supply unit 110, a second supply unit 120, an in-line mixer 130, a hydrocyclone 140, a first pump 170, and a second pump 180. Note that the adsorption unit 10 may further include pumps, valves, pressure gauges, flow meters, etc. (not shown) as needed.

[0014] The first supply unit 110 is, for example, a portion that supplies the first slurry. In this embodiment, the first supply unit 110 is a storage tank (tank) that stores the first slurry. As shown in FIG. 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 transfer line 151, and is connected to the first liquid transfer line 151. In this embodiment, a first pump 170 that promotes liquid transfer from the first supply unit 110 is provided upstream of the confluence portion 152X on the first liquid transfer line 151.

[0015] The first slurry contains a carrier having a ligand that binds to the protein. The protein may be the protein to be purified as described above. The carrier having the ligand may be, for example, dispersible in a liquid. The carrier having the ligand is preferably one in which the ligand and the carrier are not easily separated when the apparatus 1 is used, and more preferably one in which they do not separate. Any carrier used for this type of application may 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 polymer compound or a synthetic polymer compound.

[0016] 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.

[0017] 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.

[0018] The second supply unit 120 is, for example, a portion that supplies the second slurry. In this embodiment, the second supply unit 120 is a storage tank (tank) that stores the second slurry. As shown in FIG. 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 transfer line 152 and is connected to the second liquid transfer line 152. In this embodiment, a second pump 180 that promotes liquid transfer from the second supply unit 120 is provided upstream of the confluence portion 152X on the second liquid transfer line 152. Here, the second slurry is a cell culture solution containing a protein. The protein and the cell culture solution are as described above.

[0019] The in-line mixer 130 is a mixer that mixes, for example, a first slurry supplied from the first supply unit 110 and a second slurry supplied from the second supply unit 120. As shown in FIG. 2 , the in-line mixer 130 is provided downstream of the first supply unit 110 and the second supply unit 120. The in-line mixer 130 is provided in the second liquid feed line 152, downstream of a junction 152X with the first liquid feed line 151. Here, the in-line mixer 130 has a conduit 131, an inlet 132, and an outlet 133. The conduit 131 is tubular (here, cylindrical), is the main body of the in-line mixer 130, and is a flow path for the mixed slurry of the first slurry and the second slurry. The inlet 132 is, for example, a portion where the first slurry and the second slurry that have joined at the joining portion 152X flow into the pipeline 131. In the embodiment shown in FIG. 2 , the inlet 132 is provided at one end of the pipeline 131. The inlet 132 is connected to the second liquid feed line 152. The outlet 133 is, for example, a portion where the mixed slurry is discharged from the in-line mixer 130. In the embodiment shown in FIG. 2 , the outlet 133 is provided at the other end of the pipeline 131. Here, the outlet 133 is connected to a pipe 161.

[0020] In this specification, the term "in-line mixer" refers to a static mixer that does not have a driving part and mixes at least two types of liquids by using energy generated by the flow rate of the liquids flowing inside the device.

[0021] The mixed slurry contains, for example, a carrier and a protein. In this embodiment, the mixed slurry is prepared to contain 10 to 25% by volume of the carrier, with the total volume of the mixed slurry being 100% by volume. In this embodiment, the mixed slurry is prepared to contain 5 to 20 mg of protein per 1 ml of the carrier, with the total volume of the mixed slurry being 100% by volume.

[0022] In this embodiment, the length of the in-line mixer 130 is set so that the first slurry and the second slurry can be mixed for 0.6 to 60 seconds. From the viewpoint of increasing the adsorption of the protein to the carrier, the length of the in-line mixer 130 is set so that the first slurry and the second slurry can be mixed for, 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 reducing the space required for the apparatus 1 and shortening the protein purification time using the apparatus 1, the length of the in-line mixer 130 is set so that the first slurry and the second slurry can be mixed for preferably 55 seconds or less, more preferably 50 seconds or less. The length of the in-line mixer 130 is set so that the first slurry and the second slurry can be mixed for preferably 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 in-line mixer 130 so that the above-mentioned mixing time of the first slurry and the second slurry can be achieved, 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, the "length of the in-line mixer 130" refers to the length of the pipe 131 of the in-line mixer 130. Although one in-line mixer 130 is shown in the embodiment shown in Fig. 2, the apparatus 1 may include a plurality of in-line mixers 130. In this case, the "length of the in-line mixer 130" refers to the total length of the pipe 131 of each in-line mixer 130.

[0024] The hydrocyclone 140 is a separator that separates the protein-carrier complex from, for example, the mixed slurry of the first slurry and the second slurry obtained by the in-line mixer 130. In this embodiment, by passing through the hydrocyclone 140, the mixed slurry is separated into liquid A and liquid B. Liquid A contains, for example, the complex. Liquid A may also contain, for example, the carrier that has not formed a complex with the protein. Liquid B is, for example, the remainder of the mixed slurry after the complex and the carrier that has not formed a complex with the protein have been removed. In this embodiment, in the configuration shown in FIG. 2, the hydrocyclone 140 is connected to the in-line mixer 130 downstream of the in-line mixer 130. As shown in FIG. 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, a portion through which liquid A is discharged from the hydrocyclone 140. In the embodiment shown in FIG. 2 , the first outlet 141 is connected to a pipe 162. In this embodiment, the first outlet 141 is connected to the first cleaning unit 20 via the pipe 162. The second outlet 142 is a portion through which liquid B is discharged from the hydrocyclone 140. The second outlet 142 is connected, for example, to a drain line outside the apparatus 1 or to another liquid supply line. The inlet 143 is a portion through which liquid sent from upstream is introduced into the hydrocyclone 140. In this embodiment, the inlet 143 is connected to the pipe 161. In the embodiment shown in FIG. 2 , an in-line mixer 130 is disposed upstream of the hydrocyclone 140. The pipe 161 may directly or indirectly connect the in-line mixer 130 and the hydrocyclone 140. In this specification, the term "hydrocyclone" refers to a separator that does not have a driving part and that can separate, classify, concentrate, etc., liquid introduced into it while it is stationary.

[0025] 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 embodiment shown in Fig. 2, the first pump 170 is provided on the first liquid supply line 151. Here, the first pump 170 is provided on the first liquid supply line 151 closer to the first supply unit 110 than a junction 152X where the first liquid supply line 151 joins with the second liquid supply line 152. The first pump 170 is provided between the connection port 111 and the junction 152X.

[0026] 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 embodiment shown in Fig. 2, the second pump 180 is provided on the second liquid feed line 152. Here, the second pump 180 is provided on the second liquid feed line 152, closer to the second supply unit 120 than the junction 152X where the second liquid feed line 152 and the first liquid feed line 151 join. The second pump 180 is provided between the connection port 121 and the junction 152X.

[0027] The first washing unit 20 is a functional block connected to, for example, the adsorption unit 10 and washes the complex using a first washing liquid. As shown in FIG. 1 , the first washing unit 20 includes a first washing liquid supply unit 210, a pump 220, an in-line mixer 230, and a hydrocyclone 240. In the embodiment shown in FIG. 1 , the first washing unit 20 includes, from the adsorption unit 10 side, the in-line mixer 230 and the hydrocyclone 240. Here, the adsorption unit 10 and the in-line mixer 230 are connected to each other by a pipe 162. The in-line mixer 230 and the hydrocyclone 240 are connected to each other by a pipe 261. Pipes 262 and 263 extend from the hydrocyclone 240. The pipe 262 is connected to the elution unit 30. The pipe 263 is connected to, for example, a drain line (not shown).

[0028] 1 , a pipe 250 extends from the first cleaning liquid supply unit 210. Here, the pipe 250 merges with the pipe 162 at a junction 250X. In this embodiment, the pump 220 is provided on the pipe 250. As the first cleaning liquid, for example, the buffer solution used for the first slurry can be preferably used. Note that the first cleaning unit 20 may further include a pump, a valve, a pressure gauge, a flow meter, and the like (not shown) as needed.

[0029] The elution unit 30 is a functional block connected to, for example, the first washing unit 20 and uses an eluent to elute proteins from the complex that has passed through the first washing unit 20. As shown in FIG. 1 , the elution unit 30 includes an eluent supply unit 310, a pump 320, an in-line mixer 330, and a hydrocyclone 340. In the embodiment shown in FIG. 1 , the elution unit 30 includes, from the first washing unit 20 side, the in-line mixer 330 and the hydrocyclone 340. Here, the first washing unit 20 and the in-line mixer 330 are connected to each other by a pipe 262. The in-line mixer 330 and the hydrocyclone 340 are connected to each other by a pipe 361. Pipes 362 and 363 extend from the hydrocyclone 340. Pipe 362 is connected to the second washing unit 40. Pipe 363 is connected to, for example, a recovery line (not shown).

[0030] In the embodiment shown in FIG. 1 , a pipe 350 extends from the eluate supply unit 310. Here, the pipe 350 merges with the pipe 262 at a junction 350X. In this embodiment, the pump 320 is provided on the pipe 350. The eluate is preferably an eluate having a pH of 5 or less, more preferably a buffer solution having a pH of 2 to 5, and even more preferably an eluate having a pH of 2.5 to 3.5. Examples of the eluate include acetate buffer, citrate buffer, phosphate buffer, and a dilute solution of phosphoric acid or hydrochloric acid. If necessary, the buffer may contain 0.1 M to 1 M (preferably 0.1 M to 0.5 M, more preferably 0.1 M to 0.3 M) sodium chloride, potassium chloride, or the like. The elution unit 30 may further include pumps, valves, pressure gauges, flow meters, and the like (not shown) as needed.

[0031] 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 with a second washing solution. As shown in FIG. 1 , the second washing section 40 includes a second washing solution supply section 410, a pump 420, an in-line mixer 430, and a hydrocyclone 440. In the embodiment shown in FIG. 1 , the second washing section 40 includes, from the elution section 30 side, the in-line mixer 430 and the hydrocyclone 440. Here, the second washing section 40 and the in-line mixer 430 are connected to each other by a pipe 362. The in-line mixer 430 and the hydrocyclone 440 are connected to each other by a pipe 461. Pipes 462 and 463 extend from the hydrocyclone 440. The pipe 462 is connected to the equilibration section 50. The pipe 463 is connected to, for example, a drain line (not shown).

[0032] 1, a pipe 450 extends from the second cleaning liquid supply unit 410. Here, the pipe 450 merges with the pipe 362 at a junction 450X. In this embodiment, the pump 420 is provided on the pipe 450. For example, the buffer solution used for the first slurry can be preferably used as the second cleaning liquid. Note that the second cleaning unit 40 may further include a pump, a valve, a pressure gauge, a flow meter, and the like (not shown) as needed.

[0033] The equilibration unit 50 is a functional block connected to, for example, the second washing unit 40 and uses an equilibration solution to equilibrate the ligand (ligand attached to the carrier) after washing in the second washing unit 40. As shown in FIG. 1 , the equilibration unit 50 includes an equilibration solution supply unit 510, a pump 520, an in-line mixer 530, and a hydrocyclone 540. In the embodiment shown in FIG. 1 , the equilibration unit 50 includes, from the second washing unit 40 side, the in-line mixer 530 and the hydrocyclone 540. Here, the equilibration unit 50 and the in-line mixer 530 are connected to each other by a pipe 462. The in-line mixer 530 and the hydrocyclone 540 are connected to each other by a pipe 561. Pipes 562 and 563 extend from the hydrocyclone 540. In this embodiment, the pipe 562 is connected to the first supply unit 110 (not shown). The pipe 563 is connected to, for example, a drain line (not shown).

[0034] In the embodiment shown in FIG. 1 , a pipe 550 extends from the equilibration liquid supply unit 510. Here, the pipe 550 merges with the pipe 462 at a junction 550X. In this embodiment, the pump 520 is provided on the pipe 550. For example, the buffer solution used in the first slurry can be preferably used as the equilibration liquid. Note that the equilibration unit 50 may further include a pump, a valve, a pressure gauge, a flow meter, and the like (not shown) as needed.

[0035] The apparatus 1 is configured to operate, for example, as follows. First, when the switch of the apparatus 1 is turned on, a first slurry is supplied from the first supply unit 110. The first slurry enters the first liquid feed line 151 through the connection port 111 and flows downstream, where its flow rate is adjusted by the operation of the first pump 170. The first slurry, flowing through the first liquid feed line 151 while being operated by the first pump 170, reaches the confluence 152X. Meanwhile, a second slurry is supplied from the second supply unit 120. The second slurry enters the second liquid feed line 152 through the connection port 121 and flows downstream, where its flow rate is adjusted by the operation of the second pump 180. The second slurry, flowing through the second liquid feed line 152 while being operated by the second pump 180, reaches the confluence 152X.

[0036] The first slurry and the second slurry join at the joining portion 152X and continue to flow downstream. In the embodiment shown in FIG. 2 , the first slurry and the second slurry join at the joining portion 152X and flow toward the in-line mixer 130. The first slurry and the second slurry flow through the inlet 132 into the conduit 131 of the in-line mixer 130. The first slurry and the second slurry flow downstream while being mixed in the conduit 131. At this time, the mixed slurry of the first slurry and the second slurry achieves a carrier concentration within the above-mentioned range and a protein concentration within the above-mentioned range. The mixing time of the first slurry and the second slurry is set so as to be within the above-mentioned range.

[0037] The mixed slurry then flows out of the in-line mixer 130 through outlet 133 and into pipe 161. The mixed slurry then flows into the hydrocyclone 140 provided downstream of pipe 161. In this embodiment, the mixed slurry flows from pipe 161 into the hydrocyclone 140 through inlet 143. The mixed slurry is then separated into liquid A and liquid B by the hydrocyclone 140. Liquid B enters pipe 163 through, for example, second outlet 142 and is sent to a drain line. Liquid A includes, for example, a support comprising a complex and a support comprising an unreacted ligand.

[0038] In this embodiment, the solution A passes through the first outlet 141, enters the pipe 162, and is sent to the first washing unit 20. The solution A passes through the pipe 162 and reaches the confluence 250X. Meanwhile, the first washing solution is supplied from the first washing solution supply unit 210. The first washing solution passes through the pipe 250 and reaches the confluence 250X. In this embodiment, the solution A and the first washing solution join at the confluence 250X. The solution A and the first washing solution joined at the confluence 250X pass through the pipe 162 and are mixed by the in-line mixer 230. At this time, for example, by washing the complex with the first washing solution, components nonspecifically attached to the carrier, the complex, etc. (e.g., proteins derived from the culture medium) are removed and float in the liquid. Then, the mixture of Liquid A and the first cleaning liquid (hereinafter also referred to as "Mixed Liquid A") passes through the in-line mixer 230 and is sent from the pipe 261 to the hydrocyclone 240. The mixed liquid A is separated into Liquid C and Liquid D by the hydrocyclone 240. Liquid D is, for example, a waste liquid. In this embodiment, Liquid D enters the pipe 263 from the hydrocyclone 240 and is sent to a waste liquid line. Liquid C includes, for example, a carrier having a complex and a carrier having an unreacted ligand.

[0039] In this embodiment, liquid C enters pipe 262 from hydrocyclone 240 and is sent to elution section 30. Liquid C passes through pipe 262 to reach confluence 350X. Meanwhile, eluate is supplied from eluate supply section 310. The eluate passes through pipe 350 to reach confluence 350X. In this embodiment, liquid C and eluate merge at confluence 350X. Liquid C and eluate merged at confluence 350X pass through pipe 262 and are mixed by in-line mixer 330. At this time, for example, proteins are eluted from the complex into the liquid by the eluate. Then, the mixture of liquid C and eluate (hereinafter also referred to as "mixed liquid B") passes through in-line mixer 330 and is sent to hydrocyclone 340 from pipe 361. Mixed liquid B is separated into liquid E and liquid F by hydrocyclone 340. In this embodiment, liquor E may contain the eluted protein. liquor E enters piping 363 from hydrocyclone 340 and is sent to a protein recovery process. liquor F may contain the carrier after the protein has been eluted.

[0040] In this embodiment, the F solution enters the pipe 362 from the hydrocyclone 340 and is sent to the second cleaning unit 40. The F solution passes through the pipe 362 and reaches the confluence 450X. Meanwhile, the second cleaning solution is supplied from the second cleaning solution supply unit 410. The second cleaning solution passes through the pipe 450 and reaches the confluence 450X. In this embodiment, the F solution and the second cleaning solution merge at the confluence 450X. The F solution and the second cleaning solution that have merged at the confluence 450X pass through the pipe 362 and are mixed by the in-line mixer 430. At this time, the support is washed with, for example, the second cleaning solution. Then, the mixed solution of the F solution and the second cleaning solution (hereinafter also referred to as "mixed solution C") passes through the in-line mixer 430 and is sent to the hydrocyclone 440. The mixed solution C is separated into the G solution and the H solution. In this embodiment, the G solution is discharged. The G solution leaves the hydrocyclone 440 and enters line 463, which is sent to the drain line. The H solution, in this embodiment, may include a carrier.

[0041] In this embodiment, the H solution enters the pipe 462 from the hydrocyclone 440 and is sent to the equilibration unit 50. The H solution passes through the pipe 462 to reach the confluence 550X. Meanwhile, the equilibration solution is supplied from the equilibration solution supply unit 510. The equilibration solution passes through the pipe 550 to reach the confluence 550X. In this embodiment, the H solution and the equilibration solution merge at the confluence 550X. The H solution and the equilibration solution that have merged at the confluence 550X pass through the pipe 462 and are mixed by the in-line mixer 530. At this time, for example, the ligand (ligand attached to the carrier) is equilibrated by the equilibration solution. Then, the mixture of the H solution and the equilibration solution (hereinafter also referred to as "mixed solution D") passes through the in-line mixer 530 and is sent to the hydrocyclone 440. The mixed solution D is separated into a liquid I and a liquid J. In this embodiment, the I liquid is the waste liquid. The I liquid enters the hydrocyclone 540 through the pipe 563 and is sent to the waste line. The J liquid may include a carrier in this embodiment. The J liquid enters the hydrocyclone 540 through the pipe 562 and is sent to the first supply unit 110.

[0042] The operation of the apparatus 1 has been described above with reference to Figures 1 and 2. The configuration of the apparatus 1 is not limited to that shown in Figures 1 and 2. For example, the number of in-line mixers, hydrocyclones, pumps, etc. in each functional block is not limited. 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.

[0043] As described above, the apparatus 1 is an apparatus for purifying a protein from a cell culture solution. The apparatus 1 includes a first supply unit 110, a second supply unit 120, an in-line mixer 130, and a hydrocyclone 140. The first supply unit 110 supplies a first slurry. The first slurry contains a carrier having a ligand that binds to the protein. The second supply unit 120 supplies a second slurry. The second slurry is a cell culture solution containing the protein. The in-line mixer 130 mixes the first slurry supplied from the first supply unit 110 with 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 the protein-carrier complex from the mixed slurry of the first slurry and the second slurry obtained by the in-line mixer 130.

[0044] The mixed slurry is prepared to contain 10 to 25% by volume of the carrier and 5 to 20 mg of protein per ml of the carrier, with the total volume of the mixed slurry being 100% by volume. The length of the in-line mixer 130 is set so that the first slurry and the second slurry can be mixed for 0.6 to 60 seconds.

[0045] 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, it is possible to separate and produce a target protein from a cell culture solution without using an affinity column or the like. This allows the apparatus itself to have a simpler and more continuous configuration, and shortens the time required to purify the protein. In the apparatus 1, the mixed slurry in the in-line mixer 130 is prepared to contain 10% to 25% by volume of the carrier, assuming the total volume to be 100%, and 5 mg to 20 mg of protein per ml of carrier. This allows the first and second slurries to be mixed in the in-line mixer 130, creating conditions favorable for the protein to be adsorbed onto the carrier. At the same time, when separation is performed using the hydrocyclone 140 in the subsequent process, the mixed slurry after passing through the in-line mixer 130 (here, the mixed slurry containing the protein-carrier complex formed during passage through the in-line mixer 130) can be prevented from clogging when sent to the hydrocyclone 140 through the pipe 161. Therefore, in the apparatus 1, conditions are set that can achieve high adsorption efficiency between the protein and the carrier in the in-line mixer 130 while favorably preventing clogging when the slurry is sent to the hydrocyclone 140. Furthermore, in the apparatus 1, the length of the in-line mixer 130 is set so that the mixing time of the first slurry and the second slurry in the in-line mixer 130 is 0.6 seconds to 60 seconds. This allows the first slurry and the second slurry to be mixed in the in-line mixer 130 while appropriately maintaining a favorable condition for the protein to be adsorbed onto the carrier. Therefore, during this mixing time, approximately 70% or more of the protein to be purified contained in the second slurry can be adsorbed onto the carrier in the in-line mixer 130. This makes it possible to increase the efficiency of adsorption of a protein derived from a cell onto a carrier having a ligand that binds to the protein in the in-line mixer 130 provided in the device 1.

[0046] From another perspective, an adsorption method can be carried out using the apparatus 1. As described above, the apparatus 1 includes an in-line mixer 130 and a hydrocyclone 140 connected to the in-line mixer 130 downstream. The adsorption method involves adsorbing a cell-derived protein onto a carrier equipped with a ligand that binds to the protein. The adsorption method involves mixing a first slurry containing a carrier and a second slurry containing a protein in the in-line mixer 130, and adsorbing the protein onto the carrier in the mixed slurry of the first and second slurries to produce a protein-carrier complex. The mixed slurry in this adsorption method is prepared to contain 10% to 25% by volume of the carrier, with the total mixed slurry being 100% by volume, and 5 mg to 20 mg of protein per ml of carrier. The mixing time using the in-line mixer 130 in this adsorption method is 0.6 to 60 seconds.

[0047] The above-described effects can be achieved with the apparatus 1. As a result, by implementing the adsorption method disclosed herein, it is possible to increase the efficiency of adsorption of a protein and a carrier when a cell-derived protein is adsorbed onto a carrier equipped with a ligand that binds to the protein in the in-line mixer 130 included in the apparatus 1. At the same time, the mixed slurry that has passed through the in-line mixer 130 can be sent to the hydrocyclone 140 without causing clogging in the pipe 161.

[0048] A method for purifying a protein from a cell culture solution containing the protein can be carried out using the apparatus 1. This purification method includes preparing a complex by the above-described adsorption method and separating, in a hydrocyclone 140, the complex from a mixed slurry continuously fed from the in-line mixer 130 to the hydrocyclone 140.

[0049] The apparatus 1 can achieve the above-described effects. By implementing the adsorption method disclosed herein, the adsorption efficiency between the protein and the carrier can be increased when the cell-derived protein is adsorbed onto a carrier equipped with a ligand that binds to the protein in the in-line mixer 130 included in the apparatus 1. This ultimately allows the target protein to be purified efficiently. At the same time, the mixed slurry that has passed through the in-line mixer 130 can be sent to the hydrocyclone 140 without clogging the piping 161.

[0050] Below, test examples conducted by the inventors regarding the technology disclosed herein will be described, but it is not intended that the technology disclosed herein be limited to the following test examples.

[0051] <Test 1 - Mixing test using a stirring blade> In Test 1, a human IgG antibody and silica beads (manufactured by Osaka Soda Co., Ltd.) were stirred using a stirring blade. The antibody used in this test was an antibody sample solution prepared with a concentration of 20 mg / mL. The silica beads used in this test were silica beads to which protein A was immobilized as a ligand, and had an average particle size of 50 μm.

[0052] [Group 1] For Group 1, five 50 ml slurries containing 5, 10, 20, 25, and 50% carrier by volume, based on 100% by volume of the first slurry, were prepared. Five 50 ml slurries were prepared as second slurries, each containing 5 mg of antibody per 1 ml of carrier. Five mixed slurries were then prepared by stirring and mixing 50 ml of each first slurry with 50 ml of each second slurry. Each of the five mixed slurries contained 2.5, 5, 10, 12.5, or 25% carrier by volume, based on 100% by volume of the mixed slurry. All five mixed slurries contained 5 mg of antibody per 1 ml of carrier. The first slurry and the second slurry were stirred using a stirring blade (Tornado SMT-101 manufactured by AS ONE Corporation) at a rotation speed of 200 rpm.

[0053] Next, the adsorption rate of the antibody to the silica beads after a predetermined stirring time (hereinafter also referred to simply as "adsorption rate") was measured. Portions of the mixed slurry were sampled at the start of stirring, and 6, 13, 19, 26, 32, 60, and 120 seconds after the start of stirring. The antibody adsorbed to the silica beads was eluted with acetate buffer, and the absorbance of the eluate at a wavelength of 280 nm was measured to quantify the amount of antibody adsorbed to the carrier. The results are shown in Table 1.

[0054]

[0055] Table 1 shows the adsorption rate (%) after each stirring time for each volume fraction of the carrier in 100 ml of mixed slurry. Based on the obtained data, the results were plotted for each volume fraction of the carrier, with the adsorption rate (%) on the vertical axis and time (seconds) on the horizontal axis, and an approximation formula for the obtained curve was determined. Then, using this approximation formula, the time (seconds) at which the adsorption rate (%) reached 70% for each volume fraction of the carrier was calculated. The results are shown in the corresponding columns of Table 1.

[0056] [Group 2] Five types of slurries were prepared as the second slurries, each containing 50 ml of antibody per ml of carrier contained in each first slurry. All five mixed slurries prepared using this second slurry contained 10 mg of antibody per ml of carrier. Data for Group 2 was obtained using the same materials and procedures as Group 1. The results are shown in Table 2.

[0057]

[0058] [Group 3] Five types of slurries were prepared as the second slurries, each containing 50 ml of antibody per ml of carrier contained in each first slurry. All five mixed slurries prepared using this second slurry contained 15 mg of antibody per ml of carrier. Data for Group 3 were obtained using the same materials and procedures as Group 1. The results are shown in Table 3.

[0059]

[0060] [Group 4] Five types of slurries were prepared as the second slurries, each containing 50 ml of antibody per ml of carrier contained in each first slurry. All five mixed slurries prepared using this second slurry contained 20 mg of antibody per ml of carrier. Data for Group 4 was obtained using the same materials and procedures as Group 1. The results are shown in Table 4.

[0061]

[0062] <Test 2 - Mixing Test Using an In-Line Mixer> In Test 2, the above-described human IgG antibody and the above-described silica beads were stirred using an in-line mixer. Three types of in-line mixers were prepared for Test 2. The first in-line mixer was In-Line Mixer A, which had an outer diameter of 14 mm, an inner diameter of 10 mm, and a length adjusted so that the mixing time (liquid-passing time) was 13 seconds. The second in-line mixer was In-Line Mixer B, which had an outer diameter of 14 mm, an inner diameter of 10 mm, and a length adjusted so that the mixing time (liquid-passing time) was 19 seconds. The third in-line mixer was In-Line Mixer C, which had an outer diameter of 14 mm, an inner diameter of 10 mm, and a length adjusted so that the mixing time (liquid-passing time) was 32 seconds.

[0063] [Group 1] For Group 1, 250 ml of the first slurry was prepared, containing 20% ​​by volume of carrier, with the total volume of the first slurry being 100% by volume. 250 ml of the second slurry was prepared, containing 5 mg of antibody per 1 ml of carrier contained in the first slurry. Next, 250 ml of the first slurry and 250 ml of the second slurry were mixed to prepare a mixed slurry. The mixed slurry contained 10% by volume of carrier, with the total volume of the mixed slurry being 100% by volume. The mixed slurry contained 5 mg of antibody per 1 ml of carrier. In-line mixer A was used to mix the first and second slurries.

[0064] Next, the adsorption rate of the antibody to the silica beads was measured after the stirring time in the in-line mixer had elapsed. Portions were taken from the mixed slurry at the start of stirring and after passing through in-line mixer A, and the antibody adsorbed to the silica beads was eluted with an acetate buffer. The absorbance of the eluate at a wavelength of 280 nm was measured to quantify the amount of antibody adsorbed to the carrier. The results are shown in Table 5. Note that there were two samples in Group 1 (n = 2). The adsorption rates shown in Table 5 are the average values ​​of the two samples in Group 1.

[0065]

[0066] [Group 2] A 250 ml slurry containing 15 mg of antibody per 1 ml of carrier contained in the first slurry was prepared as the second slurry. The mixed slurry contained 15 mg of antibody per 1 ml of carrier. Data for Group 2 was obtained using the same materials and procedures as Group 1. The results are shown in Table 5. Note that Group 2 had two samples (n=2). The adsorption rates shown in Table 5 are the average values ​​of the two samples in Group 2.

[0067] [Group 3] In-line mixer B was used as the in-line mixer. Data for Group 3 was obtained using the same materials and procedures as Group 1. The results are shown in Table 5. Note that Group 3 had four samples (n=4). The adsorption rates shown in Table 5 are the average values ​​of the four samples in Group 3.

[0068] [Group 4] 250 ml of a first slurry containing 30% by volume of carrier, with the total volume of the first slurry being 100% by volume, was prepared. The mixed slurry contained 15% by volume of carrier, with the total volume of the mixed slurry being 100% by volume. In-line mixer B was used as the in-line mixer. Data for Group 4 was obtained using the same materials and procedures as for Group 1. Note that the number of samples in Group 4 was one (n=1). The adsorption rates shown in Table 5 are the measured values ​​for one sample in Group 4.

[0069] [Group 5] In-line mixer C was used as the in-line mixer. Data for Group 5 was obtained using the same materials and procedures as Group 1. The results are shown in Table 5. Note that Group 5 had three samples (n=3). The adsorption rates shown in Table 5 are the average values ​​of the three samples in Group 5.

[0070] From the data shown in Tables 1 to 5 obtained in Test 1 and Test 2, it was found that, from the viewpoint of better realizing the effects of the technology disclosed herein, the mixed slurry of the first slurry and the second slurry should be prepared so as to contain 10% to 25% by volume of the carrier, and 5 mg to 20 mg of protein per ml of the carrier, assuming the total mixed slurry to be 100% by volume. In addition, it was found that the mixing time using the in-line mixer should be set to 0.6 to 60 seconds.

[0071] REFERENCE SIGNS LIST 1 Apparatus 10 Adsorption section 110 First supply section 120 Second supply section 130 In-line mixer 140 Hydrocyclone 170 First pump 170 180 Second pump 20 First washing section 30 Elution section 40 Second washing section 50 Equilibration section

Claims

1. A method for adsorbing a cell-derived protein onto a carrier having a ligand that binds to the protein, using a protein purification apparatus comprising: an in-line mixer; and a hydrocyclone connected to the in-line mixer downstream of the in-line mixer, the method comprising: mixing a first slurry containing the carrier and a second slurry containing the protein in the in-line mixer; and adsorbing the protein onto the carrier in a mixed slurry of the first and second slurries to produce a complex of the protein and the carrier, wherein the mixed slurry is prepared to contain 10% by volume or more and 25% by volume or less of the carrier, with the total mixed slurry being 100% by volume, and 5 mg to 20 mg of the protein per ml of the carrier, and the mixing time using the in-line mixer is 0.6 seconds to 60 seconds.

2. A method for purifying a protein from a cell culture solution containing the protein, comprising: preparing the complex by the adsorption method according to claim 1; and separating, in the hydrocyclone, the complex from the mixed slurry that has been continuously pumped from the in-line mixer to the hydrocyclone.

3. An apparatus for purifying a protein from a cell culture solution, comprising: a first supply unit that supplies a first slurry containing a carrier having a ligand that binds to the protein; a second supply unit that supplies a second slurry, which is a cell culture solution containing the protein; an in-line mixer that mixes the first slurry supplied from the first supply unit and the second slurry supplied from the second supply unit; and a hydrocyclone connected to the in-line mixer downstream of the in-line mixer, that separates a complex between the protein and the carrier from a mixed slurry of the first slurry and the second slurry obtained by the in-line mixer, wherein the mixed slurry is prepared to contain 10% by volume or more and 25% by volume or less of the carrier, and 5 mg to 20 mg of the protein per ml of the carrier, assuming the entire mixed slurry to be 100% by volume, and the length of the in-line mixer is set so that the first slurry and the second slurry can be mixed for 0.6 seconds to 60 seconds.