JP6991312B2 - An apparatus for continuously producing an antibody from a cell culture medium using a plurality of modules in which an in-line mixer and a hydrocyclone are integrated, and a method for producing an antibody using the apparatus. - Google Patents

Description

The present invention relates to an apparatus composed of a module in which an in-line mixer and a hydrocyclone are combined for separating and purifying an antibody or other protein produced by a cell culture technique, and a method for producing an antibody or other protein using the apparatus.

Proteins, glycoproteins, peptides, nucleic acids, etc. that are recently used as biopharmacy are conventionally separated and purified from blood, other body fluids, homogenates of biological tissues, etc. by precipitation operation, filtration, centrifugation, or chromatographic separation operation. Has been done. Recently, proteins and peptides have been expressed by genetic recombination of microorganisms such as Escherichia coli (abbreviation: E. coli) and produced as inclusion body granules (inclusion bodies) in the cells, followed by denaturation and solubilization. The target product regenerated by refolding has been purified by centrifugation, filtration, chromatographic operation, or the like. Especially recently, in the case of glycoprotein drugs such as monoclonal antibodies, after the production by culturing transgenic Chinese Hamster Ovary (CHO) cells, continuous centrifugation, depth filter and membrane filter are added. After removing and clarifying the cells, various separating agents such as affinity chromatography, ion exchange chromatography, and hydrophobic interaction chromatography were finely packed in a tubular housing (column) for chromatography and clarified. It has been purified by flowing a solution containing glycoprotein or protein in combination with a chromatographic separation operation or a membrane separation operation.

However, the chromatographic separation operation that has been performed so far is time-consuming, and the amount of the target substance that can be purified to use a column of a limited size in which a separating agent is packed in a cylindrical housing is required. Limited. Therefore, after adding the sample to the first column using a device that operates multiple columns in sequence, add the sample to the next column while performing the separation operation, and then perform the separation operation on the second column. While the first column is being regenerated and washed, the sample is continuously added to the third column by repeating the operation of so-called Simulated Moving Bed (SMB) type separation and purification. There is an example in which a continuous purification process can be efficiently performed by sequentially switching between a sample to be treated and a regenerated washing solution in a plurality of columns (Non-Patent Document 1).

However, in all of these conventional separation and purification operations, a chromatographic column in which the column housing is densely filled with a separating agent, a housing, or a filter filled in a cartridge container is used, so that cells, cell debris, some aggregates, precipitates, etc. are generated. If there is, clogging (Fouling) occurs, so that a solution or suspension containing proteins, glycoproteins, peptides, nucleic acids, etc. cannot flow, and the work must be interrupted, which makes continuous operation difficult.

Therefore, in recent years, a method for purifying a protein without using a column has attracted attention (Non-Patent Document 2). As a protein purification device that avoids the use of a resin-filled column, "a system for purifying a target substance from a liquid source, a container for contacting the liquid source with a chromatography resin, and chromatography. A cross-flow filter (CFF or tangential filter: also referred to as TFF) for filtering the resin to recover the target substance from the chromatographic resin, and the container and the cross-flow filter are interconnected and flowed. A flow circuit for flowing a liquid containing the chromatographic resin through a circuit, a chromatographic resin in the flow circuit, the container, and the cross-flow filter, and the liquid supply connected so as to be sent to the container. A system (Patent Document 1) comprising a source and a means for circulating a liquid and a chromatographic resin through the flow circuit is known, but maintenance is complicated due to clogging of the cross flow filter. Is. Further, there is known a backflow cross-flow chromatography module (Patent Document 2) in which a chromatographic resin slurry and an unrefined standard are continuously mixed by a mixer, and the mixed solution is concentrated and separated by a filter. However, it is not possible to avoid clogging of the filter and complicated maintenance.

Cyclone separators that use the centrifugal force of the flow to separate large substances from two-phase fluids have been used for a long time, but they are used in systems that separate slurry containing chemical substances from methanol for cleaning. Although it is known (Patent Document 3), it is not used for the purpose of collecting the target substance in the slurry using hydrocyclone, and is not used for the purification of proteins such as antibodies. Further, a method of mixing the slurry separated by the hydrocyclone with an in-line mixer is known, but a device for combining the in-line mixer with the hydrocyclone, mixing and reacting with the in-line mixer, and separating the slurry with the hydrocyclone is known. do not have.

In the present invention, the culture solution containing cells and fragments thereof is directly or the solid component in the suspension is precipitated and removed (however, it is not necessary to completely remove it) using a hydrocyclone, and then an affinity chromatography separating agent or an affinity chromatography separating agent is used. After performing an adsorption reaction with a slurry such as an ion exchange chromatography separator in an in-line mixer, the separator in which the unadsorbed component and the target protein component are bound is separated by a hydrocyclone. Further, the separated and discharged protein binding separating agent is guided to the next in-line mixer and a device (module) in which hydrocyclone is bound and washed, and then a solution that inhibits the binding between the target protein and the resin or a solution that can be dissociated. At the same time, the target protein and the adsorption separator are separated by being guided to a device consisting of a third in-line mixer and a hydrocyclone, and the target protein overflows from the upper part of the hydrocyclone and is recovered. On the other hand, the separating agent dissociated from the protein is guided to the next device consisting of an in-line mixer and a hydrocyclone, regenerated with a washing and regeneration solution, and then introduced into the first in-line mixer to continuously introduce the target protein such as a cell culture solution. Subject to reaction with the containing suspension.

As described above, the present invention is based on a solution containing proteins, glycoproteins, peptides, nucleic acids, etc. without causing clogging (Fouling) even in a suspension containing cells, cell debris, or some aggregates or precipitates. It is a method capable of continuously separating components containing a target protein, and is an apparatus that can be used for such a continuous separation and purification method.

Special Table 2003-512594 Gazette US2010 / 0193434A Japanese Unexamined Patent Publication No. 5-140044

Seiki Sugiyama, Noriko Yoshimoto, Shuichi Yamamoto, "Continuous Chromatographic Separation Operation by Column Switching", Food Engineering Journal, 2017, Vol. 18, No. 2, pp. A9-A12 Petra Gronemeyer, Reinhard Ditz and Jochen Strub, “Trends in Upstream and Downstream Process Development for Antibody Manufacturing”, Bioengineering, (2014), Vol1, 188-212

The target protein expressed by culturing animal cells or microorganisms has been made into a clarified solution from which solids have been removed by centrifugation or multi-step filter filtration, and then a chromatographic separating agent is finely filled in the column housing. It has been purified by using a multi-step chromatographic filling column, a filter filled in a container called a cartridge or a housing, and the like. However, it is difficult to continuously operate a column or filter that is tightly packed in a closed container because it causes clogging.

Further, the present invention, as shown in FIGS. 1 and 2, one aspect thereof is shown in FIG.
[1] A device for continuously purifying proteins from cell culture medium using a module in which an in-line mixer and a cyclone are combined.
(a) At the inlet of the in-line mixer (6) of the first module (A: reaction module), a culture tank (1) or a pipe (3) connecting the cell separation device (D) and the first module, The pipe (4) connecting the first storage tank (2) and the first module is connected via the branch valve (5), and the discharge port of the in-line mixer (6) is the introduction of the hydrocyclone (7). A pipe (8) connecting the first reaction module and the second module is connected to the lower discharge port of the hydrocyclone (7), which is connected to the port, and a pipe (pipe) is connected to the upper discharge port of the hydrocyclone (7). With the first module to which 9) is combined,
(b) At the introduction port of the in-line mixer (13) of the second module (B: elution module), a pipe (8) connecting the first module and the second module and a second storage tank (10) The pipe (11) connecting the module and the second module is connected via the branch valve (12), the outlet of the inline mixer (13) and the inlet of the hydrocyclone (14) are connected, and the exhaust of the lower part of the hydrocyclon is connected. A pipe (15) connecting the second module and another module is connected to the outlet, and a second module to which the pipe (16) is connected is configured at the upper discharge port of the hydrocyclone (14). Regarding the device.

The present invention also has, as one embodiment of which is shown in FIG.
[2] Another module, as a third module (C: regeneration module), is a pipe (15) connecting the second module (B) and the third module to the introduction port of the inline mixer (20). The third storage tank (17) and the pipe (18) connecting the third module are connected via the branch valve (19), and the discharge port of the inline mixer (20) and the introduction port of the hydrocyclone (21) are connected. Is connected, a pipe (23) connecting the third module and the first storage tank (2) is connected to the lower discharge port of the hydrocyclon (21), and the upper discharge port of the hydrocyclon (21) is connected to the pipe (23). The device according to [1], which is the third module to which the pipe (22) is connected.

Further, as shown in FIG. 4, one aspect of the present invention is shown.
[3] In the cell separation device (D), the pipe (24) connecting the culture tank (1) and the cell separation device is connected to the introduction port of the hydrocyclone (25), and the lower discharge port of the hydrocyclon (25) is connected. Is a cell separation unit composed of a pipe (26) connecting the cell separation device and the culture tank (1), and the upper outlet is a pipe (3) connecting the cell culture device and the first module. The device according to [1] or [2].

The present invention also has one aspect shown in FIG.
[4] One or two fourth modules (E: cleaning modules) for cleaning between the pipe (8) connecting the first module (A) and the second module (B) described in [1]. The device according to any one of [1] to [3], which is characterized by being inserted, and [5] the fourth module is the introduction port of the in-line mixer (30) of the fourth module (E). The pipe (8) connecting the first module and the fourth module and the pipe (28) connecting the fourth storage tank (27) and the fourth module are connected to each other via a branch valve (29). , The discharge port of the in-line mixer (30) and the introduction port of the hydrocyclone (31) are connected, and the pipe (8) connecting to the second module or a new cleaning module is connected to the discharge port at the bottom of the hydrocyclone. The device according to claim 4, wherein a pipe (32) for transferring the cleaning liquid to the discharge port is connected to the upper discharge port of the hydrocyclone (31).

Further, the present invention
[6] A method for continuously producing a protein from a cell culture medium using a plurality of modules in which an in-line mixer and a cyclone are combined.
(a) In the in-line mixer of the first module, mix the cell culture solution containing the protein transferred from the culture tank and the slurry containing the resin transferred from the first storage tank, and mix the protein in the cell culture solution. A step of combining with a resin in a slurry to generate a slurry containing a resin bonded with a protein,
(b) A step of transferring the slurry containing the resin bound to the protein obtained in the mixing step of the in-line mixer of the first module to the cyclone and separating the slurry containing the resin bound to the protein and the cell culture medium. ,
(c) A step of transferring the slurry containing the resin bound to the protein obtained in the separation step of the first module with a cyclone to the second module and transferring the cell culture medium to the first storage tank.
(d) In the in-line mixer of the second module, the slurry containing the resin bound to the protein obtained in the above step and the eluate transferred from the second storage tank are mixed to generate an elution slurry. Process, and
(e) A method comprising a step of transferring the elution slurry obtained in the mixing step of the second module with an in-line mixer to a cyclone and separating the slurry into a slurry containing a protein and a resin not bound to the protein.
[7] A method comprising a cleaning step using a third module between step b and step c according to [6].
[8] In the cleaning method according to [7], the slurry containing the resin bound to the protein and the cleaning liquid transferred from the fourth storage tank are mixed in the in-line mixer of the third module, and after cleaning. A method characterized by a step of transferring the mixed solution of the above to a cyclone and separating the mixture.
[9] In the step of transferring the protein-containing cell culture medium from the culture tank to the in-line mixer of the first module, the cells and the protein-containing cell culture medium are separated via a cyclone, and the separated cells are cultured. The method according to any one of [6] to [8] and [10], wherein the cell culture medium containing the purified protein is transferred to the in-line mixer of the first module. The method according to any one of [6] to [9], wherein the resin is an affinity resin.
The present invention also
[11] A device for continuously purifying a protein from a cell culture medium using a plurality of modules in which an in-line mixer and a cyclone are combined.
[12] The apparatus according to [1] mixes a cell culture solution and a slurry containing a resin with an in-line mixer, binds the protein in the cell culture solution to the resin in the slurry, and the slurry containing the resin bound to the protein. The reaction module, which has the function of separating the resin-containing slurry and the resin-free cell culture solution by cyclone, and the protein-bound resin-containing slurry and eluent are mixed with an in-line mixer and hydrocyclone is used. A device consisting of an elution module that separates into protein and resin slurry by
[13] Cells existing in the cell culture medium containing the protein flowing out of the culture tank are separated by hydrocyclone, the collected cells are transferred to the culture tank, and the cell culture medium containing the protein excluding the cells is used. The device according to [11] or [12], which is equipped with a cell separation device to be transferred to the next purification means.
[14] Any of [11] to [13], wherein the slurry containing the resin bound to the protein and the cleaning liquid are mixed with an in-line mixer, and a cleaning module for separating the slurry containing the resin bound to the protein by a cyclone and the waste liquid is attached. The device described in one.
[15] In the apparatus according to any one of [12] to [14] in which the reaction module and the elution module are combined, the resin slurry discharged by the elution module of the apparatus is transferred to the reaction module or the storage tank of the resin slurry. The present invention relates to an apparatus that enables cycle utilization of the resin slurry, and a method for producing a protein using the apparatus according to any one of [16] [11] to [15].
The present invention further
[17] A device for continuously purifying a protein from a cell culture solution using a module in which an in-line mixer and a cyclone are bound, and the protein is obtained by mixing a cell culture solution containing a protein and a slurry containing a resin. A second module (reaction module) that produces a slurry bound to the resin is mixed with a slurry purified by the first module and an eluent, and the protein is eluted from the slurry. A protein purification apparatus, characterized by having a module (dissolution module).
[18] The claim is characterized in that it has a third module (regeneration module) for producing a regenerated resin slurry containing a resin by mixing the slurry from which the protein has been separated and the regenerated liquid in the second module. 17. The protein purification apparatus according to 17.
[19] The invention according to claim 17, wherein the first module has a cell separation module that separates cells from the cell culture solution supplied from the culture tank and supplies the cell culture solution containing the protein to the first module. Protein Purifier,
[20] A slurry containing a resin bound to the protein produced by the first module and a washing liquid, which are interposed between the first module and the second module, are mixed and bound to the protein. The protein purification apparatus according to claim 17, further comprising a fourth module (cleaning module) for cleaning the prepared resin slurry.
[21] The first module mixes a cell culture solution and a slurry containing a resin, binds the protein in the cell culture solution to the resin in the slurry, and produces a slurry containing the resin bound to the protein. The protein purification apparatus according to claim 17, further comprising one in-line mixer and a first hydrocyclone that separates a resin-containing slurry and a resin-free cell culture solution.
[22] The second module is characterized by having a second in-line mixer that mixes a slurry containing a resin bound to a protein and an eluate, and a second hydrocyclone that separates the protein into a resin slurry. 17. The protein purification apparatus according to claim 17.
[23] A storage tank for storing the slurry containing the resin is provided.
The protein purification apparatus according to claim 18, wherein the third module transfers the regenerated resin slurry to the storage tank.
[24] The cell separation module separates cells existing in a cell culture medium containing a protein flowing out of the culture tank, transfers the collected cells to the culture tank, and cells containing the protein excluding the cells. The apparatus according to claim 19, wherein the apparatus has a hydrocyclone that transfers a culture solution to the first module, and [25] the fourth module contains a slurry containing a resin bound to the protein and the washing liquid. The apparatus according to claim 20, further comprising a fourth in-line mixer for mixing the cells, a slurry containing a resin bound to a protein, and a fourth hydrocyclone for separating into a waste liquid.

This specification includes the disclosure content of Japanese Patent Application No. 2018-058853, which is the basis of the priority of the present application.

The present invention provides a method and an apparatus thereof capable of continuously recovering and purifying a target protein from a suspended sample containing a small amount of cells, cell fragments, host microorganisms, etc. in a short time. do. By continuously recovering and purifying the target protein etc. directly from the sample containing a small amount of solid matter, it becomes possible to obtain a large amount of product relatively easily even in a small-scale culture facility depending on the operating time of the facility. High productivity and continuous production enable efficient manufacturing with reduced manufacturing cost.
(1) According to the present invention, a target protein such as an antibody molecule can be continuously separated without highly separating and removing cells, cell debris, or a precipitate from the culture medium of animal cells.
(2) According to the present invention, proteins can be continuously separated without using a chromatography column packed with a separating material conventionally used.
(3) The present invention enables perfusion culture of animal cells without using hollow fibers or a continuous centrifuge, and continuously separates the target protein from the extracted culture solution without using a filtration filter or a chromatography column. Can be done.
(4) By using the apparatus of the present invention, proteins can be continuously separated without using equipment such as a large membrane separation device, a chromatography device, and a large chromatography column.
(5) By using the apparatus of the present invention, the adsorption separator can be regenerated and washed in the flow path of the apparatus and circulated, so that continuous separation of proteins and the like can be repeated with a small amount of the adsorption separator.
(6) In the present invention, the target protein is dissociated within a short period of time after reacting with the slurry-like adsorption separator in the flow path of the apparatus to separate it from impurities without filling the column with the adsorption separator. It is possible to suppress structural denaturation due to adsorption of target proteins and the like.
(7) In the present invention, the adsorbent-separating agent can be reacted with a target protein or the like in a slurry state without being filled in a container such as a column, and can be washed in the slurry state. Therefore, impurities such as host protein, host DNA or RNA can be obtained. By avoiding unnecessary adsorption with, a high-purity target protein or the like can be obtained.

FIG. 1 is a reaction module A for adsorbing and separating an antibody on a resin. FIG. 2 is an elution module B that separates the antibody from the resin. FIG. 3 is a regeneration module C for recycling the resin. FIG. 4 is a cell separation device D for cell separation during reflux culture. FIG. 5 is a cleaning module E for cleaning the resin to which an antibody or the like is bound. FIG. 6 is a continuous purification device for antibodies from a culture solution. FIG. 7 is the reaction module of the present invention. FIG. 8 is a reaction module equipped with a cell sorting device. FIG. 9 is a schematic diagram of a hydrocyclone evaluation module. FIG. 10 is a schematic diagram of an evaluation module when the first hydrocyclone and the second hydrocyclone are secondarily connected. FIG. 11 is a schematic diagram of a module used in the test in which it was confirmed that the antibody can be recovered by mixing the antibody sample solution and the chromatographic resin for purification with an in-line mixer and then separating and recovering the resin with a hydrocyclone.

In the present invention, the in-line mixer may be any static mixing device without a driving part, but preferably a static mixer without a moving part or a fluidized bed type mixer in consideration of damage to cells and resin. It is preferable to use.

Since the in-line mixer has a simple structure in which two fluids that are normally introduced are mixed and discharged by the flow of liquid, one of the cylindrical shapes has an introduction port and a discharge port in the liquid outflow direction, which is used in the present invention. The in-line mixer has a shape in which a mixture of two fluids is introduced into one inlet. Further, the discharge port may have one discharge port in the liquid discharge direction.

In the present invention, the hydrocyclone refers to a liquid cyclone, and any device having no drive unit capable of separating, classifying, and concentrating by injecting a treatment liquid into a device in a stationary state of the liquid cyclone may be used. However, various types of hydrocyclones such as Mozley type, Dorr-Oliver type, Bradley type, Rietema type, Krebs type, and CBV / DEMCO type are typically used, but Bradley is considered for damage to cells and resin. It is preferable to use a type or Rietema type hydrocyclone.
These hydrocyclones are usually funnel-shaped, with a funnel-shaped upper wall surface that separates the mixture inlet, a funnel-shaped upper ceiling that separates and purifies the upper discharge port, and a funnel-shaped upper wall. At the lower end is the lower outlet for the mixture, excluding the purified material.

The feature of the present invention is that a plurality of modules in which an in-line mixer and a cyclone are combined are used, and each module has individual functions of adsorption and separation of an antibody to a resin, separation of an antibody from the resin, and regeneration of the resin. In addition, it is possible to provide a simple and trouble-free antibody purification device.

First, the feature of the present invention is that the cell culture solution and the slurry containing the resin are mixed by an in-line mixer, the protein in the cell culture solution is bound to the resin in the slurry, and the slurry containing the resin bound to the protein is obtained. The present invention relates to a reaction module having a function of being generated and separated into a resin-containing slurry and a resin-free cell culture medium by a cyclone.

The reaction module may be used alone, but a device in which multiple reaction modules are arranged in parallel to increase the capacity, a device in which reaction modules are arranged in series to increase efficiency, or a device in which multiple reaction modules are arranged in parallel and It can also be used as a device arranged in series.

The proteins in the present invention include antibodies, hematopoietic proteins such as erythropoetin, enzymes such as t-PA, cytokines such as G-CSF and interferon β, hormones such as insulin and human growth hormone, vaccines such as hepatitis B vaccine, and blood coagulation. Any substance used for pharmaceuticals such as factors may be used, but it is particularly preferable to target an antibody. As the antibody, it is preferable to use a monoclonal antibody, and it is particularly preferable to target a mouse antibody, a humanized antibody, a human antibody, or a chimeric antibody. The cell culture medium may be any culture medium as long as it is a culture medium in which the cells that produce the protein are cultured and the protein is produced. Examples of cells that produce the protein include microbial cells such as Escherichia coli, yeast, and animal cells such as CHO cells, BHK cells, NSO cells, and SP2 / 0 cells in addition to insect cells such as Sf9 and Sf21. It is preferable to use animal cells.

Examples of the resin in the present invention include resins used for affinity chromatography or ion exchange chromatography, and resins to which a ligand for binding and separating the protein is bound are preferable. When the protein to be bound is an antibody as the ligand for affinity chromatography, it is preferable to use protein A, protein G, protein L and derivatives thereof, and it is particularly preferable to use protein A and its derivatives. In the case of proteins other than antibodies, synthetic ligands such as HIS tags and GST tags, antibodies corresponding to various proteins, receptors for various proteins, various proteins and their small or high molecular weight inhibitors and the like can be used as ligands. .. Further, a metal chelate affinity resin or the like carrying a metal ion such as Cu2 + or Zn2 + can also be used. Any resin may be used as long as it has these ligands bound to it and has excellent fluidity. However, in order to improve the separation by hydrocyclone, a resin or synthetic polymer based on an inorganic material having a high density to some extent is used. Alternatively, it is preferable to use a resin based on an organic polymer such as a natural polymer and a resin in which an inorganic material is combined with a polymer organic material. Examples of the inorganic material include glass, silica, zirconium, and alloys using stainless steel or tungsten. On the other hand, examples of the synthetic polymer include polystyrene resin and methacrylate resin, and examples of the natural polymer include resins composed of agarose, dextran, cellulose, chitin, chitosan, mannan and the like. In particular, it is composed of a base material in which an inert metal such as stainless steel or a tungsten alloy is mixed with a natural polymer such as agarose or a synthetic polymer such as a vinyl polymer in order to increase the specific gravity in consideration of separation by hydrocyclone. The resin to be used is mentioned.

The resin used for ion exchange chromatography used in the present invention may be any anion exchange resin or cation exchange resin, but it is particularly preferable to use a strong anion exchange resin or a strong cation exchange resin. .. As the anion exchange resin, as a ligand, a diethylaminoethyl (DEAE) group, a trimethylaminoethyl (TAME) group, a quaternary aminoethyl (QAE) group, and a primary amino (-NH ₂ ) or a secondary (-NHR), Examples thereof include resins having a substituent such as a tertiary amino group (-NR1R2) and a quaternary amino (Q) group, and examples of the cation exchange resin include carboxymethyl (CM), sulfomethyl (SM), and sulfoethyl (SE) as ligands. Examples thereof include resins having substituents such as sulfopropyl (SP), phosphate (P), and sulfonate (S) groups. The resin used for ion exchange may be any resin to which these ligands are bound and has excellent fluidity, but in order to improve the separation by hydrocyclone, an inorganic material having a high density to some extent is used as a base material. In addition to resins based on organic polymers such as resins or synthetic polymers or natural polymers, natural polymers and synthetic polymers are mixed with inert metals such as stainless steel and tungsten alloys to increase the specific gravity. It is preferable to use a resin composed of a base material. Examples of the inorganic material include glass, silica, zirconium, and alloys using stainless steel or tungsten. Examples of the synthetic polymer include polystyrene resin and methacrylate resin, and examples of the natural polymer include a resin composed of agarose, dextran, and cellulose.

It is preferable that the reaction module is connected to the culture tank by piping via a cell separation device, if necessary. The culture tank may be any device that can produce the protein by culturing the cells that produce the protein, and even if it is a stainless steel culture tank, it is made of plastic or resin for single use. It may be a culture tank.

Any culture method may be used, but it is preferable to use a fed-batch method, a continuous culture method, or the like, and it is particularly preferable to use the continuous culture method. A cell culture medium containing the protein produced by the culture is used in the present invention to produce the protein by culturing the cells producing the protein in a culture tank under the culture conditions and medium usually used by those skilled in the art. .. The concentration of cells in the cell culture medium is preferably such that the packed cell volume is about 20% (v / v) or less. The pipe for transferring the culture broth may be any pipe as long as it is used as a flow path for the cell culture broth to flow smoothly, but it may be a stainless steel tube, a plastic for single use, rubber or silica, etc., natural or synthetic. It may be a tube made of resin.

The reaction module is also connected to a storage tank storing the resin slurry by a pipe, and the resin slurry and the cell culture solution can be mixed. The resin slurry has a pH at which the antibody or protein to which the resin is the target substance is efficiently bound, preferably adjusted to a concentration close to physiological conditions with a pH buffer solution near neutral in order to suppress denaturation of the protein. It is preferable to use.

Piping has the same meaning as described above, and it is preferable to incorporate a pump as necessary to keep the transfer concentration constant. The pipe for transferring the cell culture medium and the pipe for transferring the resin slurry are connected to the in-line mixer of the reaction module via a branch valve, if necessary. The branch valve may be any one as long as it is usually used for protein production equipment, but it is preferable to use a three-way valve.

Secondly, the feature of the present invention is that the cells existing in the cell culture medium containing the protein flowing out of the culture tank are separated by hydrocyclone, the collected cells are transferred to the culture tank, and the cells are removed. The present invention relates to a cell separation unit that transfers a cell culture medium containing the above to the next purification means. The next purification means may be any means used for purification of ordinary proteins such as an ion exchange chromatography device and an affinity chromatography device, but it is particularly preferable to use the reaction module.

Thirdly, a feature of the present invention relates to an elution module in which a slurry containing a resin bound to a protein and an eluate are mixed by an in-line mixer and separated into a protein and a resin slurry by a hydrocyclone. Although the separated protein is stored, the separated slurry is transferred to the slurry storage tank again, and as a device for supplying the antibody bound to the protein to the elution module, a tank having a stirring device or the like is used. Any device that can mix the cell culture solution and the resin slurry and bind the protein in the cell culture solution to the resin ligand in the resin slurry may be used, but it is preferable to use the reaction module of the present invention. ..

The elution module may be used alone, but a device in which multiple elution modules are arranged in parallel to increase capacity, a device in which elution modules are arranged in series to increase efficiency, or a device in which multiple elution modules are arranged in parallel and It can also be used as a device arranged in series.

The eluate may be any eluate that is usually used for protein affinity chromatography or ion exchange chromatography.
In the case of a resin bound to a ligand used for affinity chromatography such as protein A, the pH of the eluate is preferably 5 or less, preferably pH 4 to pH 2, and particularly preferably pH 2.5 to pH 2. As the medium, an acetate buffer solution, a citric acid buffer solution, a phosphoric acid buffer solution, or a dilute solution such as phosphoric acid or hydrochloric acid is used. In the case of a resin bound to a ligand used for ion exchange chromatography, it is preferable to increase the salt concentration of the eluate, and sodium chloride, potassium chloride or the like is used to reduce the salt concentration to 1 M or less, preferably 0.1 M to 0. It is used at .5 M, particularly preferably 0.1 M to 0.3 M. As the medium, Tris-hydrochloric acid buffer solution, acetate buffer solution, citric acid buffer solution, phosphate buffer solution and the like are used.

Fourth, the feature of the present invention relates to a cleaning module in which a slurry containing a resin bound to a protein and a cleaning liquid are mixed by an in-line mixer and separated into a slurry containing a resin bound to the protein by a cyclone and a waste liquid. The washing module is preferably installed between the reaction module and a device for separating proteins bound to the resin, preferably between the reaction modules. As the cleaning solution, it is preferable to use an aqueous medium having a pH of 5 to 8, preferably pH 6.5 to 8.0, and as the medium, a Tris-hydrochloric acid buffer solution, an acetate buffer solution, a citric acid buffer solution, a phosphate buffer solution or the like is used. .. If necessary, 0.1 to 1 M salt of sodium chloride, potassium chloride or the like can be added to the medium.

Fifth, in the present invention, by combining the reaction module and the elution module, the apparatus for producing protein from the cell culture solution transfers the resin slurry discharged by the elution module of the apparatus to the reaction module or the storage tank of the resin slurry. , The present invention relates to a device for producing a protein from a cell culture solution, which enables cycle utilization of a resin slurry. A feature of the present invention relates to a device for producing a protein from a cell culture medium having a cell circulation system by combining these manufacturing devices with a cell separation device.

Further, by incorporating a wash module between the reaction module and the elution module of these devices, a manufacturing device for proteins requiring a washing step is provided.

Sixth, in the present invention, the cell culture solution and the slurry containing the resin are mixed by an in-line mixer, and the protein in the cell culture solution is bound to the resin in the slurry to generate a slurry containing the resin bound to the protein. The present invention relates to a method for separating a resin-containing slurry and a resin-free cell culture solution by a cyclone. The definitions of the in-line mixer, the cell culture solution, the slurry containing the resin, and the hydrocyclone in the present invention are synonymous with the above.

Seventh, the present invention contains cells present in a cell culture medium containing proteins flowing out of the culture tank by hydrocyclone, the collected cells are transferred to the culture tank, and the cells excluding the cells are contained. The present invention relates to a method of transferring a cell culture medium to the next purification means. The next purification means may be any means used for purification of ordinary proteins such as an ion exchange chromatography device and an affinity chromatography device, but it is particularly preferable to use the reaction module.

Eighth invention relates to a method in which a slurry containing a resin bound to a protein and an eluate are mixed by an in-line mixer and separated into a protein and a resin slurry by a cyclone. The definition of eluate is synonymous with the above. By combining the present invention with the sixth invention and, if necessary, the seventh invention, a method for purifying a protein from a cell culture medium is constructed.

Ninth, the present invention relates to a cleaning method in which a slurry containing a resin bound to a protein and a cleaning liquid are mixed by an in-line mixer and separated into a slurry containing a resin bound to the protein by a cyclone and a waste liquid. The definition of the cleaning liquid is synonymous with the above, and the cleaning temperature is 4 ° C to 40 ° C. By using the present invention in combination with the sixth invention, the eighth invention, and if necessary, the seventh invention, a method for purifying a protein from a cell culture medium is constructed.

An aspect of purification of the protein of the present invention by the apparatus for producing the protein is shown below in FIG.
Cell separation step In the protein production apparatus of FIG. 6, the culture tank (101) provided with the storage tank (102) for supplying the medium is used to purify the cell culture solution produced in the culture tank. Is connected to a cell separator (103) for circulating use. In the cell separator, only the cells are separated from the cell culture medium and sent to the culture tank (101), and the cell culture medium containing the protein from which the cells are separated is transferred to the in-line mixer (105) of Module A used in the capture step. To.
Capturing step The capturing step refers to the initial purification step when protein is produced using a plurality of purification means.

Since the cell separator (103) is connected to the in-line mixer (105) of the module A, and the in-line mixer (105) of the module A is also connected to the storage tank (104) for the adsorbent resin (A) such as protein A. The cell culture medium in which the cells were separated in the cell separation step is mixed with the resin (A) in an in-line mixer, and the protein in the cell culture solution and the resin (A) bind to each other. Form a slurry with (A). Since the outlet of the in-line mixer (105) of the module A is bound to the inlet of the cyclone (106) of the module A, the slurry of the resin (A) bound to the cell culture medium and the protein in the cyclone is separated. , The cell culture medium is discharged from the upper discharge port, and the resin (A) bound to the protein is discharged from the lower discharge port. Since the lower discharge port of the cyclone (106) of the module A is connected to the in-line mixer (108) of the module B, and the in-line mixer is also connected to the storage tank (104) for the resin (A) eluate, the module B is connected. In the in-line mixer (108) of the above, the resin (A) bound to the protein and the eluate are mixed and the protein is separated from the resin (A). Since the outlet of the in-line mixer is connected to the inlet of the cyclone (109) of the module B, the protein and the resin (A) are separated in the cyclone (109) of the module B, and the protein is discharged from the cyclone upper outlet. The resin (A) is discharged from the lower discharge port. The protein is sent via the pipe (110) to the in-line mixer (111) of module D to be used in the next polishing step.
On the other hand, since the lower discharge port of the cyclone (109) of the module B is connected to the inline mixer (116) of the module C, and the inline mixer is also connected to the resin regeneration liquid storage tank (117), the cyclone of the module B (116). 109) The resin (A) discharged from the lower discharge port is mixed with the regenerating liquid in the in-line mixer (116) of the module C, and is mixed with the regenerating liquid in the cyclone (118) of the module C connected to the in-line mixer. To separate. The separated resin A is sent from the cyclone lower discharge port of the module C to the resin (A) storage tank (104) via the pipe (119).

Polishing process The polishing process refers to the purification process after the mid-term process when protein is produced using a plurality of purification means.
Polishing process 1
Since the in-line mixer (111) of the module D is connected to the resin (B) storage tank (123) such as the cyclone and the ion exchange resin of the module B of the capture step by the pipe (110), it is obtained in the capture step. The resulting protein and the resin (B) are mixed in the in-line mixer to form a slurry of the resin (B) bound to the protein. Since the in-line mixer (111) outlet of the module D is connected to the inlet of the cyclone (112) of the module D, the slurry of the resin (B) bound to the protein in the cyclone (112) of the module D and the residue. The solution is separated, the waste liquid is discharged from the upper discharge port, and the slurry of the resin (B) bound to the protein is discharged from the lower discharge port. The lower discharge port of the cyclone (112) of the module D is connected to the inline mixer (121) of the module E, and the inline mixer is also connected to the eluate storage tank (124) for the resin (B). The resin (B) slurry bound to the protein in the above is mixed with the eluate, and the protein is released from the resin (B). Since the in-line mixer (121) discharge port of the module E and the introduction port of the cyclone (122) of the module E are connected, the protein and the resin (B) are separated in the cyclone. The protein is discharged from the upper outlet of the cyclone (122) of the module E, and the resin (B) is discharged from the lower outlet. The protein is transferred via the pipe (115) to the in-line mixer of module E performing the polishing step 2. The resin (B) slurry discharged from the cyclone lower discharge port is transferred to the resin (B) storage tank via the pipe (125) and reused.

Polishing process 2
Since the in-line mixer (113) of the module F is connected to the cyclone of the module E of the polishing step 1 and the resin (C) storage tank (133) such as the hydrophobic resin by the pipe (115), it is obtained in the polishing step 1. The crudely purified protein and the resin (C) are mixed in the in-line mixer to form a slurry of the resin (C) bound to the protein. Since the discharge port of the in-line mixer (113) of the module F is connected to the introduction port of the cyclone (114) of the module F, the slurry and the residue of the resin (C) bound to the protein in the cyclone (114) of the module F. The solution of the above is separated, the waste liquid is discharged from the upper discharge port, and the slurry of the resin (C) bound to the protein is discharged from the lower discharge port. Since the lower discharge port of the cyclone (114) of the module F is connected to the in-line mixer (131) of the module G, and the in-line mixer is also connected to the eluate storage tank (134) for the resin (C), the in-line of the module G is connected. The resin (C) slurry bonded to the protein in the mixer is mixed with the eluate, and the protein is released from the resin (C). Since the in-line mixer (131) discharge port of the module G and the introduction port of the cyclone (132) of the module G are connected, the protein and the resin (B) are separated in the cyclone. The obtained purified protein becomes a purified protein through a high-level purification step such as virus filtration and a UF / DF membrane from the upper discharge port of the cyclone (132) of the module G via a pipe (120). The resin (C) slurry discharged from the cyclone (132) lower discharge port of the module G is transferred to the resin (C) storage tank via the pipe (135) and reused.

The above aspect is the positional aspect of the present invention, and the present invention is not limited to this, and it is also possible to add a resin conventionally used for protein purification, its elution, a regeneration method, and the like. be.

In the present invention, by adopting the configuration of such a device, it is possible to repeat and reuse the expensive resin.

Example 1
FIG. 7 is a module of the present invention. Connect the three-way joint (202) to the introduction port of the in-line mixer (201, outer diameter 8 mm, inner diameter 5 mm, total length 145 mm, product name static mixer N40-172-0, manufactured by Noritake Company Limited), and mix with the in-line mixer. Two types of fluid are introduced through the other two fittings. The outlet of the in-line mixer is connected to the inlet of the hydrocyclone (204, outer diameter 8 mm, inner diameter 5 mm, total length 190 mm, self-made) via a connection tube (203). A three-way cock (205) that can be connected to two flow paths for discharging the separated slurry and cleaning liquid is connected to the lower part of the cyclone, and a flow rate adjusting device that adjusts the flow rate of the liquid is connected to the discharge port at the upper part of the cyclone. (206, air control slurry, single type, manufactured by Azu Japan Co., Ltd.) is installed.

Example 2
FIG. 8 is a reaction module equipped with a cell separation unit. The cell separation unit HydroCyclone (301, outer diameter 16 mm, inner diameter 13 mm, total length 150 mm, made of polyacrylic acid, self-made) is provided with an introduction port (302) for injecting cell culture medium from the culture tank, and the lower end. Is connected to a three-way joint (303) that can be switched to a outlet for circulating cells in the culture tank again and an outlet for waste liquid. The outlet on the upper part of the hydrocyclone is connected to the pipe (305) for transferring the culture supernatant to the reaction module via the junction (304). The in-line mixer of the reaction module (307, outer diameter 8 mm, inner diameter 5 mm, total length 145 mm, product name static mixer N40-172-0, manufactured by Noritake Company Limited) has a three-way joint (306) connected to its inlet and is inline. Two kinds of fluids, a cell supernatant liquid and a gel slurry, to be mixed by a mixer are introduced through the three-way joint (306). The outlet of the in-line mixer is connected to the inlet of the hydrocyclone (309, outer diameter 8 mm, inner diameter 5 mm, total length 190 mm, self-made) via a connection tube (308). A three-way joint (310) that can be connected to two flow paths for discharging the separated slurry and cleaning liquid is connected to the lower part of the hydrocyclone, and the flow rate adjustment that adjusts the flow rate of the liquid is connected to the discharge port at the upper part of the cyclone. The device (311; air control bus, single type, manufactured by Azu Japan Co., Ltd.) is installed.

Example 3
In this example, it was confirmed that the resin can be concentrated and separated from the resin slurry liquid by hydrocyclone. FIG. 9 is a schematic diagram of the module used at that time. As the resin, silica gel (Osaka Soda Co., Ltd.) having an average particle size of 50 μm was used. A 25% slurry liquid was prepared by mixing 250 mL of the resin and 750 mL of the solvent (pure water). This slurry liquid is injected into the introduction port of the hydrocyclone using a pump (Model RD-05 manufactured by Iwaki Co., Ltd.), and the slurry liquid flowing out from the upper outlet and the lower outlet of the hydrocyclone within a certain period of time is discharged. The degree of separation of the resin was confirmed by recovering and measuring the volumes of the resin and the solvent (pure water) contained therein. The situation of the experiment was videotaped and analyzed after the experiment to determine the time taken for each task. As the hydrocyclone, those having a total length of 75 mm and 100 mm were used, the flow velocity was adjusted by changing the strength of the pump, and the difference in the degree of separation depending on the size of the cyclone and the flow velocity was confirmed. The degree of separation was indexed by the flow rate of the resin and solvent at the lower and upper discharge ports of the hydrocyclone and the ratio of the resin. Table 1A shows the results of the hydrocyclone having a total length of 75 mm, and Table 1B shows the results of the hydrocyclone having a total length of 100 mm.

For example, when flowing through a 100 mm cyclone with a strength of 4, 0.7 L of resin and 0.3 L of solvent (pure water) flow out from the lower outlet in 1 minute (resin ratio: 70%), and the upper outlet 0.04 L of resin and 3.32 L of solvent (pure water) flowed out from the resin (resin ratio: 1.2%). In this way, it was possible to separate the resin, the flow velocity was high, and the larger cyclone (100 mm) had a better degree of separation.

Example 4
In this embodiment, two hydrocyclones are connected, and the slurry liquid flowing out from the lower outlet of the first cyclone upstream and the solvent (pure water) are used as an ejector (model manufactured by Asahi Organic Materials Industry Co., Ltd .: DAEJH18012). ), And it was sent to the second cyclone downstream, where it was confirmed that the resin could be separated. FIG. 10 is a schematic diagram of the module used at that time. As the resin slurry liquid, a 25% slurry liquid of silica gel (Osaka Soda Co., Ltd.) was used as in Example 3. The strength of the pump for supplying the resin slurry to the first cyclone is set to the maximum, and the strength of the pump for supplying the solvent (pure water) to the second cyclone is also set to the maximum, and the liquid is sent for a certain period of time. The slurry liquid flowing out from the upper outlet and the lower outlet of the second cyclone is collected during the period, and the volume of the resin and the solvent (pure water) contained in the slurry is measured to separate the resin. I checked the degree. The situation of the experiment was videotaped and analyzed after the experiment to determine the time taken for each task. Table 2 shows the results of calculating the flow rate of the resin, the flow rate of the solvent, and the ratio of the resin in the first cyclone upper discharge liquid, the second cyclone lower discharge liquid, and the second cyclone upper discharge liquid.

As shown in Table 2, the resin was supplied to the second cyclone, and the resin was separated there. That is, 0.55 L of resin and 0.6 L of solvent (pure water) flowed out from the lower outlet in 1 minute (resin ratio: 47.9%), and 0.02 L of resin and 2 from the upper outlet. .49 L of solvent (pure water) was flowing out (resin ratio: 1.0%). In the connection between the effluent from the first cyclone and the solvent, if a normal three-way joint is used, the resin may not be supplied to the second cyclone downstream, so it is desirable to use an ejector.

Example 5
In this example, it was confirmed that the antibody can be recovered by mixing the antibody sample solution and the chromatographic resin for purification with an in-line mixer and then separating and recovering the resin with a hydrocyclone. FIG. 11 is a schematic diagram of the module used at that time. In this example, an antibody solution (1 mg / mL) diluted with a phosphoric acid buffer was used as the antibody sample solution, and silica gel (average particle size 50 μm, Osaka Soda Co., Ltd.) on which protein A was immobilized was used as a chromatographic resin for antibody purification. .. The antibody sample solution (1 mg / mL: 1 L) and the resin slurry solution (25% resin: 1 L) are merged at a three-way joint, then introduced into an in-line mixer and thoroughly mixed, and then a hydrocyclone for separation is introduced. I tried to flow into my mouth. After each solution began to flow steadily, each of the slurry liquids flowing out from the upper outlet and the lower outlet of the hydrocyclone was recovered for about 16 seconds. Regarding the slurry liquid flowing out from the lower outlet, the resin and the solvent (antibody sample liquid) were quickly separated by filter filtration immediately after the outflow, and only the resin was recovered. The recovered resin was washed twice with 100 mL of phosphate buffer, and then 100 mL of elution buffer (0.1 M glycine hydrochloric acid, pH 2.5) was added to elute the adsorbed antibody. This operation was repeated 7 times, the eluted antibody was collected, and the antibody was quantified by measuring the absorbance at 280 nm. The situation of the experiment was videotaped and analyzed after the experiment to determine the time taken for each task. However, in this embodiment, as an in-line mixer, a hydrocyclone that blocks the upper outlet is installed upside down. In addition, by setting a pinch cock in the upper outlet of the hydrocyclone for separation, the outflow of the solution from the upper outlet was suppressed.

Table 3 shows the flow rate of the slurry solution by the pump before the introduction of the hydrocyclone, the flow rate of the resin at the lower discharge port and the upper discharge port of the cyclone, the flow rate of the solution, and the ratio of the resin.

Table 4 shows the antibody recovery rate, the adsorption amount, the antibody liquid feed amount, and the treatment time.

As a result, as shown in Tables 3 and 4, the resin was separated by the hydrocyclone for separation, and about 66% of the input amount of the antibody was recovered from the resin.

Example 6.
In this example, it was confirmed that the antibody can be purified from the culture supernatant (the supernatant obtained by removing cells from the antibody-expressing cell culture solution) using the module shown in FIG. As the culture supernatant, a culture supernatant having an antibody concentration of 1.16 mg / mL was used, and as a chromatographic resin for purification, the same protein A-immobilized silica gel as in Example 5 was used. The experiment was carried out in the same manner as in Example 5, but the recovered resin was washed 5 times with 200 mL of phosphate buffer, and the elution after washing was carried out 5 times with 200 mL of elution buffer. Table 5 shows the flow rate of the slurry solution by the pump before the introduction of the hydrocyclone, the flow rate of the resin at the lower discharge port and the upper discharge port of the cyclone, the flow rate of the solution, and the ratio of the resin.

Table 6 shows the antibody recovery rate, the adsorption amount, the antibody liquid feed amount, and the treatment time.

As a result, the resin was separated by the hydrocyclone for separation as in Example 5, and about 69.4% of the input amount of the antibody was recovered from the resin (Tables 5 and 6).

Example 7.
In this example, it was confirmed that the antibody can be purified from the culture medium (including antibody-expressing cells) using the module shown in FIG. As the culture solution, a culture solution having an antibody concentration of 3.15 g / L and a cell number of 34 × 10 ⁶ cells / mL was used, and the same protein A-immobilized silica gel as in Example 5 was used as the chromatographic resin for purification. The experiment was carried out in the same manner as in Example 5, but the recovered resin was washed 12 times with 300 mL of phosphate buffer, and the elution after washing was carried out 7 times with 100 mL of elution buffer, and the recovery was quantified. Table 7 shows the flow rate of the slurry solution by the pump before the introduction of the hydrocyclone, the flow rate of the resin at the lower discharge port and the upper discharge port of the cyclone, the flow rate of the solution, and the ratio of the resin.

Table 8 shows the antibody recovery rate, the adsorption amount, the antibody liquid feed amount, and the treatment time.

As a result, the resin was separated by the hydrocyclone for separation as in Example 5, and about 78.4% of the input amount of the antibody was recovered from the resin (Tables 7 and 8).

INDUSTRIAL APPLICABILITY The present invention provides a module suitable for continuous production of a protein, maintenance-free, and having improved resin regeneration efficiency, and is used for a protein, particularly a biopharmaceutical production apparatus.

1. 1. Culture tank, 2. Storage tank, 3. Plumbing, 4. Plumbing, 5. Branch valve, 6. Inline mixer, 7. Hydro cyclone, 8. Piping, 9. Piping, 10. Storage tank, 11. Plumbing, 12. Branch valve, 13. Inline mixer, 14. Hydro cyclone, 15. Plumbing, 16. Plumbing, 17. Storage tank, 18. Plumbing, 19. Branch valve, 20. Inline mixer, 21. Hydro cyclone, 22. Plumbing, 23. Plumbing, 24. Plumbing, 25. Hydro cyclone, 26. Plumbing, 27. Storage tank, 28. Plumbing, 29. Branch valve, 30. Inline mixer, 31. Hydro cyclone, 32. Plumbing, 101. Culture tank, 102. Reservoir, 103 cell separator, 104. Storage tank, 105. Inline mixer, 106. Hydro Cyclone, 107. Storage tank, 108. Inline mixer, 109. Hydro cyclone, 110. Plumbing, 111. Inline mixer, 112. Hydro Cyclone, 113. Inline mixer, 114. Hydro Cyclone, 115. Plumbing, 116. In-line mixer 117. Storage tank, 118. Hydro cyclone, 119. Plumbing, 120. Plumbing, 121. Inline mixer, 122. Hydro cyclone, 123. Storage tank, 124. Storage tank, 125. Plumbing, 131. Inline mixer, 132. Hydro cyclone 133. Storage tank, 134. Storage tank, 135. Plumbing, 201. Inline mixer, 202. Three-way fitting, 203. Connection tube, 204. Hydro Cyclone, 205. Three-way cook, 206. Flow rate regulator, 301. Hydro cyclone, 302. Introductory port, 303. Three-way fitting, 304. Joining member, 305. Pipe, 306. Three-way fitting, 307. Inline mixer, 309. Hydro cyclone, 310. Three-way joint, 311. Flow control device All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

Claims (10)

A method for continuously producing an antibody from a CHO cell culture medium using a plurality of modules in which an in-line mixer and a cyclone are bound.
(a) The in-line mixer of the first module contains the cell culture medium containing the antibody transferred from the culture tank and the resin used for affinity chromatography or ion exchange chromatography transferred from the first storage tank. The slurry is mixed, the antibody in the cell culture solution is bound to the resin used for affinity chromatography or ion exchange chromatography in the slurry, and the resin used for affinity chromatography or ion exchange chromatography bound to the antibody is obtained. The process of producing the containing slurry,
(b) The slurry containing the resin used for affinity chromatography or ion exchange chromatography obtained in the mixing step of the in-line mixer of the first module is transferred to the cyclone , and the affinity bound to the antibody . Separation of resin-containing slurry and cell culture medium used for chromatography or ion exchange chromatography ,
(c) The slurry containing the resin used for affinity chromatography or ion exchange chromatography bound to the antibody obtained in the separation step with the cyclone of the first module is transferred to the second module, and the cell culture solution is transferred. The process of transferring to the first storage tank,
(d) In the in-line mixer of the second module, the slurry containing the resin used for affinity chromatography or ion exchange chromatography bound to the antibody obtained in the above step was transferred from the second storage tank. The process of mixing the eluate to produce an elution slurry, and
(e) The elution slurry obtained in the mixing step of the in-line mixer of the second module is transferred to a cyclone, and contains an antibody and a resin used for affinity chromatography or ion exchange chromatography that is not bound to the antibody . A method comprising the step of separating into a slurry. A slurry containing a resin used for affinity chromatography or ion exchange chromatography bound to an antibody in an in-line mixer of a third module between steps b and c according to claim 1 and a fourth. A method characterized in that a cleaning step is entered by mixing the cleaning liquid transferred from the storage tank of the above, transferring the mixed liquid after cleaning to a cyclone, and separating the mixture . In the step of transferring the cell culture solution containing the antibody from the culture tank to the in-line mixer of the first module, the CHO cells and the cell culture solution containing the antibody are separated via the cyclone, and the separated CHO cells are separated in the culture tank. The method according to claim 1 or 2 , wherein the cell culture medium containing the purified antibody is transferred to the in-line mixer of the first module. The method according to any one of claims 1 to 3 , wherein the resin used for affinity chromatography is a resin bound to protein A gand , and the eluate has a pH of 5 or less . The method according to claim 1 to 4, wherein the cyclone is a Bradley type or Rietema type hydrocyclone. It has a funnel-shaped shape with the discharge port of an in-line mixer, which is a static mixing device that has a shape in which a mixture of two fluids is introduced into one inlet and has no drive unit, and is separated on the upper wall surface of the funnel type. Connect the inlet of the mixture, the upper outlet of the purified substance separated into the upper ceiling of the funnel type, and the lower outlet of the mixture excluding the purified substance at the lower end of the funnel type. In a device in which a plurality of modules having the above-mentioned structures are connected, the cell culture solution and the slurry containing the resin are mixed by an in-line mixer, the antibody in the cell culture solution is bound to the resin in the slurry, and the resin bound to the antibody is obtained. By generating and discharging the contained slurry and introducing the resin into the cyclone, the cell culture solution is separated from the upper discharge port, and the resin slurry bound to the purified antibody is separated from the lower discharge port. The resin slurry bound to the antibody and the eluent are mixed with an in-line mixer to separate and discharge the antibody and the resin, and the slurry in which the resin and the antibody are separated is introduced into the cyclone to produce the antibody purified from the upper discharge port. A device characterized by being composed of two types of modules, an elution module in which resin slurry is separated and discharged from a lower discharge port. The introduction port of the in-line mixer of the reaction module is connected to the culture device, and the cell culture solution discharged from the culture tank is discharged from the upper discharge port and the culture solution is discharged from the lower discharge port and transferred to the culture tank. The apparatus according to claim 6, wherein the apparatus is linked to an upper outlet of a cyclone having a function of separating cells into cells. The introduction port is connected to the lower discharge port of the reaction module, and the slurry and cleaning solution bound to the antibody are separated.
An in-line mixer that mixes and discharges the mixed liquid, and a cleaning module that separates and discharges the waste liquid from the upper discharge port and the resin slurry bound to the antibody washed from the lower discharge port by introducing the resin into the cyclone. The device according to claim 6 or 7, characterized in that it is attached. The apparatus according to any one of claims 6 to 8, wherein a Bradley type or Rietema type hydrocyclone is used. The apparatus according to any one of claims 6 to 9, wherein the in-line mixer is a static mixer having no moving part or a fluidized bed type mixer.

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