JP2023184194A - Protein purification methods using chromatography support - Google Patents

Abstract

To provide a method of protein purification using a chromatography support having both excellent adsorption and separation performance at a high flow rate region and a property hard to cause pore clogging.SOLUTION: Disclosed is at least one chromatography support selected from the group consisting of an anion exchange material, a cation exchange material, chelating material, and hydrophobic material, where these materials respectively comprise an anion exchange group, a cation exchange group, a chelating group and a hydrophobic group introduced to substrates through graft polymer by graft polymerization, where the substrate is monofilament system or multifilament system, and each support is in a form of wind filter cartridge module, thereby smooth protein purification can be achieved due to the high adsorption capacity and a property hard to cause pore clogging, of the support.SELECTED DRAWING: Figure 1

Description

The present invention relates to a method for purifying proteins using a chromatography carrier suitable for purifying proteins such as monoclonal antibodies.

Various therapeutic proteins such as monoclonal antibodies are manufactured from biological materials such as cell culture fluid through multiple chromatographic purification steps. In these chromatography steps, for example, a column filled with an ion exchange chromatography resin based on porous beads is used. Porous beads have a large specific surface area and can achieve a high density of ion-exchange groups, so they can achieve a high adsorption capacity for the therapeutic protein of interest. However, the diffusion rate of the therapeutic protein into the pores of the porous bead is dependent on the adsorption characteristics. As a result, the adsorption capacity decreases significantly in the high flow rate region. Furthermore, a column filled with porous beads cannot be used in a high flow rate region because the beads are compressed and deformed, resulting in an increase in pressure. For these reasons, when therapeutic proteins are commercially produced, large columns with a diameter of more than 1 m and large quantities of chromatography resins of more than 100 L are used. Therefore, the chromatography purification process using porous beads is a process that is expensive and takes a long time.

On the other hand, a chromatography membrane based on a porous membrane has the characteristic of maintaining a high adsorption capacity even in a high flow rate range because the liquid flows directly through its pores. In particular, anion exchange membranes are becoming popular in the production of monoclonal antibodies because they can significantly improve the processing speed of the purification process using a flow-through mode in which impurities are selectively adsorbed onto the anion exchange membrane rather than therapeutic proteins. However, chromatography membranes based on porous membranes also have the disadvantage of being susceptible to membrane clogging.

Patent Document 1 and Non-Patent Document 1 describe a chromatography carrier in which ion exchange groups are introduced into a base material of cellulose fibers, which are natural fibers, by radiation graft polymerization.

Patent Document 2 describes a chromatography medium that uses a specially shaped nylon fiber with a star-shaped cross-section to increase the surface area as a base material, and has an ion exchange group introduced into the base material by graft polymerization. There is.

Patent Document 3 and Patent Document 4 describe a nonwoven fabric filter in which ion exchange groups are introduced into a nonwoven fabric made of nylon, polyethylene, or polypropylene fibers using an emulsion graft polymerization method.

Patent Document 5 describes a hollow fiber porous membrane into which ion exchange groups are introduced using a graft polymerization method.

US Patent Application Publication No. 2010/0065499 International Publication No. 2012/015908 Patent No. 5082038 Patent No. 5013333 International Publication No. 2009/054226

Processes2015, 3, 204-221

According to the technology described in Patent Document 1 and Non-Patent Document 1, by filling a column with a fiber-based carrier, it has better adsorption and separation performance in a high flow rate region than porous beads, and also It is possible to create a column that is less likely to clog even in the presence of However, cellulose fibers generally have high water absorption and swell significantly in buffer solutions, and their wet strength also decreases significantly, making it difficult to stably pack them into columns.

Further, regarding the technology described in Patent Document 2, synthetic fibers such as nylon generally have poor water absorption and do not swell compared to cellulose fibers, and therefore have excellent wet strength. However, such specially shaped fibers are not the best in terms of supply stability and cost as a base material. Furthermore, although nylon fibers with a special shape having a large surface area are employed to obtain high adsorption capacity, the adsorption capacity cannot be said to be sufficiently high compared to a porous bead-based medium.

Furthermore, in the nonwoven fabric filters described in Patent Document 3 and Patent Document 4, the protein adsorption/separation performance of the nonwoven fabric filters has not been verified.

Further, in the hollow fiber porous membrane described in Patent Document 5, although the amount of protein adsorption is large, there is a problem in that the pore diameter narrows as the protein is adsorbed and eventually becomes clogged.

The present invention solves the above problems and efficiently binds proteins such as antibody drugs by using a material that has both adsorption and separation performance in a high flow rate range and pore-resistant performance. The objective is to provide a method by which purification can be carried out.

As a result of extensive studies, the present inventors found that a base material that is a monofilament yarn or a multifilament yarn into which a graft chain having an ion exchange group or a hydrophobic group has been introduced through graft polymerization is used as a core material. We have discovered that by using a wound filter cartridge module, which is a wound chromatography carrier, unnecessary proteins can be removed in flow-through mode and single-use because it has low permeation pressure and is inexpensive.

<Aspect 1>
A method for purifying a target protein in a liquid to be processed containing the target protein and impurities, the method comprising:
The target protein in the liquid to be treated is passed through a chromatography carrier that is one or more selected from the group consisting of an anion exchange material, a cation exchange material, a chelate-forming material, and a hydrophobic material. including a chromatography step for purification;
Each of the anion exchange material, the cation exchange material, the chelate forming material, and the hydrophobic material is grafted with a graft chain having each of an anion exchange group, a cation exchange group, a chelate forming group, and a hydrophobic group. is a material that is introduced into the substrate via polymerization,
The base material is a monofilament yarn or a multifilament yarn,
The method for purifying proteins, wherein the carrier is in the form of a wind filter cartridge module, which is formed by winding the base material into which the graft chains have been introduced around a core material, and optionally removing the core material.
<Aspect 2>
The protein purification method according to aspect 1, wherein the grafting rate of the chromatography carrier is 20% or more and 300% or less.
<Aspect 3>
The protein purification method according to aspect 1 or 2, wherein the wind filter cartridge module has a pore diameter of 0.5 μm or more and 500 μm or less.
<Aspect 4>
The protein purification method according to any one of aspects 1 to 3, wherein in the chromatography step, the liquid to be treated is permeated through the wind filter cartridge module at a permeation pressure of 100 kPa or less.
<Aspect 5>
Any of aspects 1 to 4, further comprising a step of flowing the liquid to be treated in a flow-through mode to a hollow fiber ultrafiltration (UF) membrane module disposed downstream of the chromatography carrier after the chromatography step. The method for purifying the protein according to item 1.
<Aspect 6>
The protein purification method according to any one of aspects 1 to 5, wherein the wind filter cartridge module is single-use.
<Aspect 7>
According to any one of aspects 1 to 6, the method further includes a membrane filtration step in which the liquid to be treated is passed through a filtration membrane having a molecular weight cutoff of 10,000 or more and 800,000 or less, after the chromatography step. Protein purification methods.
<Aspect 8>
The protein purification method according to aspect 7, further comprising a virus removal step of passing the liquid to be treated through a virus removal filter after the membrane filtration step.
<Aspect 9>
The method for purifying a protein according to any one of aspects 1 to 8, wherein the target protein is produced in a tangential flow continuous culture using a hollow fiber membrane module.
<Aspect 10>
The hollow fiber membrane module has the following characteristics:
・Membrane material: polyvinylidene fluoride,
・Pore diameter: 0.05-0.7μm,
・Inner surface aperture ratio: 25-50%,
・Inner diameter: 1 to 5 mm, and ・Inner surface membrane area: 1 m2 ^or more,
The method for purifying a protein according to aspect 9, which is a hollow fiber membrane module having the following.
<Aspect 11>
11. The protein purification method according to aspect 10, wherein the hollow fiber membrane module is single-use.
<Aspect 12>
the chromatography support comprises the anion exchange material;
The method for purifying a protein according to any one of aspects 1 to 11, wherein the anion exchange group contains one or more selected from the group consisting of a tertiary amino group and a quaternary amino group.
Method.
<Aspect 13>
the chromatography support comprises the cation exchange material;
The method for purifying a protein according to any one of aspects 1 to 12, wherein the cation exchange group contains one or more selected from the group consisting of a sulfonic acid group and an acrylic acid group.
<Aspect 14>
the chromatography support comprises the chelate-forming material;
The method for purifying a protein according to any one of aspects 1 to 13, wherein the chelate-forming group contains one or more selected from the group consisting of an iminodiacetic acid group and an iminodiethanol group.
<Aspect 15>
the chromatography carrier comprises the hydrophobic material;
The method for purifying a protein according to any one of aspects 1 to 14, wherein the hydrophobic group contains one or more selected from the group consisting of a phenyl group and a C4 to C18 alkyl group.

According to the present invention, an efficient protein purification method is provided by employing a material that has both adsorption and separation performance in a high flow rate range and performance that prevents pores from clogging.

FIG. 2 is a diagram showing a flow of a protein purification method according to one embodiment of the present invention.

Hereinafter, preferred embodiments (hereinafter referred to as "embodiments") of the present invention will be described in detail. The embodiments shown below are illustrative of devices and methods for embodying the technical idea of this invention, and the technical idea of this invention is not limited to specific combinations of component parts, etc. It's not something you do. The technical idea of this invention can be modified in various ways within the scope of the claims.

In the following description, an upper limit value or a lower limit value in a numerical range described in a step-by-step manner may be replaced with an upper limit value or a lower limit value in a numerical range described in a step-by-step manner. Furthermore, in the following description, the upper limit or lower limit in a certain numerical range may be replaced with the values described in the examples. Furthermore, regarding the term "step" in the following description, not only an independent step but also a step that cannot be clearly distinguished from other steps can be included in the term as long as the function of the "step" is achieved.

The chromatography carrier for protein purification according to the embodiment is one or more selected from the group consisting of an anion exchange material, a cation exchange material, a chelate forming material, and a hydrophobic material. Each of these materials has graft chains having each of anion exchange groups, cation exchange groups, chelating groups, and hydrophobic groups introduced into a substrate that is a monofilament yarn or a multifilament yarn through graft polymerization. It is the material.
In one embodiment, the carrier is prepared by winding the base material into which the graft chain has been introduced around a core material, and optionally removing the core material, i.e., an ion exchange group (IEX) or a hydrophobic group (HIC). ) in the form of a chromatography wind filter cartridge module (WFC). Graft chains with ion exchange groups or hydrophobic groups are introduced into the substrate via graft polymerization.
Thus, in grafted monofilament or multifilament yarns, the graft chains with ion exchange or hydrophobic groups are covalently bonded to the substrate. For example, grafted monofilament or multifilament yarns are produced by contacting a substrate activated by radiation with one or more reactive monomers to form grafted chains on the substrate. Ru. At least one type of reactive monomer has an ion exchange group or an ion exchange group introduction precursor, or a hydrophobic group or a hydrophobic group precursor. Grafted monofilament or multifilament yarns are produced by introducing ion exchange or hydrophobic groups onto the substrate simultaneously with or after the formation of the graft chains.

The IEX-WFC or HIC-WFC according to the embodiment is used to purify a target protein in a liquid to be processed containing the target protein and impurities. The target protein to be purified is, for example, an antibody protein monomer or a protein drug such as insulin or albumin. Impurities include, for example, aggregates of antibody protein dimers or more, other protein impurities, HCP, DNA, endotoxin, and the like. Purification of the protein of interest may be achieved by removing at least a portion of impurities from the liquid to be treated, recovering at least a portion of the protein of interest from the liquid to be treated, or a combination thereof.

Antibody protein, which is an example of a physiologically active substance, is a glycoprotein molecule (also called γ-globulin or immunoglobulin) produced by B lymphocytes as a mechanism for preventing infection in vertebrates, as generally defined in biochemistry. For example, the antibody protein purified by IEX-WFC or HIC-WFC according to the embodiment is used as a human drug and has substantially the same structure as the antibody protein present in the human body to which it is administered.

The antibody protein may be a human antibody protein or an antibody protein derived from a non-human mammal such as a cow or a mouse. Alternatively, the antibody protein may be a chimeric antibody protein with human IgG and a humanized antibody protein. A chimeric antibody protein with human IgG is an antibody protein in which the variable region is derived from a non-human organism such as a mouse, but the other constant regions are substituted with human-derived immunoglobulin. Furthermore, a humanized antibody protein is one in which the complementarity-determining regions (CDRs) of the variable regions are derived from a non-human organism, but the other framework regions (FRs) are derived from humans. is an antibody protein. Humanized antibody proteins have even reduced immunogenicity than chimeric antibody proteins.

The class (isotype) and subclass of the antibody protein, which is an example of the object to be purified by the chromatography carrier according to the embodiment, are not particularly limited. For example, antibody proteins are classified into five classes: IgG, IgA, IgM, IgD, and IgE, depending on the structure of their constant regions. However, the antibody protein to be purified by the ion exchange chromatography carrier according to the embodiment may belong to any of the five classes. Furthermore, in human antibody proteins, IgG has four subclasses, IgG1 to IgG4, and IgA has two subclasses, IgA1 and IgA2. However, the antibody protein to be purified by IEX-WFC or HIC-WFC according to the embodiment may be of any subclass. Note that antibody-related proteins such as Fc fusion proteins binding proteins to the Fc region may also be included in the antibody proteins to be purified by IEX-WFC or HIC-WFC according to the embodiment.

Furthermore, antibody proteins can also be classified by origin. However, the antibody protein to be purified by the chromatography carrier according to the embodiment may be a natural human antibody protein, a recombinant human antibody protein produced by genetic recombination technology, a monoclonal antibody protein, or a polyclonal antibody protein. There may be. Among these antibody proteins, human IgG is preferable as the antibody protein to be purified by IEX-WFC or HIC-WFC according to the embodiment, from the viewpoint of demand and importance as an antibody drug. but not limited to.
When the target protein becomes a protein drug, Pichia yeast or Escherichia coli that has been transformed to mass-produce the target protein (insulin, albumin, etc.) is cultured in an incubator, crushed and/or sterilized, and then the target protein is produced. Insulin and albumin are purified. In this process, a chromatography carrier plays a part in the purification process, and IEX-WFC or HIC-WFC is preferably used at that time.

In one aspect, the base material of the IEX-WFC or HIC-WFC according to the embodiment is made of synthetic fiber. Synthetic fibers may have a regular cylindrical shape. The synthetic fibers are preferably fibers made from polyolefin, polyamide, polyester, or the like, and more preferably polyamide fibers.
Further, the base material may be a monofilament yarn or a multifilament yarn, but a multifilament yarn is preferable because it can secure a surface area. Note that monofilament yarn refers to a structure made of single fibers. Furthermore, multifilament yarn refers to a structure in which a large number of single fibers are twisted together. The base material according to the embodiment is a monofilament yarn or a multifilament yarn before graft chains are introduced. Monofilament yarn or multifilament yarn can be used by being molded into braided cords, woven fabrics, and the like.

Examples of polyolefins include olefin homopolymers such as ethylene, propylene, butylene, and vinylidene fluoride, copolymers of two or more of these olefins, or one or more olefins and perhalogenated olefins. Examples include copolymers of , and the like. Examples of perhalogenated olefins include tetrafluoroethylene and/or chlorotrifluoroethylene. Among these, polyethylene or polypropylene is preferred because it has excellent mechanical strength and a high adsorption capacity for impurities such as proteins.

Examples of polyamides include nylon 6 (polycondensate of ε-caprolactam), nylon 11 (polycondensate of undecane lactam), nylon 12 (polycondensate of lauryllactam), and nylon 66 (polycondensate of hexamethylene diamine and adipic acid). Nylon 610 (co-condensed polymer of hexamethylene diamine and adipic acid), Nylon 6T (co-condensed polymer of hexamethylene diamine and terephthalic acid), Nylon 9T (co-condensed polymer of nonanediamine and terephthalic acid) ), nylon M5T (cocondensation polymer of methylpentanediamine and terephthalic acid), nylon 621 (cocondensation polymer of caprolactam and lauryllactam), cocondensation polymer of p-phenylenediamine and terephthalic acid, and m-phenylenediamine Examples include cocondensation polymers of isophthalic acid and isophthalic acid.

Examples of polyesters include polyethylene terephthalate, polytrimethylene terephthalate, polybutylene terephthalate, polyethylene naphthalate, and polybutylene naphthalate.

The graft chains covalently bonded to the substrate are composed of monomer units. In one embodiment, the monomer unit has an ion exchange group or a hydrophobic group. The ion exchange group is selected from a cation exchange group (CEX), an anion exchange group (AEX), or a chelate forming group (CHE) depending on the object to be purified and the purification conditions.

The cation exchange group may be a strong cation exchange group or a weak cation exchange group as long as impurities can be removed.

Examples of strong cation exchange groups include sulfonic acid groups. Almost all strong cation exchange groups are charged in the pH range of a practical antibody solution when purifying antibody proteins, so the amount of charge is constant. Therefore, the presence of a strong cation exchange group on the base material always guarantees a certain amount of charge or more. Further, it is possible to prevent the performance of the CEX material from being greatly influenced by slight changes in pH.

Examples of weak cation exchange groups include carboxylic acid groups (eg, acrylic acid groups), phosphonic acid groups, and phosphoric acid groups. The amount of charge of the weak cation exchange group can be changed by changing the pH of the mobile phase. Therefore, by changing the pH of the mobile phase, it is possible to adjust the charge density of the cation exchange chromatography carrier. Therefore, any impurity can be removed by adjusting the pH according to the characteristics of the impurity to be removed.

From the viewpoint of ease of production, the cation exchange group is preferably a sulfonic acid group or an acrylic acid group, and more preferably a sulfonic acid group.

The anion exchange group only needs to be able to adsorb negatively charged proteins etc. in the liquid. For example, although not particularly limited, examples of the anion exchange group include diethylamino group (DEA, Et ₂ ) which is a tertiary amino group. N-), triethylene diamino group (TEDA, Et ₃ N ₂ -), quaternary ammonium group (Q, R ₃ N+-), quaternary aminoethyl group (QAE, R ₃ N+-(CH ₂ ) ₂ -), diethylaminoethyl group (DEAE, Et ₂ N-(CH ₂ ) ₂ -), and diethylaminopropyl group (DEAP, Et ₂ N-(CH ₂ ) ₃ -). Here, R is not particularly limited, and R's bonded to the same N may be the same or different, and preferably represent a hydrocarbon group such as an alkyl group, a phenyl group, or an aralkyl group. Examples of the quaternary ammonium group include trimethylamino group (trimethylammonium group, Me ₃ N+-) and the like. Note that the tertiary amino group and the quaternary amino group are also referred to as a dialkyl-substituted amino group and a trialkyl-substituted amino group, respectively. From the viewpoint of easy chemical fixation to the substrate and high adsorption capacity, the anion exchange group is preferably DEA, TEDA, and trimethylamino group, and DEA is more preferable.

The chelate-forming group only needs to be capable of adsorbing metals, and for example, it can also adsorb proteins containing metals. Since the chelate-forming group forms a complex with a specific metal and adsorbs it, it becomes possible to adsorb a specific metal (eg, copper, iron, nickel, etc.) or a protein containing a specific metal.
Examples of the chelate-forming group include groups having two or more oxygens and nitrogen and/or sulfur in the molecule, with iminodiacetic acid groups and iminodiethanol groups being more preferred.

When the chromatography carrier contains an anion exchange material, the anion exchange group preferably contains one or more selected from the group consisting of a tertiary amino group and a quaternary amino group, and the anion exchange group preferably contains a diethylamino group and a trimethyl More preferably, it contains one or more selected from the group consisting of an amino group and a triethylenediamino group.
When the chromatography carrier contains a cation exchange material, it is preferred that the cation exchange group contains one or more selected from the group consisting of sulfonic acid groups and acrylic acid groups.
When the chromatography carrier contains a chelate-forming material, it is preferred that the chelate-forming group contains one or more selected from the group consisting of iminodiacetic acid groups and iminodiethanol groups.

Examples of hydrophobic groups (HIC) include C4 to C18 aliphatic hydrocarbon groups (preferably alkyl groups) and aromatic hydrocarbon groups (preferably phenyl groups), and the chromatography carrier is a hydrophobic material. When included, it is preferred that the hydrophobic group contains one or more selected from the group consisting of a phenyl group and a C4 to C18 alkyl group.

The chromatography carrier according to the embodiment includes one or more selected from the group consisting of an anion exchange material, a cation exchange material, a chelate forming material, and a hydrophobic material. In one preferred embodiment, the chromatographic support comprises an anion exchange material, a cation exchange material and a chelate forming material. In another preferred embodiment, the chromatography support comprises a hydrophobic material.
Each of the anion exchange material, cation exchange material, chelate-forming material, and hydrophobic material according to the embodiment has an anion exchange group, a cation exchange group, and a chelate-forming group on a base material that is a monofilament yarn or a multifilament yarn. This is a material in which graft chains having each of a group and a hydrophobic group are introduced through graft polymerization.
The ion exchange group (cation exchange group, anion exchange group, or chelate forming group) or hydrophobic group used in the protein purification method according to the embodiment may be used singly or in combination. . Moreover, the ion exchange group or the hydrophobic group may be used singly or in combination.
Generally, anion exchange groups are preferable for removing impurities such as protein contaminants in the solution to be processed, while hydrophobic groups are preferable for selectively removing multimers such as dimers and trimers of the target protein. is preferred. Further, when the ionic strength of the liquid to be treated is high, a cation exchange group that does not reduce the adsorption capacity is preferable.
Furthermore, chelate-forming groups are preferred for selectively removing specific metals or proteins containing specific metals.
In a typical embodiment, when the liquid to be processed is passed through a chromatography carrier, impurities are removed using an anion exchange group, then multimers of the target protein are selectively removed using hydrophobic groups, and then cation exchange is performed. Although it is preferable to selectively recover the target protein using a group, depending on the embodiment, an ion exchange group (a cation exchange group, an anion exchange group, or a chelate forming group) or It is also possible to change the order of the hydrophobic groups.

In a typical embodiment, upon introducing the graft chain into the substrate, the substrate is activated by radiation. Although any means can be used to generate radicals on the base material, irradiating the base material with ionizing radiation is preferable because uniform radicals are generated throughout the base material. As types of ionizing radiation, gamma rays, electron beams, beta rays, neutron beams, etc. can be used, but electron beams or gamma rays are preferable for implementation on an industrial scale. Ionizing radiation can be obtained from radioactive isotopes such as cobalt-60, strontium-90, and cesium-137, or by an X-ray imaging device, an electron beam accelerator, an ultraviolet irradiation device, and the like.

The irradiation dose of ionizing radiation is preferably 1 kGy or more and 1000 kGy or less, more preferably 2 kGy or more and 200 kGy or less, and still more preferably 5 kGy or more and 50 kGy or less. When the irradiation dose is 1 kGy or more, radicals tend to be generated uniformly. Further, from the viewpoint of the physical strength of the base material, the irradiation dose is preferably 1000 kGy or less.

Graft polymerization methods using ionizing radiation irradiation generally include a pre-irradiation method in which radicals are generated on a base material and then brought into contact with a reactive monomer; It is broadly divided into simultaneous irradiation method that generates radicals. In the embodiment, any method can be applied, but a pre-irradiation method that produces less oligomers is preferred. Oligomer here refers to free oligomer. Free oligomers are not incorporated into the grafted chains and are therefore preferably not produced.

The reactive monomer to be graft-polymerized onto the base material may be a reactive monomer having an ion exchange group or a reactive monomer having an "IEX or HIC introduction precursor". If a reactive monomer with an "IEX or HIC-introducing precursor" is used, the IEX- or HIC-introducing precursor is converted into IEX or HIC after the reactive monomer is copolymerized.

The term "IEX or HIC-introducing precursor" as used herein refers to a functional group capable of imparting IEX or HIC, and includes, for example, an epoxy group, but is not limited thereto. A reactive monomer having a "precursor for introducing IEX or HIC" refers to a reactive monomer having a functional group capable of imparting IEX or HIC. Furthermore, the "IEX or HIC-introducing precursor" may include the "precursor of IEX or HIC." The "precursor of IEX or HIC" is, for example, an ion exchange group with a protecting group attached. Examples of the reactive monomer having a "precursor of IEX or HIC" include, but are not limited to, phenyl vinyl sulfonate.

Examples of reactive monomers having a strong cationic group include (meth)acrylamidoalkylsulfonic acid, vinylsulfonic acid, acrylamide t-butylsulfonic acid, styrenesulfonic acid, which are structural units of polymers having a sulfonic acid group, and their Examples include metal salts.

Examples of reactive monomers having weak cationic groups include acrylic acid, 2-acryloyloxyethylsuccinic acid, 2-acryloyloxyethylhexahydrophthalic acid, 2-acryloyloxyethyl phthalic acid, methacrylic acid, 2-methacrylic acid, Roxyethylsuccinic acid, 2-methacryloyloxyethylhexahydrophthalic acid, 2-methacryloyloxyethyl phthalic acid, 2-vinylbenzoic acid, 3-vinylbenzoic acid, 4-vinylbenzoic acid, and metal salts thereof, etc. Can be mentioned.

Examples of reactive monomers having anionic groups include 2-(dimethylamino)ethyl acrylate, 2-(diethylamino)ethyl acrylate, 2-(dimethylamino)ethyl acrylate, and 2-(diethylamino)ethyl acrylate, and vinylbenzyl Examples include trimethylammonium chloride.

Examples of the reactive monomer having an ion exchange group (IEX)-introducing precursor include glycidyl methacrylate, glycidyl ether, styrene, chloromethylstyrene, acrylonitrile, acrolein, and vinylbenzyl.

When the reactive monomer having an ion exchange group-introducing precursor is glycidyl methacrylate, some of the epoxy groups of glycidyl methacrylate are polymerized on the substrate to form a graft chain, and then reacted with sodium sulfite. Or all can be converted to sulfonic acid groups.
Furthermore, by polymerizing glycidyl methacrylate on a substrate to form a graft chain, and then reacting with diethylamine, part or all of the epoxy groups of glycidyl methacrylate can be converted to diethylamino groups.
Furthermore, after polymerizing glycidyl methacrylate on a substrate to form a graft chain, it is possible to convert some or all of the epoxy groups of glycidyl methacrylate into iminodiacetic acid groups by reacting with disodium iminodiacetate. can.

Examples of the reactive monomer having a hydrophobic group (HIC)-introducing precursor include glycidyl methacrylate, glycidyl ether, and styrene.

The binding rate (grafting rate) of graft chains of the chromatography carrier according to the embodiment is preferably 20% or more from the viewpoint of adsorption capacity, etc., more preferably 25% or more, and still more preferably 30% or more. In addition, from the viewpoint of ensuring mechanically stable strength, it is preferably 300% or less, more preferably 200% or less, still more preferably 150% or less, particularly preferably 120% or less. However, since a suitable grafting ratio depends on the base material used and the monomer unit introduced, it is desirable to perform optimization as appropriate, and it is not limited to the above range. The grafting rate is calculated by the following formula (1).

Grafting rate: dg (%) = (w1-w0)/w0×100...Formula (1)
Here, w0 is the mass of the base material before the graft chain is introduced, and w1 is the mass of the base material after the graft chain is introduced, that is, the chromatography carrier.

Usually, it is thought that the reactive monomer enters the amorphous region inside the base material and undergoes graft polymerization, and a graft chain is also formed inside the base material. However, the molecules to be adsorbed by the graft chains are not large enough to fit into the amorphous region inside the base material. Therefore, the graft chains formed inside the base material cannot be used to adsorb molecules to be adsorbed. When the irradiation dose of radiation that generates initiation points for graft polymerization is high, there are many initiation points, and when it is low, there are few initiation points. Therefore, at the same grafting rate, the lower the irradiation dose, the fewer the number of starting points and the formation of longer graft chains. Therefore, by lowering the irradiation dose, many graft chains are formed on the base material, and more graft chains can be effectively used for adsorption of molecules to be adsorbed. The irradiation dose at this time is preferably 10 to 50 kGy.

The graft chains formed inside the base material are called internal graft chains or polymer roots. The grafted chains formed on the substrate are called surface grafted chains or polymer brushes. In the chromatography carrier according to the embodiment, the proportion of surface graft chains to the total graft chains is, for example, 70% or more, preferably 80% or more, more preferably 85% or more, and still more preferably 90% or more. The proportion of surface graft chains can be determined by creating a graph showing the relationship between the molar conversion rate of an ion-exchange group-introduced precursor such as an epoxy group to an ion-exchange group and the saturated adsorption amount of the target molecule to be adsorbed by the ion-exchange group. This corresponds to the molar conversion at the inflection point that appears. Preferably, in the chromatography carrier according to the embodiment, substantially no graft chains are formed inside the base material.

IEX for protein purification can be obtained by winding a base material, which is a monofilament yarn or multifilament yarn according to an embodiment, into which a graft chain having IEX or HIC is introduced via graft polymerization, around a core material. Alternatively, a HIC wind filter cartridge module (IEX-WFC, HIC-WFC) can be produced. The core material (eg, cylindrical core material) may be porous. Further, after the filament yarn is wound around the cylindrical core material, the cylindrical core material may be pulled out.
The chromatography carrier according to the embodiment has the form of a wind filter cartridge module formed by winding a base material into which graft chains have been introduced around a core material, and optionally removing the core material.

A wind filter cartridge module (WFC) can be suitably used for single use. In other words, IEX-WFC or HIC-WFC can be suitably used for single use.

According to the IEX-WFC or HIC-WFC according to the embodiment, it is possible to achieve both protein adsorption and separation performance in a high flow rate region and performance that prevents pores from clogging. Further, the chromatography carrier according to the embodiment has a high protein adsorption capacity and has excellent wet strength.

Usually, a core material with a diameter of about 30 mm is often used. The thickness of the wound body is set to about 20 mm to 100 mm. The thicker the layer, the more impurities can be adsorbed without escaping, but if the layer is too thick, the permeation pressure will increase when the liquid is permeated. If the thickness is 20 mm or more, the minimum adsorption performance can be ensured, and if the thickness is 100 mm or less, the permeation pressure will not increase so much.

Further, the diameter of captured particles of WFC is usually measured by passing a liquid containing homogeneous particles of silica or latex and determining whether or not they are detected on the permeate side. The particle size that captures 90% or more of the homogeneous particles is defined as the pore size of the WFC.
The pore diameter of the WFC can be controlled by the diameter of the monofilament yarn or multifilament yarn; the thinner the yarn is wound, the smaller the pore diameter is, and the thicker the yarn is, the larger the pore diameter is. In this embodiment, the pore diameter of the WFC is preferably 0.5 μm or more and 500 μm or less, more preferably 1 μm or more and 150 μm or less. If the pore size is small, the permeation pressure will be high, and if the pore size is too large, the dynamic adsorption performance will be poor. If the pore diameter is 0.5 μm or more, the permeation pressure is within the permissible range, and if the pore diameter is 500 μm or less, it does not significantly affect the dynamic adsorption performance.

In the chromatography step, the permeation pressure when the liquid to be treated is permeated through the WFC is preferably 100 kPa or less, more preferably 50 kPa or less. More preferably, it is 35 kPa or less.

The WFC according to the embodiment can be used in a method for purifying a protein of interest. The purification method according to the embodiment includes a chromatography step for purifying the target protein in a liquid to be processed containing the target protein and impurities, and optionally includes a membrane filtration step and/or a virus removal step. The membrane filtration step and the virus removal step are each preferably performed after the chromatography step. When performing both the membrane filtration step and the virus removal step, the preferred order is the membrane filtration step, followed by the virus removal step.
An exemplary flow of a protein purification method will be described below with reference to FIG. Although FIG. 1 illustrates the case where the liquid to be treated is obtained by continuous cell culture, the type and preparation method of the liquid to be treated can be selected as appropriate depending on the purpose.

[Process of preparing the liquid to be treated]
First, cells that produce the target protein are continuously cultured by extracting the culture solution from the culture tank while supplying a medium to the culture tank (FIG. 1(A)). Note that continuous culture is a method in which a culture medium is continuously supplied to a culture tank and the same amount of culture solution is simultaneously withdrawn. At this time, the cells are returned to the culture tank from the extracted culture solution, and the cells in the culture solution in the tank are cultured in a steady state. The culture solution obtained by continuous culture is passed through a separation membrane to separate and remove cells from the culture solution to obtain a solution to be treated containing the target protein and impurities (FIG. 1(B)).

[Chromatography process]
Next, the target protein is purified by passing the liquid to be treated through a chromatography carrier (chromatography step). Purification may be the separation and removal of impurities from the liquid to be processed and/or the separation and recovery of the target protein from the liquid to be processed. In FIG. 1, the liquid to be treated is passed through an anion exchange material (FIG. 1(C)), then a hydrophobic material (FIG. 1(D)), and then a cation exchange material (FIG. 1(C)). E)) Although an example of the treatment is shown, one or more chromatography carriers may be used. Alternatively, the liquid to be treated may be passed through a chelate forming material. That is, the treatment in the chromatography step may be carried out using a single chromatography carrier, or may be carried out using two or more chromatography carriers. Note that the order in which the liquid to be treated passes is not particularly limited and can be changed depending on the embodiment.

[Membrane filtration process and virus removal process]
The liquid to be treated obtained in the chromatography step is subjected to a membrane filtration step (FIG. 1 (F)) and/or a virus removal step (FIG. 1 (G)) as desired to further purify the target protein. It's fine.

The target protein purified by the above procedure is recovered (FIG. 1(H)).

The above membrane filtration step and virus removal step can be performed for the purpose of further removal of impurities and/or desalting when there are impurities that cannot be removed by the chromatography step. The membrane filtration step may be provided after the chromatography step, and can be performed using, for example, a hollow fiber ultrafiltration (UF) membrane module. If there are impurities with a higher molecular weight than the target protein, by installing a UF membrane module with a molecular weight cut-off slightly larger than the target protein, the target protein will pass through the membrane, but the impurities will not be able to pass through the membrane. On the other hand, in the case of an impurity with a molecular weight lower than that of the target protein, a molecular weight cutoff is selected that allows the impurity to pass through and blocks the target protein. When the target protein is an antibody, its molecular weight is around 150,000, so the molecular weight cutoff of the UF membrane is preferably selected from the range of 10,000 to 800,000. The membrane filtration step is a step subsequent to the chromatography step in which the liquid to be treated is passed through a filtration (UF) membrane having a molecular weight cutoff of 10,000 or more and 800,000 or less.

Moreover, the above-mentioned virus removal step is a step of passing the liquid to be treated through a virus removal filter, and is preferably performed after the membrane filtration step. A hollow fiber ultrafiltration (UF) membrane module can be used as the virus removal filter. When a liquid to be treated is passed through a hollow fiber ultrafiltration (UF) membrane module, target proteins pass through the membrane, but viruses cannot pass through the membrane, so viruses can be efficiently removed.

After the chromatography step, if the IEX-WFC and HIC-WFC include a step in which the liquid to be treated flows down in a flow-through mode, a UF membrane module (in one embodiment, the membrane filtration step and /or modules used in the virus removal process), it becomes possible to smoothly perform the filtration operation without placing a tank or the like in between. Furthermore, according to the embodiment, since the permeation pressure of the WFC is low, the IEX-WFC and the HIC-WFC can be connected in series, and the UF membrane module can also be connected as is.

The material of the UF membrane used in the membrane filtration step and the virus removal step is preferably a hydrophilic material so that non-specific adsorption of the target protein to the membrane surface does not occur. Examples of the hydrophilic material include PAN (polyacrylonitrile) and CTA (cellulose triacetate).

When the liquid to be treated is obtained by cell culture, the cell culture is preferably continuous culture. As mentioned above, continuous culture means that a medium is continuously supplied to a culture tank and the same amount of culture solution is simultaneously withdrawn. In this method, cells are returned to the culture tank from the extracted culture solution, and the cells in the culture solution in the tank are cultured in a steady state. It is preferable to employ continuous culture because the process from cell culture to purification of the target protein can be carried out consistently and continuously. In particular, the target protein is preferably produced by tangential flow continuous culture using a hollow fiber membrane module, for example, a microfiltration (MF) membrane as described below.

The hollow fiber membrane module is preferably single-use.

According to the embodiment, since the permeation pressure of IEX-WFC and HIC-WFC in the chromatography process is low, IEX-WFC and HIC-WFC are directly connected to the filtrate outlet of continuous culture to perform chromatography from continuous culture. The entire process can be operated in flow-through mode.

A separation membrane is usually used when extracting the culture solution from the culture tank. In one embodiment, the separation membrane is composed of a hollow fiber membrane module. A separation membrane (microfiltration (MF) membrane) with a pore diameter of about 0.05 to 0.7 μm is used to block cells and allow only the culture solution (liquid to be treated) to pass through. At this time, cross-flow filtration is often used to prevent the separation membrane from clogging, but even then, it is necessary to prevent clogging and non-specific adsorption of the target protein to the separation membrane may occur.

From the viewpoint of avoiding this clogging problem, the separation membrane preferably has a three-dimensional network structure, and the membrane material is preferably polyvinylidene fluoride (PVDF), polyethylene (PE), or the like. Further, the inner surface aperture ratio is preferably 25% or more, and preferably 50% or less. By employing such a membrane, cross-flow filtration can be performed efficiently and non-specific adsorption to the separation membrane can be suppressed.
Further, a separation membrane with an inner diameter of 1 to 5 mm can be suitably used because the permeation pressure of the crossflow flow is low. In addition, from the viewpoint of production efficiency, the inner surface membrane area is preferably 1 m ² or more.

The embodiments will be described in more detail below based on examples, but these are not intended to limit the embodiments in any way.
(a) A carrier (wind filter cartridge module) for protein purification was produced by the method shown below.
(1) Radiation graft polymerization of glycidyl methacrylate (GMA) A nylon fiber with a diameter of approximately 35 μm was irradiated with 20 kGy of gamma rays. Next, it was immersed in a monomer solution of glycidyl methacrylate/methanol (=1/9 weight ratio) that had been previously deoxidized by nitrogen bubbling, and reacted at 45° C. for 6 hours. After the reaction, the fibers were immersed in a dimethylformamide solution and then in methanol for washing. By measuring the weight after drying, the mass increase rate (grafting rate) was 104%.

(2) Radiation graft polymerization of vinylbenzyltrimethylammonium chloride (VBTAC) (VBTAC/NVP-AEX)
A nylon fiber with a diameter of about 14 μm was irradiated with 20 kGy of gamma rays. Next, it was immersed in a monomer solution of VBTAC/N-vinylpyrrolidone/pure water (=1/1/8 weight ratio) that had been previously deoxidized by nitrogen bubbling, and reacted at 45° C. for 6 hours. After the reaction, the fibers were immersed in a dimethylformamide solution and then in methanol for washing. By measuring the weight after drying, the mass increase rate (grafting rate) was 40%.
Calculating from the mass increase rate, the amount of trimethylamino group introduced was 0.8 mmol/g.

(3) Introduction of triethylene diamino (TEDA) group into GMA grafted fiber (TEDA-AEX)
The GMA grafted fiber obtained in (1) was immersed in a 10% by mass TEDA aqueous solution adjusted to pH 9 with hydrochloric acid, and heated at 70° C. for 6 hours to perform a TEDA introduction reaction. After washing the fibers after the reaction with methanol, the dry mass was measured and the mass increase rate was 40.1%. The amount of TEDA introduced was calculated from the mass increase rate to be 1.9 mmol/g.

(4) Introduction of diethylamino (DEA) groups into GMA grafted fibers (DEA-AEX)
The GMA grafted fiber obtained in (1) was immersed in a 50v/v% diethylamine aqueous solution at 40°C for 15 hours to introduce diethylamino groups as anion exchange groups into the epoxy groups derived from glycidyl methacrylate, and then thoroughly washed with methanol. did. The amount of DEA introduced was calculated from the mass increase rate to be 1.6 mmol/g.

(5) Introduction of sulfonic acid groups into GMA grafted fibers (CEX)
The GMA grafted fiber obtained in (1) was immersed in a taurine solution (concentration: 50% by mass, solvent: dioxane/water = 1/1, pH: 13) at 40°C for 15 hours to remove the epoxy groups derived from glycidyl methacrylate. After introducing a sulfonic acid group as a cation exchange group, the mixture was thoroughly washed with methanol. The amount of sulfonic acid group introduced was calculated from the mass increase rate to be 1.6 mmol/g.

(6) Introduction of chelate-forming groups into GMA grafted fibers (CHE)
The GMA grafted fiber obtained in (1) was immersed in a disodium iminodiacetate solution (concentration: 0.5M, solvent: isopropyl alcohol/water = 1/1) at 50°C for 15 hours, and the epoxy derived from glycidyl methacrylate was After introducing an iminodiacetic acid group as a chelate-forming group, the mixture was thoroughly washed with methanol. The amount of CHE introduced was calculated from the mass increase rate to be 1.5 mmol/g.

(7) Introduction of hydrophobic groups to GMA grafted fibers (HIC)
The GMA grafted fiber obtained in (1) was immersed in a 10% by mass dodecylamine-isopropanol solution at 70°C for 6 hours, and after introducing a dodecyl group as a hydrophobic group into the epoxy group derived from glycidyl methacrylate, methanol was sufficient. Washed. The amount of dodecyl group (hydrophobic group) introduced was calculated from the mass increase rate to be 1.5 mmol/g.

(8) Preparation of wind filter cartridge module (WFC) The graft fibers (carriers) containing each functional group obtained in (2) to (7) were placed on a polypropylene core material with a diameter of 30 mm and a length of 250 mm. After winding to a thickness of 20 mm, the core material was removed to produce wind filter cartridge modules in which VBTAC/NVP-AEX, TEDA-AEX, DEA-AEX, CEX, CHE, and HIC were introduced.

(9) Measuring the pore size of the prepared WFC An aqueous solution containing 0.1% of homogeneous particles of uniform latex: PM010UM (particle size: 10 μm, Magsphere) is permeated through the prepared WFC, and whether or not it is detected on the permeate side is determined. When we checked, we found that it was 100% effective. On the other hand, when homogeneous particles of PM005UM (particle size: 5 μm, manufactured by Magsphere) were similarly transmitted, the rejection rate was 10%. From this result, the pore diameter of the produced WFC was determined to be 10 μm.

[Example 1]
A buffer solution containing a metal salt was prepared by adding NaCl to 20 mM Tris-HCl (pH 8.0) to a concentration of 0.17 M. BSA (pI: 5.6) was dissolved in this buffer at a concentration of 1 g/L to prepare a protein solution. 20 L of this protein solution was passed from the center to the outside of the WFC into which VBTAC/NVP-AEX, TEDA-AEX, DEA-AEX and HIC were introduced at a rate of 1 mL/min/mL-Bed to absorb protein. We conducted an experiment. The adsorption capacities of VBTAC/NVP-AEX, TEDA-AEX, DEA-AEX, and HIC were 68, 73, 80, and 65 mg/mL-Bed, respectively. In addition, the permeation pressures when 20 L of protein solution was completely flowed were 29, 30, 32, and 30 kPa, respectively, indicating that operation can be performed at low pressure despite the high adsorption capacity, making it suitable for use in flow-through mode. I found out that

[Comparative example 1]
The protein solution prepared in Example 1 was passed through 150 mL of Qyu Speed D (manufactured by Asahi Kasei Medical Co., Ltd., hollow fiber membrane, tertiary amino group) at a rate of 1 mL/min/mL-Bed from the inner surface to the outer surface of the membrane. Then, a protein adsorption experiment was conducted. The adsorption capacity was 70 mg/mL, and the permeation pressure when 20 L of protein solution was completely drained was 150 kPa, which increased with adsorption.

[Example 2]
A protein solution was prepared by dissolving lysozyme (pI: 11.35) at a concentration of 1 g/L in 150 mM phosphate (pH 6.0) buffer. A protein adsorption experiment was conducted by passing 20 L of this protein solution outward from the center of the WFC into which CEX had been introduced at a rate of 1 mL/min/mL-Bed. Even under a high ionic strength of 150 mM, the adsorption capacity was 80 mg/mL-Bed. In addition, the permeation pressure when 20 L of protein solution was completely flowed was 35 kPa, indicating that it can be operated at low pressure despite the high adsorption capacity, and is suitable for use in flow-through mode.
[Example 3]
A 10 mM nickel sulfate solution (10 mM acetate buffer, pH: 5.0) was permeated from the center of the WFC into which CHE was introduced to the outside, and Ni ions were fixed to the WFC by forming a chelate with CHE.
In order to express the histidine-tagged green fluorescent protein in E. coli, E. coli DH10B transformed with pUC19-GFPuv/His into which the His-tag fused GFP gene had been introduced was cultured in LB medium at 37° C. for 12 hours. Bacteria were collected by centrifugation, frozen and thawed three times using liquid nitrogen, lysed, and the centrifuged supernatant was passed through the Ni ion-immobilized CHE-WFC at a rate of 1 mL/min/mL-Bed. , conducted protein adsorption experiments. After that, it was eluted with a 0.5M NaCl solution (10mM acetate buffer, pH: 5.0), and it was confirmed by SDS-PAGE that only the histidine-tagged green fluorescent protein was eluted. The permeation pressure was 35 kPa, and it was found that the system could be operated at low pressure.

(b) A microfiltration (MF) hollow fiber membrane mini module for continuous culture was produced by the method shown below.

40% by mass of PVDF resin (manufactured by Kureha Co., Ltd., KF-W#1000) as a thermoplastic resin, 23% by mass of fine powder silica (primary particle size: 16 nm), and 32% by mass of bis-2-ethylhexyl adipate (DOA) as a non-solvent. A melt-kneaded product was prepared using 9% by mass and 4.1% by mass of acetyl tributyl citrate (ATBC, boiling point 343°C) as a poor solvent. The temperature of the obtained melt-kneaded product was 240°C. The obtained melt-kneaded product was passed through a hollow fiber-like extrudate for an empty running distance of 120 mm using a spinning nozzle with a double-tube structure, and then solidified in water at 30°C, and the porous structure was created using a thermally induced phase separation method. developed. The obtained hollow fiber extrudate was taken off at a speed of 5 m/min and wound into a skein. The wound hollow fiber extrudate was immersed in isopropyl alcohol to extract and remove DOA and ATBC, then immersed in water for 30 minutes to replace the hollow fiber membrane with water, and then soaked in 20% by mass NaOH aqueous solution for 70 minutes. The membrane was immersed for 1 hour at 0.degree. C., and washed with water repeatedly to extract and remove the fine silica powder, thereby producing a porous hollow fiber membrane.

The obtained porous hollow fiber membrane was placed in a module case with an effective length of 500 mm so that the membrane area was 1.9 ^m2 , and both ends were potted with epoxy resin, and a microfiltration (MF) hollow fiber for continuous culture with openings at both ends was placed. A thread membrane mini module was created.

(c) A hollow fiber ultrafiltration (UF) membrane module was produced by the method shown below.

A copolymer polymerized with 91.5% by mass of acrylonitrile, 8.0% by mass of methyl acrylate, and 0.5% by mass of sodium methallylsulfonate as monomers (intrinsic viscosity: 1.2), 18% by mass, and a weight average molecular weight of 600. 21.0% by mass of polyethylene glycol (manufactured by Wako Pure Chemical Industries, Ltd., PEG600) was dissolved in a mixed solvent of 9.15% by mass of propylene carbonate and 51.85% by mass of dimethyl sulfoxide to form a homogeneous solution. The amount of water in the solution was measured with a Karl Fischer moisture meter and was 600 ppm or less. This solution was kept at 60°C, and was mixed with an internal liquid consisting of a mixed solution of 50% by mass of tetraethylene glycol and 50% by mass of water (viscosity of 24cP at 20°C) through a spinneret (double annular nozzle 0.5mm-0.5mm). 7 mm - 1.3 mm), passed through a 20 mm air gap, and passed through a coagulation bath with a total length of 5 m made of 43° C. water to obtain a porous hollow fiber filtration membrane.
At this time, the area from the spinneret to the coagulation bath was surrounded by a cylindrical tube, and the relative humidity in the air gap inside the tube was controlled to 100%. Further, the spinning speed was fixed at 10 m/min. The obtained hollow fiber filtration membrane was soaked in pure water at 25° C. for one day to sufficiently remove the residual solvent in the membrane. The remaining amounts of polyethylene glycol, propylene carbonate, and dimethyl sulfoxide in the wet film were 1 ppm or less.
Further, the obtained hollow fiber filtration membrane was immersed in pure water at 20°C, heated at a rate of temperature increase of 15°C/hour, and immersed in water with a final temperature of 55°C for 2 hours.

The porous hollow fiber filtration membrane obtained so that the membrane area is 1.9 ^m2 is placed in a module case with an effective length of 20 cm, and both ends are potted with epoxy resin to create a hollow fiber type ultrafiltration (UF) with openings at both ends. A membrane mini module was created.

[Example 4]
A CHO serum-free cell culture solution containing 0.5 g/L of γ-globulin as the target protein was dead-end filtered using the microfiltration (MF) hollow fiber membrane module prepared in (b), and the turbid solution was removed. Obtained. The solution is passed in this order through the WFC in which the DEA-AEX and HIC prepared in (a) have been introduced, and then flowed through the hollow fiber ultrafiltration (UF) membrane module prepared in (c). γ-globulin was continuously purified as the target protein by filtration in the following mode. At this time, since the permeation pressure of the WFC into which DEA-AEX and HIC had been introduced was as low as 35 kPa, it was possible to feed it directly to the hollow fiber ultrafiltration membrane.
The concentrations of HCP and DNA, which are typical impurities, are 351 μg/mL and 8200 ng, respectively, in WFC introduced with DEA-AEX and HIC, and in the cell culture supernatant before permeation through the hollow fiber ultrafiltration membrane. /mL, but this significantly decreased to 36 μg/mL and 53 ng/mL, respectively, in the permeate, indicating that they were excellent in removing these dissolved impurity proteins.
It was confirmed that γ-globulin, on the other hand, could be purified by SDS-PAGE. In this membrane filtration step, since a hollow fiber ultrafiltration membrane made of polyacrylonitrile was used, non-specific adsorption of proteins to the membrane could be suppressed and high fractionation performance was achieved.

[Comparative example 2]
Commercially available membranes with anion exchange groups include SartobindQ MA75 (manufactured by Sartorius, flat membrane, membrane volume 2.06 mL, quaternary amino group), MustangQ Acrodisc (manufactured by Pall, flat membrane, membrane volume 0.18 mL, quaternary amino group). ) and BioCap25Filter 90ZA (manufactured by Kyuno, flat membrane, membrane volume 5 mL or more, quaternary amino group) in the same manner as in Example 4. Impurities were removed by passing a volume of supernatant from serum cell culture equivalent to 100 times the volume of each membrane. To confirm whether γ-globulin had been purified, evaluation by SDS-PAGE was performed in the same manner as in Example 4.
The concentrations of HCP and DNA in the cell culture supernatant before permeation with the anion exchange membrane module were 351 μg/mL and 8200 ng/mL, respectively, whereas the concentrations of HCP and DNA in the cell culture supernatant after permeation were 259 μg/mL and 482 ng with SartobindQ MA75, respectively. /mL, 244 μg/mL and 3816 ng/mL for MustangQ Acrodisc, and 330 μg/mL and 6600 ng/mL for BioCap25Filter 90ZA, and it was found that the impurity removal performance was lower than that of the method of the embodiment.

[Example 5]
Using the microfiltration (MF) hollow fiber membrane mini module for continuous culture prepared in (b), a 15 L (effective volume: 8.5 L) glass bio CHO-K1 cells containing a gene expressing a monoclonal antibody were introduced into a reactor (manufactured by Repligen), and a continuous culture experiment was conducted. A basal medium containing glucose and an antifoaming agent was continuously added to adjust the cell concentration to be maintained at 75±10×10 ⁶ Cells/mL. At this time, 3/4 of the effective volume was replaced in one day. The precision filtration hollow fiber membrane mini module was set at a permeation flux of 1 L/m ² /h, allowing permeation from the inner surface to the outer surface of the hollow fiber membrane, and the shear rate of the culture solution flowing through the hollow part was 1,250 s ^-1 It was set to The culture was carried out for about 40 days, during which time stable operation was possible with the permeation pressure of the microfiltration hollow fiber membrane being about 10 kPa.

The permeated liquid was shortened to a length of 1 cm through the WFC prepared in (a) into which DEA-AEX and HIC had been introduced, and the solution was passed in this order to continuously purify the monoclonal antibody. The liquid flow rate was set to 1 mL/min/mL-Bed. The concentrations of HCP and DNA, which are typical impurities, were 951 mg/mL and 82 μng/mL, respectively, in the solution before passing through the microfiltration hollow fiber membrane, but they were 951 mg/mL and 82 μng/mL, respectively, in the solution after purification. They were significantly reduced to 156 μg/mL and 43 ng/mL, indicating that these dissolved impurities were excellently removed. It was confirmed that one of the monoclonal antibodies could also be purified by SDS-PAGE.
Since the permeation pressure of the filter module produced in (a) was low, the permeate of the continuous culture microfiltration hollow fiber membrane could be passed through as it was, and smooth production of antibodies was possible.

In addition, the pore diameter, surface aperture ratio, water permeability, etc., tensile breaking strength test, etc. listed in Table 1 were measured or conducted under the following conditions.

Pore structure Using an electron microscope SU8000 series (manufactured by HITACHI), electron microscope (SEM) images of the surface and cross section of the hollow fiber membrane produced at an accelerating voltage of 3 kV were taken at 5000 times. By observing the photographed surface and cross-section, it was determined that the polymer trunk had a three-dimensional network structure without spherulites and had a three-dimensional network structure.

Pore diameter of membrane The pore diameter of the produced hollow fiber membrane was measured before and after the chemical resistance test under the following conditions.
The pore diameter was determined by adding up the pore area of each pore existing on the surface in order from the smallest pore diameter, and the pore diameter of the pore whose sum reached 50% of the total pore area of each pore. .

Surface Aperture Ratio Using an electron microscope SU8000 series (manufactured by HITACHI), electron microscope (SEM) images of the surface and cross section of the hollow fiber membrane produced at an accelerating voltage of 3 kV were taken at a magnification of 5000 times. Cross-sectional electron microscopy samples were obtained by cutting membrane samples frozen in ethanol into circular slices. Next, using the image analysis software Winroof 6.1.3, "noise removal" of the SEM image was performed using the numerical value "6", and then binarization was performed using a single threshold value. Valued. The aperture ratio of the membrane surface was determined by determining the area occupied by the pores in the binarized image thus obtained.

Membrane Outer Diameter/Inner Diameter The produced hollow fiber membrane was sliced thinly with a razor, and the outer diameter and inner diameter were measured using a 100x magnifying glass. For one sample, measurements were performed at 60 locations at 30 mm intervals.

Membrane Area The membrane area of a hollow fiber membrane refers to the inner surface area of the hollow part of the hollow fiber membrane.

Measurement of Tensile Breaking Strength and Tensile Breaking Elongation The tensile breaking strength and tensile breaking elongation of the produced hollow fiber membrane before and after the chemical liquid resistance test were measured under the following conditions.
The tensile breaking strength test was conducted using AGX-V (manufactured by Shimadzu Corporation) under the conditions of a distance between chucks of 50 mm and a tensile speed of 10 mm/min.
A tensile elongation at break test was conducted, and the tensile elongation at break was calculated from the obtained results according to JIS K7161.
The tensile elongation at break test was conducted using an AGS-5D: Instron type tensile testing machine (manufactured by Shimadzu Corporation) under the conditions of a chuck distance: 5 cm and a tensile speed: 20 cm/min.

Measurement of water permeability The water permeability of the produced hollow fiber membranes before and after the chemical resistance test was measured under the following conditions.
After immersing the hollow fiber membrane in ethanol, the membrane was repeatedly immersed in pure water several times to seal one end of the approximately 10 cm long wet hollow fiber membrane, and a syringe needle was inserted into the hollow part of the other end in an environment of 25°C. Pure water at 25°C was injected into the hollow part from the syringe needle at a pressure of 0.1 MPa, the amount of pure water permeating from the outer surface was measured, and the pure water flux was determined using the formula below to evaluate water permeability. did.
Pure water flux [L/m ² /h] = 60 × (permeated water amount [L]) / {π × (membrane outer diameter [m]) × (membrane effective length [m]) × (measurement time [min]) }

Note that the effective membrane length here refers to the net membrane length excluding the portion into which the injection needle is inserted.

The obtained porous hollow fiber filtration (MF) membrane had a three-dimensional network structure. In addition, chemical resistance was measured by immersing the hollow fiber membrane in a 0.5 mass% sodium hypochlorite (NaClO) aqueous solution containing 1 mass% sodium hydroxide (NaOH) at 40°C for 7 days. As a result, no change was observed in the tensile strength at break and tensile elongation at break of the membrane before and after the chemical resistance test. Furthermore, no change or decrease in water permeability or pore size was observed before and after the test.
The results are shown in Table 1.

When the obtained porous hollow fiber filtration (UF) membrane was observed using an electron microscope, it showed a gradient structure in which the pore diameters increased continuously from both the inner and outer surfaces toward the center of the membrane, and the size exceeded 10 μm. It had a sponge structure that did not contain any defective parts of the polymer. No pores larger than 0.02 μm were observed on the outer surface of the membrane. On the other hand, numerous slit-like streaks and slit-like holes were observed on the inner surface.
In addition, the hollow fiber membrane was immersed in a 0.1 mass% sodium hypochlorite (NaClO) aqueous solution containing 0.4 mass% sodium hydroxide (NaOH) at 25°C for 5 days to test its chemical resistance. When measured, no change was observed in the tensile strength at break and tensile elongation at break of the membrane before and after the chemical resistance test. The results are shown in Table 1.
Furthermore, no change or decrease in water permeability was observed before and after the test.
The composition, test conditions, and various physical properties of the obtained porous hollow fiber membrane are shown in Table 1 below.

According to the protein purification method using a wind filter cartridge module according to the present invention, proteins such as antibody drugs can be efficiently purified.

Claims (15)

A method for purifying a target protein in a liquid to be processed containing the target protein and impurities, the method comprising:
The target protein in the liquid to be treated is passed through a chromatography carrier that is one or more selected from the group consisting of an anion exchange material, a cation exchange material, a chelate-forming material, and a hydrophobic material. including a chromatography step for purification;
Each of the anion exchange material, the cation exchange material, the chelate forming material, and the hydrophobic material is grafted with a graft chain having each of an anion exchange group, a cation exchange group, a chelate forming group, and a hydrophobic group. is a material that is introduced into the substrate via polymerization,
The base material is a monofilament yarn or a multifilament yarn,
The method for purifying proteins, wherein the carrier is in the form of a wind filter cartridge module, which is formed by winding the base material into which the graft chains have been introduced around a core material, and optionally removing the core material. The protein purification method according to claim 1, wherein the grafting rate of the chromatography carrier is 20% or more and 300% or less. The protein purification method according to claim 1 or 2, wherein the wind filter cartridge module has a pore diameter of 0.5 μm or more and 500 μm or less. 3. The protein purification method according to claim 1, wherein in the chromatography step, the liquid to be treated is permeated through the wind filter cartridge module at a permeation pressure of 100 kPa or less. 3. The method according to claim 1, further comprising a step of flowing the liquid to be treated in a flow-through mode to a hollow fiber ultrafiltration (UF) membrane module disposed downstream of the chromatography carrier after the chromatography step. Methods for purifying the described proteins. The protein purification method according to claim 1 or 2, wherein the wind filter cartridge module is single-use. The protein purification method according to claim 1 or 2, further comprising a membrane filtration step in which the liquid to be treated is passed through a filtration membrane having a molecular weight cutoff of 10,000 or more and 800,000 or less, subsequent to the chromatography step. . 8. The protein purification method according to claim 7, further comprising a virus removal step of passing the liquid to be treated through a virus removal filter after the membrane filtration step. 3. The protein purification method according to claim 1, wherein the target protein is produced by tangential flow continuous culture using a hollow fiber membrane module. The hollow fiber membrane module has the following characteristics:
・Membrane material: polyvinylidene fluoride,
・Pore diameter: 0.05-0.7μm,
・Inner surface aperture ratio: 25-50%,
・Inner diameter: 1 to 5 mm, and ・Inner surface membrane area: 1 m2 ^or more,
The protein purification method according to claim 9, which is a hollow fiber membrane module having the following. The protein purification method according to claim 10, wherein the hollow fiber membrane module is single-use. the chromatography support comprises the anion exchange material;
The protein purification method according to claim 1 or 2, wherein the anion exchange group contains one or more selected from the group consisting of a tertiary amino group and a quaternary amino group. the chromatography support comprises the cation exchange material;
The protein purification method according to claim 1 or 2, wherein the cation exchange group contains one or more selected from the group consisting of a sulfonic acid group and an acrylic acid group. the chromatography support comprises the chelate-forming material;
The protein purification method according to claim 1 or 2, wherein the chelate-forming group contains one or more selected from the group consisting of an iminodiacetic acid group and an iminodiethanol group. the chromatography carrier comprises the hydrophobic material;
The method for purifying a protein according to claim 1 or 2, wherein the hydrophobic group contains one or more selected from the group consisting of a phenyl group and a C4 to C18 alkyl group.

Images