JP2013103189A - Method for processing protein adsorbent, method for recovering protein adsorption capability of protein adsorbent, and method for producing protein adsorbent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for processing of sufficiently improving protein adsorption capability (adsorption capacity) of a protein adsorbent.SOLUTION: The method of processing for improving a protein adsorption capability of a protein adsorbent, wherein the protein adsorbent includes a polymer substrate and a polymer chain containing a functional group binding to a surface of the polymer substrate and having the protein adsorption capability, includes a step of heating the protein adsorbent in a state that the protein adsorbent is moistened by an organic solvent or an aqueous solution containing the organic solvent.

Description

  The present invention relates to a method for treating a protein adsorbent, a method for recovering protein adsorption capacity of a protein adsorbent, and a method for producing a protein adsorbent. The present invention also relates to a protein adsorbent and a column or module using the same.

  In recent years, practical mass purification of proteins has become an important issue in the biotechnology industry. That is, establishment of a technique capable of efficiently mass-producing and mass-purifying proteins is desired. As one means for that purpose, development of protein adsorbents such as protein adsorbing films or beads has been studied.

  In general, since proteins are produced by cell culture using animal-derived cell lines, it is necessary to separate and purify the target protein (hereinafter sometimes simply referred to as “target protein”). is there. In particular, in order to commercialize antibodies using antibodies (antibody drugs), turbid components such as cell debris and non-turbid components such as dissolved proteins derived from cells are removed from the cell culture medium to treat humans. It needs to be purified to a purity sufficient for the application. A protein adsorbent is used in the purification step.

  In particular, recently, the demand for antibody drugs is rapidly increasing, and mass production of proteins to be antibody drugs is directed. With the rapid progress of culture technology, it is desired to improve the capacity of the purification process. That is, there is a demand for further improvement in the performance of the protein adsorbent.

  For example, Patent Literatures 1 to 5 disclose techniques related to a protein adsorbent for mass protein purification.

  Patent Document 1 discloses a protein purification method including a step of filtration using a porous membrane having a graft chain on the pore surface and an anion exchange group fixed to the graft chain.

  Patent Document 2 discloses a porous adsorption medium in which a porous substrate is covered with an adsorbing material having a crosslinked polymer having a primary amine group bonded thereto.

  Patent Document 3 includes a support member that has a plurality of holes extending therein, and a macroporous crosslinked gel that is disposed in the hole of the support member and fills the holes of the support member. A composite material is shown which is suitable for use as a separation of substances by adsorption.

  Patent Document 4 provides a crosslinked film having a cationic functional group and a positively charged porous membrane made of a porous substrate, and can be used for filtration and purification of biomolecules such as proteins, nucleic acids, and endotoxins. It is shown that there is.

  Patent Document 5 discloses an adsorbent in which a functional group having protein adsorption ability is immobilized on the surface of a polymer base particle through a polymer chain and capable of high-speed adsorption purification of proteins and the like, and a method for producing the same. ing.

  Patent Document 6 shows an example in which the surface of a base material is modified with a coating having an amino group or a sulfonic acid group, which is generally known as a functional group capable of interacting with a protein. In Patent Document 6, a film of an N-halogenated compound (polymer or polymer precursor) is formed on the surface of a polymer porous substrate film, and a graft initiator and a monomer are brought into contact with each other to form a substrate. The method of graft polymerization is shown above. Furthermore, glycidyl methacrylate (GMA) is used as the monomer for graft polymerization, and then the tertiary amino group / quaternary ammonium group is introduced into the epoxy group of GMA by a secondary / tertiary amine, or a sulfonic acid ion is used. Membranes obtained by sulfonation of epoxy groups by treatment are described.

International Publication No. 2009/054226 JP 2009-53191 A JP 2006-519273 A US Pat. No. 6,780,327 JP 2008-45906 A US Pat. No. 5,547,575

Kyoichi Saito, CHARGED POLYPER BRUSH GRAFTED ONTO POROUS HOLDLOW-FIBER MEMBRANE IMPROVES SEPARATION AND REACTION IN BIOTECNOLOGY, Separation

  Generally, a protein adsorbing material is expected to be reproducible (protein adsorbing ability can be restored) by eluting the adsorbed protein after protein adsorption. As disclosed in Patent Document 1, a salt solution is generally used when eluting adsorbed proteins.

  However, when protein is adsorbed by repeating regeneration by such treatment, the adsorption capacity (the amount of protein adsorbed per unit adsorbent volume) tends to gradually decrease. At present, when mass production and purification of a target protein is desired for efficient mass production of antibody drugs, such a decrease in adsorption capacity is a problem to be solved.

  The protein adsorption form on the adsorbent is not necessarily specified, but can be inferred as follows, for example. That is, protein acts on the functional group of the adsorbent and is adsorbed, but when adsorbed on the surface of the adsorbent coating layer (hereinafter referred to as surface adsorption), in addition to surface adsorption, protein is deeply contained in the adsorbent coating layer. May be adsorbed. The adsorbed protein molecules can be adsorbed in a single layer or in multiple layers. As an example in which protein is adsorbed deep in the adsorbent coating layer, an adsorbent described in Patent Document 2 can be mentioned. In Patent Document 2, a film polymer covering a base material is described as “proteins and other impurities are trapped in the bottom of the film, and so on”. In addition, as an example in which protein molecules are stacked and adsorbed in multiple layers, in Non-Patent Document 1, a porous hollow fiber membrane in which a polymer brush having an ion exchange group (linear graft polymer chain) is introduced on the pore surface Have been reported to adsorb proteins in multiple layers.

  For such adsorbents that adsorb proteins deep in the adsorbent coating layer in addition to surface adsorption, or adsorbents that adsorb protein molecules in multiple layers, the protein adsorbed by the salt solution is completely May not be eluted. Therefore, it is considered that the adsorption capacity is reduced due to the accumulation of the residual protein during repeated regeneration. Actually, in the example of Patent Document 3, in the desorption process using the salt solution after the adsorption process, the recovery rate of the adsorbed protein is 63 to 99%, and it is reported that it is not completely eluted. Yes. That is, in the adsorbent on which protein is adsorbed in such a form, it can be said that the reduction of the adsorption capacity is a particularly big problem.

  Then, the main objective of this invention is to provide the processing method for fully improving the protein adsorption capacity (adsorption capacity) of a protein adsorption material. Another object of the present invention is to provide a recovery method for sufficiently recovering the protein adsorption capacity (adsorption capacity) of the protein adsorbent after protein adsorption and desorption. Another object of the present invention is to provide a method for producing a protein adsorbent having a sufficiently large protein adsorption capacity (adsorption capacity).

  As a result of intensive studies to solve the problems, the present inventors have found that a method of heating a protein adsorbent in a state moistened with an organic solvent or an aqueous solution containing an organic solvent is effective for achieving the above object. I found out.

That is, the present invention relates to the following.
[1]
A treatment method for improving the protein adsorption capacity of a protein adsorbent,
The protein adsorbent comprises a polymer substrate and a polymer chain that contains a functional group that binds to the surface of the polymer substrate and has a protein adsorption ability.
A processing method comprising a step of heating the protein adsorbent in a wet state with an organic solvent or an aqueous solution containing an organic solvent.
[2]
The processing method according to [1], wherein the protein adsorbent is heated to 30 to 80 ° C. while being wetted with an organic solvent or an aqueous solution containing an organic solvent.
[3]
The processing method according to [1] or [2], wherein the organic solvent is at least one selected from the group consisting of alcohols, aromatic hydrocarbons, esters, ethers, and ketones.
[4]
The processing method according to [3], wherein the alcohol is at least one selected from the group consisting of methanol, ethanol, propanol, butanol, and pentanol.
[5]
The processing method according to any one of [1] to [4], wherein the polymer chain is bonded to the polymer substrate by a covalent bond.
[6]
The processing method according to any one of [1] to [5], wherein the polymer chain is a linear polymer chain bonded to the polymer base material by graft polymerization.
[7]
The processing method according to any one of [1] to [6], wherein the polymer substrate is a porous body.
[8]
The processing method according to any one of [1] to [7], wherein the polymer substrate is a hollow fiber porous membrane.
[9]
The processing method according to any one of [1] to [8], wherein the functional group having protein adsorption ability is an ion exchange group.
[10]
The processing method according to [9], wherein the ion exchange group is a tertiary amino group.
[11]
The processing method according to any one of [1] to [10], wherein the protein adsorbent is an adsorbent obtained by adsorbing and desorbing protein at least once.
[12]
A method for recovering the protein adsorption capacity of a protein adsorbent,
The protein adsorbent includes a polymer substrate and a polymer chain containing a functional group that binds to the surface of the polymer substrate and has a protein adsorption ability, and adsorbs a protein at least once. And desorbed,
A recovery method comprising the step of heating the protein adsorbent in a wet state with an organic solvent or an aqueous solution containing an organic solvent.
[13]
A step of obtaining an adsorbent comprising a polymer substrate, and a polymer chain that binds to the surface of the polymer substrate and contains a functional group having protein adsorption ability;
A method for producing a protein adsorbent, comprising heating the adsorbent in a state of being wetted with an organic solvent or an aqueous solution containing an organic solvent.
[14]
The production method according to [13], further comprising a step of adsorbing and desorbing protein to the adsorbent before the heating step.
[15]
A protein adsorbent obtainable by the production method according to [13] or [14].
[16]
A column comprising the protein adsorbent according to [15].
[17]
A module comprising the protein adsorbent according to [15].

  ADVANTAGE OF THE INVENTION According to this invention, the processing method for fully improving the protein adsorption capacity (adsorption capacity) of a protein adsorption material is provided. In addition, according to the present invention, there is provided a recovery method for sufficiently recovering the protein adsorption capacity (adsorption capacity) of the protein adsorbent after protein adsorption and desorption. Furthermore, according to the present invention, a method for producing a protein adsorbent having a sufficiently large protein adsorption capacity (adsorption capacity) is provided.

  According to the present invention, efficient use of a protein adsorbent is possible. Further, according to the present invention, the adsorption capacity of the protein adsorbent after being used for protein adsorption and desorption can be recovered sufficiently large.

  Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the following embodiments, and various modifications can be made within the scope of the gist.

  The method for treating a protein adsorbent according to this embodiment includes a step of heating the protein adsorbent in a state of being wetted by an organic solvent or an aqueous solution containing an organic solvent. According to the method for treating a protein adsorbent according to this embodiment, the protein adsorbing ability (adsorption capacity) of the protein adsorbent can be improved. The protein adsorbent is not particularly limited as long as it includes a polymer substrate and a polymer chain that covers the surface of the polymer substrate and contains a functional group having protein adsorption ability. Absent.

  In the present embodiment, the “adsorbent before treatment” simply means “a protein adsorbent before being subjected to a heating step in a state of being wetted with an organic solvent or an aqueous solution containing an organic solvent”. The term “adsorbent before treatment” is used to distinguish it from “protein adsorbent after treatment by a process of heating in an organic solvent or an aqueous solution containing an organic solvent in a wet state”. The “pretreatment adsorbent” is not limited to the meaning of a protein adsorbent that has not undergone protein adsorption and desorption. The number of times that the “adsorption material before treatment” has been subjected to protein adsorption and desorption before being treated by the heating process in a state of being wetted with an organic solvent or an aqueous solution containing an organic solvent is not limited. That is, the “adsorbent before treatment” is, for example, an unused one (one that has not adsorbed an adsorbate at all), an adsorbent / desorption repeated, and a reduced adsorption capacity, or a heat treatment different from the present embodiment. The adsorption capacity may be reduced.

  The organic solvent is preferably at least one selected from the group consisting of alcohols, aromatic hydrocarbons, esters, ethers, and ketones, and more preferably alcohols. These organic solvents may be used individually by 1 type, and 2 or more types may be mixed and used for them. When the organic solvent is water-soluble, an aqueous solution containing the organic solvent may be used. The aqueous solution concentration of the organic solvent is generally 10 to 99% by weight, preferably 15 to 70% by weight, and particularly preferably 20 to 40% by weight.

  As alcohols, methanol, ethanol, propanol, butanol and pentanol are preferable, and methanol and ethanol are particularly preferable. These alcohols may be used individually by 1 type, and 2 or more types may be mixed and used for them. Furthermore, the aqueous solution concentration of the alcohol is generally 10 to 99% by weight, preferably 15 to 70% by weight, and particularly preferably 20 to 40% by weight.

  The form of the protein adsorbent is not particularly limited as long as the solution in which the protein is dissolved is in contact with the surface of the pre-treatment adsorbent, and may be a non-porous body or a porous body. The form of the protein adsorbent is mainly determined by the form of the polymer substrate. Non-porous materials include films, non-porous beads, fibers, and capillaries. Examples of the porous body include a flat membrane, a nonwoven fabric, a hollow fiber membrane, a monolith, and porous beads. From the viewpoint of increasing the adsorption area per unit volume of the adsorbent and achieving a high adsorption capacity, a porous body is preferred. Furthermore, from the viewpoint of ease of handling, a film-like form, that is, a flat film, a non-woven fabric, and a hollow fiber membrane are more preferable. A hollow fiber porous membrane is more preferable because of its scale-up property and a simple flow channel structure upon module molding.

  In the present embodiment, the “hollow fiber porous membrane” means a cylindrical or fibrous porous body having a hollow portion. The hollow side (inner side) and the outer side of the hollow fiber porous membrane are continuous by pores that are through holes. The hollow fiber porous membrane has a property of allowing liquid or gas to permeate from the inside to the outside or from the outside to the inside by the pores. The outer diameter and inner diameter of the hollow fiber porous membrane are not particularly limited as long as the porous membrane can physically maintain its shape.

  The material of the polymer base material in this embodiment will be described. The polymer substrate is not particularly limited, and a known material can be used. In particular, in order to maintain the mechanical properties, it is preferably composed of a polyolefin-based polymer. Examples of the polyolefin-based polymer include homopolymers of olefins such as ethylene, propylene, butylene, and vinylidene fluoride, copolymers of two or more of the olefins, or one or two or more olefins and perhalogen. And a copolymer with a fluorinated olefin. Among these substrates, polyethylene or polyvinylidene fluoride is preferable, and polyethylene is more preferable because mechanical strength is particularly excellent.

  In the present embodiment, at least a part of the surface of the polymer substrate (including the surface inside the pores when the polymer substrate is porous) contains a polymer chain containing a functional group having protein adsorption ability. Are joined. Examples of the bonding state between the polymer substrate and the polymer chain include, but are not limited to, covalent bonding, bonding by physical adsorption, and affinity bonding.

  The method for introducing a polymer chain containing a functional group having protein adsorption ability onto the surface of the polymer substrate is not particularly limited. A method of generating radicals by irradiating the polymer substrate with radiation, graft polymerization, applying a polymer chain containing a functional group with protein adsorption ability to the substrate, and applying the polymer chain to the substrate surface with a crosslinking agent There are a method of fixing, a method of forming a polymer or polymer precursor film on the surface of a substrate, and a method of obtaining a graft polymer from polymer chains constituting the film. In particular, when a strong bond due to a covalent bond between a polymer chain in a polymer substrate and a graft chain that coats the polymer chain is expected, a functional group having a protein adsorbing ability is contained by graft polymerization from the substrate. A method of introducing a molecular chain is preferred.

  In particular, when a linear polymer chain containing a functional group having protein adsorption ability is introduced by graft polymerization from a substrate, (1) a method of directly polymerizing a monomer having a functional group having protein adsorption ability, Or (2) a method of introducing a polymer chain containing a functional group capable of introducing a functional group having protein adsorption ability by graft polymerization from a substrate, and subsequently introducing a functional group having protein adsorption ability. Can be adopted.

  (1) is a coating layer having a protein adsorption ability in a single step (a layer formed by a polymer chain containing a functional group having a protein adsorption ability, and does not necessarily cover the entire surface of the polymer substrate. This is convenient and preferable. Although it does not specifically limit as said monomer, A methacrylate derivative, a vinyl compound, an allyl compound, etc. are mentioned, For example, diethylaminoethyl methacrylate, sulfopropyl methacrylate, allylamine, etc. are selected.

  (2) is preferable from the viewpoint that it is easy to prepare variations of functional groups having various protein adsorption capacities, or to easily prepare variations of introduction ratios (details will be described later) of functional groups having protein adsorption capacities. As the monomer in the graft polymerization in the case of (2), glycidyl methacrylate (GMA) having a highly reactive epoxy group is preferable.

  In this embodiment, the state of the coating layer formed by the polymer chain containing the functional group having protein adsorption ability is not limited. For example, when a linear polymer chain is formed, a gel-like film layer is formed by loosely crosslinking the polymer chains on the substrate surface, or the polymer chain forms a crosslinked polymer. There are times. Although the details of the mechanism of this embodiment are not necessarily clear, it is considered that the heat treatment in a wet state conditions a polymer chain containing a functional group having protein adsorption ability, and increases the number of adsorption points. In particular, it is presumed that the protein adsorbed deep in the adsorbent coating layer or adsorbed by being stacked in multiple layers is efficiently discharged, and the effect of increasing the number of adsorption points is remarkably high. Therefore, an adsorbent having a linear graft polymer chain formed directly from a base material, or an adsorbent having a gel-like film in which polymer chains containing a functional group having protein adsorption ability are loosely cross-linked with each other. Preferably, an adsorbent having a linear graft polymer chain formed directly from the substrate is more preferable.

  In the present embodiment, the amount of the polymer chain containing a functional group having a protein adsorbing ability to the polymer substrate is not particularly limited as long as there is no problem such as an increase in pressure during liquid passage. In addition, an optimal amount of bonding can be selected according to the structure of the polymer base material and the combination of the functional groups contained in the polymer chain and the characteristics of the monomer. Hereinafter, with respect to the amount of binding, a polymer chain (graft chain) containing a functional group capable of introducing a functional group capable of adsorbing a protein is introduced by graft polymerization from the base material (i) mentioned above (2). Subsequently, (ii) a method for introducing a functional group having protein adsorption ability will be described as an example.

  With regard to (i), generally, the graft chain binding rate (graft rate, dg [%]) to the substrate is defined based on the weight increased by graft chain introduction, as shown in Formula (1). The

Ｗ_０：グラフト鎖導入前の高分子基材重量（ｇ）
Ｗ_１：グラフト鎖導入後の全体重量（ｇ）

W ₀ : Weight of polymer base material before graft chain introduction (g)
W ₁ : Total weight (g) after graft chain introduction

Regarding (ii), the abundance ratio of the functional group having protein adsorption ability to the functional group capable of introducing protein adsorption ability in the graft chain in the coating layer is arranged by “ligand conversion rate” represented by the formula (2). Is done. In other words, the proportion of functional groups having protein adsorption ability is represented by the number of moles of functional groups having protein adsorption ability introduced relative to the number of moles of functional groups capable of introducing protein adsorption ability in the graft chain. The For example, the graft chain is a polymer of GMA, when introduced diethylamine epoxy ring of GMA, a molecular weight 142g / mol of GMA to _{M 1} in Formula (2), substituting diethylamine molecular weight 73 g / mol to _{M 2.}

Ｗ_０：グラフト鎖導入前の高分子基材重量（ｇ）
Ｗ_１：グラフト鎖導入後の全体重量（ｇ）
Ｗ_２：タンパク質吸着能を有する官能基導入後の全体重量（ｇ）
Ｍ_１：グラフト鎖モノマー単位の分子量（ｇ／ｍｏｌ）
Ｍ_２：導入された官能基（タンパク質吸着能を有する官能基）の分子量（ｇ／ｍｏｌ）

W ₀ : Weight of polymer base material before graft chain introduction (g)
W ₁ : Total weight (g) after graft chain introduction
W ₂ : Total weight (g) after introduction of a functional group having protein adsorption ability
M ₁ : molecular weight of the graft chain monomer unit (g / mol)
M ₂ : Molecular weight of the introduced functional group (functional group having protein adsorption ability) (g / mol)

  The graft ratio is preferably 5% to 200%, more preferably 20% to 150%, and even more preferably 30% to 90% from the viewpoint of securing both higher adsorption capacity and mechanically stable strength. .

  From the viewpoint of obtaining a higher adsorption capacity, the ligand conversion rate is preferably 20% to 100%, more preferably 50% to 100%, and still more preferably 70% to 100%.

  The functional group having protein adsorption ability in this embodiment will be described. Examples of the functional group having protein adsorption ability include, but are not limited to, ion exchange type, hydrophobic interaction adsorption type, group specific affinity adsorption type, and individual specific affinity adsorption type.

  Furthermore, examples of the ion exchange group (ion exchange type functional group) include strong acid cation exchange groups, weak acid cation exchange groups, strong basic anion exchange groups, and weak basic anion exchange groups. Is not particularly limited.

For example, the strong acid cation exchange group includes a sulfonic acid group (—SO ₃ ^— ), and the weak acid cation exchange group includes a carboxylic acid group (—COO ⁻ ).

On the other hand, the strong basic anion-exchange groups, for example, quaternary ammonium groups _{^{(Q, -N + R 3)}} , quaternary aminoethyl group _{(QAE, - (CH 2)} 2 -N + R 3) and the like It is done. Here, R is not particularly limited, and R bonded to the same N may be the same or different, and preferably represents a hydrocarbon group such as an alkyl group, an aryl group, or an aralkyl group. More specifically, trimethyl amino group (TMA, ^-N + Me _3), and the like. The weakly basic anion exchange group includes a primary amino group (—NH ₂ ), a secondary amino group (—NRH), a tertiary amino group (—NR ₂ ), a diethylaminoethyl group (DEAE, — (CH ₂ ) _2. -NEt _2), diethylaminopropyl group _{(DEAP, - (CH 2)} 3 -NEt 2) , and the like. Here, R is not particularly limited, and the R bonded to the same N may be the same or different, and preferably represents a hydrocarbon group such as an alkyl group, an aryl group, or an aralkyl group.

  When a strongly acidic or strongly basic ion exchange group is selected as the ion exchange group, since almost all of the ion exchange groups are kept dissociated in a wide pH range, there is an advantage that it can be used in a wide pH range. That is, it is preferable in that it can be used regardless of the pH of the liquid to be treated. However, there is a high density of ion exchange groups with hydrated water, and in the coating layer, there is a large steric repulsion in the polymer chain and between adjacent polymer chains, and the polymer chain is extended. Therefore, in the adsorbent of the porous body, the flow path of the liquid is blocked, and there is a tendency to cause an increase in fluid pressure / a decrease in the processing amount. Therefore, when high-speed treatment is expected, a weakly acidic or weakly basic ion exchange group is preferable, and a diethylamino group or isopropylamino group is more preferable.

  Examples of the hydrophobic interaction adsorption group (hydrophobic interaction adsorption type functional group) include a phenyl group and an alkyl group.

  Examples of the county-specific affinity adsorption group (county-specific affinity adsorption-type functional group) include Cibacron Blue F3G-A, Protein A, Concanavalin A, heparin, tannin, metal chelate group and the like.

  Examples of individual specific affinity adsorption groups (individual specific affinity adsorption functional groups) include antigens and antibodies.

  A method and form of wetting the protein adsorbent in this embodiment will be described. In the present embodiment, the “wet state” refers to a state in which the surface of the protein adsorbent is wetted with a liquid. When the protein adsorbent in a dry state is wetted, it is preferably replaced with a solution for wetting (an organic solvent or an aqueous solution containing an organic solvent) after hydrophilization. The hydrophilization method is not particularly limited, but the hydrophilization can be performed with water, an aqueous alcohol solution, alcohol, or steam. Among these, hydrophilization with alcohol or an aqueous alcohol solution can be performed with various materials and shapes. Examples of alcohol hydrophilization include a method in which a protein adsorbent is brought into contact with ethanol or an ethanol aqueous solution, the ratio of water is gradually increased, and finally contacted with pure water. The method of replacing with a solution for wetting is not limited as long as the protein adsorbent is sufficiently replaced with a solution (an organic solvent or an aqueous solution containing an organic solvent). For example, in the case of using a membrane, in order to sufficiently replace the solution, a method of passing the solution is effective, and the passing amount of each solution is preferably 3 times or more of the module volume, more preferably 5 times or more. Preferably, 10 times or more is more preferable. In addition, when using alcohol or the aqueous solution containing alcohol as a solution for wetting, the step of hydrophilization may be omitted.

  The method for heating the pre-treatment adsorbent is a category in which the protein adsorbent in a state wetted by an organic solvent or an aqueous solution containing an organic solvent is sufficiently heated and does not affect the mechanical strength of the protein adsorbent. There is no particular limitation. The heat treatment of the protein adsorbent can be performed by heating from the outside of the packing container in a form in which the protein adsorbent is packed (for example, a module, a column, etc.). What is necessary is just to heat to room temperature (25 degreeC) or more as heating temperature, 30 degreeC or more is preferable and 40 degreeC or more is more preferable. Although the upper limit of heating temperature is based also on the kind of the organic solvent to be used or the aqueous solution containing an organic solvent, it is 100 degrees C or less normally, 80 degrees C or less is preferable and 70 degrees C or less is more preferable. The treatment method according to this embodiment is moistened with an organic solvent or an aqueous solution containing an organic solvent, and thus does not require a high heating temperature. The heating time is preferably 12 hours or longer, and more preferably 24 hours or longer for the purpose of sufficient heating. The upper limit of the heating time is not particularly limited, but is usually 72 hours or less.

  In the present embodiment, it is preferable to pack the protein adsorbent so that the protein adsorbent as mentioned above efficiently contacts the solution in which the protein is dissolved. Such a packing form is generally called a module when the adsorbent is a film, and a column when the adsorbent is another film, but the structure thereof is not limited. However, in the case of a hollow fiber porous membrane, a simple liquid passing method is also possible by flowing a solution in which protein is dissolved in the hollow portion and receiving the liquid leaking from the outer surface of the hollow portion. It doesn't have to be a module.

  An example of the module structure is QyuSpeed ™ D (hollow fiber type anion exchange adsorption membrane) sold by Asahi Kasei Medical Corporation. The structural features include a housing having openings at both ends and one or more hollow fiber membranes secured within the housing. At both ends of the hollow fiber membrane, the outside of the hollow fiber membrane and the inner wall of the housing are fixed, and the protein dissolved from the housing opening passes through the membrane from the inside of the hollow fiber to the outside and is attached to the side of the housing It is discharged from the nozzle.

  The present invention can also be referred to as a method for recovering the protein adsorbing ability of a protein adsorbing material, which comprises a step of heating the protein adsorbing material in a state wetted with an organic solvent or an aqueous solution containing an organic solvent. The protein adsorbent herein includes a polymer substrate and a polymer chain containing a functional group that binds to the surface of the polymer substrate and has a protein adsorption ability, and at least once. Is adsorbed and desorbed.

  According to the method for recovering protein adsorption capacity (adsorption capacity) according to the present embodiment, it is possible to recover the reduced adsorption capacity by repeating adsorption and desorption of proteins.

  In addition, the method for producing a protein adsorbent according to the present embodiment includes at least a polymer substrate and a polymer chain containing a functional group that binds to the surface of the polymer substrate and has a protein adsorption ability. And a step of heating the adsorbent in a state wetted with an organic solvent or an aqueous solution containing the organic solvent. By providing this heating step, a protein adsorbent with improved protein adsorption capacity (adsorption capacity) can be obtained. Furthermore, the manufacturing method according to the present embodiment may further include a step of adsorbing and desorbing protein (for example, bovine serum albumin) to the adsorbent before the heating step. Thereby, the protein adsorption capacity (adsorption capacity) of the protein adsorbent obtained can be further improved.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples. Furthermore, the protein adsorbent is not limited to QyuSpeed ™ D 150 mL (hollow fiber type anion exchange adsorption membrane) used in the examples.

[Evaluation Method 1] Adsorption Capacity Measuring Method Evaluation of protein adsorption capacity of 150 mL of QyuSpeedTM D, which is a hollow fiber type anion exchange adsorption membrane, was performed using a general-purpose HPLC system (GE Healthcare Japan AKTApilot). When displaying the performance of a biotechnology purification apparatus, a purified protein solution is generally used as a model. In this example, BSA (bovine serum albumin, manufactured by Sigma-Aldrich) generally used as a model protein was used. 1 g of BSA was dissolved in 1 L of 20 mM Tris-HCl buffer (hereinafter referred to as buffer) adjusted to pH 8 and passed through a 0.45 μm filter to obtain a BSA solution.

  QyuSpeedTM D 150 mL was connected to the HPLC system, and buffer solution, BSA solution, buffer solution, buffer solution containing 1 mol / L sodium chloride (hereinafter referred to as salt buffer solution), 1N sodium hydroxide, salt buffer at 800 mL / min. The liquids were passed in the order of the liquids. The first buffer solution flow was performed for the purpose of membrane equilibration, and the second buffer solution flow after supplying BSA was performed for the purpose of washing non-adsorbed BSA. Further, subsequent buffer solution containing 1M sodium chloride and 1N sodium hydroxide solution were passed for the purpose of desorption of adsorbed BSA.

  The BSA leakage trend toward the filtrate side was traced by the absorbance at a wavelength of 280 nm, and the filtrate amount at the time when the ratio of the absorbance of the filtrate to the absorbance of the BSA solution as the stock solution became 10% was confirmed. The amount of the filtrate was converted to the weight of BSA based on the concentration of 1 g / L, and the protein adsorption amount [mg] when the absorbance ratio reached 10% was calculated. Furthermore, protein (BSA) adsorption capacity per membrane volume (mg / mL) was determined by dividing the calculated value by the volume of the annular portion calculated from the inner and outer diameters of the hollow fiber and the effective yarn length. This value is called “dynamic adsorption capacity” and is commonly used in the field of biotechnology.

[Example 1]
An example is shown in which 150 mL of QyuSpeed ™ D as the pre-treatment adsorbent is heated to 40 ° C. in a state wetted with 20 wt% ethanol to increase the adsorption capacity.

  First, the protein adsorption capacity before the heat treatment was measured according to the evaluation method 1. The dynamic adsorption capacity was 51.7 mg / mL.

  Subsequently, the inside of the module was filled with 20% by weight ethanol, and then the module was sealed. In that state, the module was heated for 24 hours in a dryer controlled to 40 ° C. The module was cooled to room temperature.

  Thereafter, the protein adsorption capacity after the heat treatment was measured according to the evaluation method 1. The dynamic adsorption capacity was 54.3 mg / mL. The performance improvement rate at this time (= dynamic adsorption capacity after heat treatment / dynamic adsorption capacity before heat treatment × 100) was 105%.

[Example 2]
An example is shown in which 150 mL of QyuSpeed ™ D as the pre-treatment adsorbent is heated to 60 ° C. while being wetted with 20 wt% ethanol, thereby increasing the adsorption capacity.

  First, the protein adsorption capacity before the heat treatment was measured according to the evaluation method 1. The dynamic adsorption capacity was 50.0 mg / mL.

  Subsequently, the inside of the module was filled with 20% by weight ethanol, and then the module was sealed. In this state, the module was heated for 24 hours with a dryer controlled to 60 ° C. The module was cooled to room temperature.

  Thereafter, the protein adsorption capacity after the heat treatment was measured according to the evaluation method 1. The dynamic adsorption capacity was 54.2 mg / mL. The performance improvement rate at this time (= dynamic adsorption capacity after heat treatment / dynamic adsorption capacity before heat treatment × 100) was 108%.

[Example 3]
An example is shown in which 150 mL of QyuSpeed ™ D as the pre-treatment adsorbent is heated to 30 ° C. in a state wetted with 100 wt% ethanol to increase the adsorption capacity.

  First, the protein adsorption capacity before the heat treatment was measured according to the evaluation method 1. The dynamic adsorption capacity was 51.1 mg / mL.

  Subsequently, the inside of the module was filled with 100% by weight ethanol, and then the module was sealed. In this state, the module was heated for 24 hours with a dryer controlled to 30 ° C. The module was cooled to room temperature.

  Thereafter, the protein adsorption capacity after the heat treatment was measured according to the evaluation method 1. The dynamic adsorption capacity was 53.0 mg / mL. The performance improvement rate (= dynamic adsorption capacity after heat treatment / dynamic adsorption capacity before heat treatment × 100) at this time was 104%.

[Comparative Example 1]
As a comparative example with respect to Examples 1-3, when 150 mL of QyuSpeed ™ D as an adsorbent before treatment is stored at room temperature (25 ° C.) in a state wetted with 20 wt% ethanol, the adsorption capacity does not increase. Show.

  First, the protein adsorption capacity was measured according to the evaluation method 1. The dynamic adsorption capacity was 55.1 mg / mL.

  The module was filled with 20% by weight ethanol and sealed, and the module was stored at room temperature (25 ° C.) in that state.

  Thereafter, the protein adsorption capacity was measured according to the evaluation method 1. The dynamic adsorption capacity was 54.5 mg / mL. The performance improvement rate (= dynamic adsorption capacity after heat treatment / dynamic adsorption capacity before heat treatment × 100) at this time was 99%.

[Comparative Example 2]
An example in which the adsorption capacity does not increase when 150 ml of QyuSpeed ™ D as the pre-treatment adsorbent is heated to 40 ° C. while being wetted with water is shown as a comparative example for Examples 1-3.

  First, the protein adsorption capacity was measured according to the evaluation method 1. The dynamic adsorption capacity was 46.6 mg / mL.

  The inside of the module was filled with water and sealed, and the module was heated to 40 ° C. in that state.

  Thereafter, the protein adsorption capacity was measured according to the evaluation method 1. The dynamic adsorption capacity was 46.6 mg / mL. The performance improvement rate at this time (= dynamic adsorption capacity after heat treatment / dynamic adsorption capacity before heat treatment × 100) was 100%.

[Example 4]
An example of increasing adsorption capacity by heating 150 mL of QyuSpeedTM D as an adsorbent that has not been adsorbed (not adsorbed once) to 40 ° C. while being wetted with 20 wt% ethanol. Show.

  Prepare two unused adsorbents (QyuSpeedTM D 150 mL) that have not adsorbed adsorbents once, one after heat treatment, and the other without heat treatment. Asked.

  About the adsorbent which heat-processes, after filling the inside of a module with 20 weight% ethanol, the module was sealed. In that state, the module was heated for 24 hours in a dryer controlled to 40 ° C. The module was cooled to room temperature. Thereafter, the protein adsorption capacity of the adsorbent subjected to the heat treatment was measured according to the evaluation method 1 described above. The dynamic adsorption capacity was 54.6 mg / mL.

  Subsequently, the protein adsorption capacity of the adsorbent that was not heat-treated was measured according to the evaluation method 1 described above. The dynamic adsorption capacity was 52.0 mg / mL.

  From these results, the performance improvement rate (= dynamic adsorption capacity of adsorbent subjected to heat treatment / dynamic adsorption capacity of adsorbent not subjected to heat treatment × 100) was 105%.

  The present invention provides a method for recovering and increasing the adsorption capacity of a protein adsorbent. The present invention has industrial applicability in purifying a target protein for efficient mass production of antibody drugs.

Claims (17)

A treatment method for improving the protein adsorption capacity of a protein adsorbent,
The protein adsorbent comprises a polymer substrate and a polymer chain that contains a functional group that binds to the surface of the polymer substrate and has a protein adsorption ability.
A processing method comprising a step of heating the protein adsorbent in a wet state with an organic solvent or an aqueous solution containing an organic solvent.   The processing method according to claim 1, wherein the protein adsorbent is heated to 30 to 80 ° C. while being wetted with an organic solvent or an aqueous solution containing an organic solvent.   The processing method according to claim 1 or 2, wherein the organic solvent is at least one selected from the group consisting of alcohols, aromatic hydrocarbons, esters, ethers, and ketones.   The processing method according to claim 3, wherein the alcohol is at least one selected from the group consisting of methanol, ethanol, propanol, butanol, and pentanol.   The processing method according to any one of claims 1 to 4, wherein the polymer chain is bonded to the polymer substrate by a covalent bond.   The processing method according to any one of claims 1 to 5, wherein the polymer chain is a linear polymer chain bonded to the polymer base material by graft polymerization.   The processing method according to any one of claims 1 to 6, wherein the polymer base material is a porous body.   The processing method according to claim 1, wherein the polymer substrate is a hollow fiber porous membrane.   The processing method as described in any one of Claims 1-8 whose functional group which has the said protein adsorption ability is an ion exchange group.   The processing method according to claim 9, wherein the ion exchange group is a tertiary amino group.   The processing method according to any one of claims 1 to 10, wherein the protein adsorbent is an adsorbent obtained by adsorbing and desorbing protein at least once. A method for recovering the protein adsorption capacity of a protein adsorbent,
The protein adsorbent includes a polymer substrate and a polymer chain containing a functional group that binds to the surface of the polymer substrate and has a protein adsorption ability, and adsorbs a protein at least once. And desorbed,
A recovery method comprising the step of heating the protein adsorbent in a wet state with an organic solvent or an aqueous solution containing an organic solvent. A step of obtaining an adsorbent comprising a polymer substrate, and a polymer chain that binds to the surface of the polymer substrate and contains a functional group having protein adsorption ability;
A method for producing a protein adsorbent, comprising heating the adsorbent in a state of being wetted with an organic solvent or an aqueous solution containing an organic solvent.   The manufacturing method of Claim 13 further equipped with the process of adsorb | sucking and desorbing protein with respect to the said adsorbent before the said process to heat.   A protein adsorbent obtainable by the production method according to claim 13 or 14.   A column comprising the protein adsorbent according to claim 15.   A module comprising the protein adsorbent according to claim 15.