WO2017183536A1

WO2017183536A1 - Method for removing protein aggregates

Info

Publication number: WO2017183536A1
Application number: PCT/JP2017/014974
Authority: WO
Inventors: 菜津乃松井; 謙小木戸; 青木　裕史; 米田　正
Original assignee: 昭和電工株式会社
Priority date: 2016-04-18
Filing date: 2017-04-12
Publication date: 2017-10-26
Also published as: JP2019108274A

Abstract

The purpose of the present invention is to produce a protein monomer that is useful as a raw material for medicines and the like in an industrially and economically efficient manner with high yield and high purity. A method for removing protein aggregates, characterized by allowing a mobile phase, e.g., a salt-containing buffer solution that contains a protein monomer and the protein aggregates, to pass through a column on which a porous polymer self-supporting structure having an anion exchange group fixed thereto is carried, thereby adsorbing the aggregates onto the column to collect the monomer in a flow-through manner.

Description

Method for removing protein aggregates

The present invention relates to a method for removing protein aggregates from a solution containing protein monomers and protein aggregates.
This application claims priority based on Japanese Patent Application No. 2016-082707 filed in Japan on April 18, 2016, the contents of which are incorporated herein by reference.

In the manufacturing process of biopharmaceuticals, proteins may form aggregates such as dimers and trimers, and the formed protein aggregates may become impurities derived from the target substance. Proteins are known to associate between molecules in the manufacturing process (concentration, acidic pH exposure, heating operation) and storage processes (solution, frozen solution, freeze-drying) to form aggregates, which reduces the efficacy of the protein. There is concern that it may cause harmful effects on pharmaceuticals, such as development of immunogenicity. Therefore, it is desired to remove the aggregate which is an impurity to such an extent that harmful effects on the human body are eliminated.
Based on this, many methods for removing aggregates by chromatography have been developed for the purpose of purifying pharmaceutical proteins, particularly antibody proteins.

As a reported method, there is a method in which an egg white protein is separated using an anion exchange porous membrane (Patent Document 1). In that case, it is necessary to adsorb all proteins on the anion exchange porous membrane, and separation with a large volume is difficult. Therefore, a method that can be separated more industrially and economically has been demanded.

In addition, a method for purifying antibody monomers using a porous adsorption membrane having anion exchange groups immobilized thereon has been reported (Patent Document 2). However, in the biotechnology industry, mass purification is required not only for antibodies but also for various proteins, and a method that can be used for purification of various protein monomers has been demanded.

Japanese Patent Laid-Open No. 11-12300 JP 2010-241761 A

The present invention has been made in view of the above circumstances, and an object of the present invention is to obtain protein monomers useful as raw materials for pharmaceuticals and the like, industrially and economically in high yield and high purity. More specifically, an object of the present invention is to provide a method for removing protein aggregates from a mixture containing protein monomers and protein aggregates at a high speed to obtain protein monomers at a high yield.

The present inventors conducted extensive studies on a method for removing protein aggregates. As a result, it was found that protein monomers can be recovered under high flow rate conditions by performing chromatography using a column holding a porous polymer self-supporting structure to which an anion exchange functional group is fixed, and the present invention has been completed. .

The present invention is as follows.
[1] Using a salt-containing buffer as a mobile phase, passing through a column holding a porous polymer self-supporting structure to which an anion exchange group is fixed, to thereby remove a mixture containing protein monomers and protein aggregates. A method for removing a protein aggregate, comprising adsorbing the protein aggregate on a column and recovering the protein monomer.
[2] The mixture is a salt-containing buffer containing a protein monomer and a protein aggregate,
The salt-containing buffer containing the protein monomer and the protein aggregate is used as a mobile phase to pass through the column, thereby adsorbing the protein aggregate to the column and recovering the protein monomer by flow-through. The protein aggregate removal method [3] according to [1], wherein the mixture is a buffer containing the protein monomer and the protein aggregate,
Loading the buffer containing the protein monomer and the protein aggregate onto the column by supplying the buffer with the protein monomer and the protein aggregate;
Elution and recovery of the protein monomer while adsorbing the protein aggregate by passing the salt-containing buffer as a mobile phase through the column loaded with the protein monomer and the protein aggregate And the method for removing a protein aggregate according to [1].
[4] The removal method according to any one of [1] to [3], wherein the monomers for constituting the porous polymer self-supporting structure are glycidyl methacrylate and alkylene dimethacrylates.
[5] The removal method according to [4], wherein monomers for constituting the porous polymer self-supporting structure are glycidyl methacrylate and ethylene glycol dimethacrylate.
[6] The removal method according to any one of [1] to [5], wherein the anion exchange group is a quaternary ammonium group.
[7] The removal method according to any one of [1] to [6], wherein the porous polymer self-supporting structure has a thickness in the liquid passing direction of 1 to 100 mm.
[8] The removal method according to any one of [1] to [7], wherein the flow rate of the mobile phase is 2 CV / min or more.
[9] The removal method according to [1] to [8], wherein the protein is albumin or immunoglobulin.

According to the method for removing protein aggregates of the present invention, protein monomers useful as raw materials for pharmaceuticals and the like can be obtained industrially and economically with high yield and high purity. More specifically, the protein aggregate can be removed at a high speed from the mixture containing the protein monomer and the protein aggregate, and the protein monomer can be obtained in a high yield.

Hereinafter, preferred embodiments for carrying out the present invention will be described. In addition, embodiment described below shows an example of typical embodiment of this invention, and this invention is not limited to them.

[Method for removing protein aggregates]
The method for removing a protein aggregate of the present invention uses a salt-containing buffer as a mobile phase and passes it through a column holding a porous polymer self-supporting structure having an anion exchange group immobilized thereon, thereby The protein aggregate is adsorbed on a column from a mixture containing the protein aggregate, and the protein monomer is recovered.

<Anion exchange group>
In the present embodiment, the anion exchange group is not particularly limited as long as it can adsorb a negatively charged protein or the like in the liquid. For example, a diethylamino group, a quaternary ammonium group, a quaternary aminoethyl group, Examples thereof include a diethylaminoethyl group and a diethylaminopropyl group. As the anion exchange group, a diethylamino group and a quaternary ammonium group are preferable, and a quaternary ammonium group is more preferable.
For example, the structure of a quaternary ammonium group is illustrated by the following chemical formula (1).

(Wherein R ₁ , R ₂ , R ₃ and R ₄ are each independently H, a hydrocarbon group such as a methyl group, an ethyl group, a methylene group or an ethylene group, or a hydrocarbon having a functional group such as a hydroxyl group. An aromatic alkylene group such as a benzyl group or the like, and at least one is a divalent group such as an alkylene group.)
R ₁ , R ₂ , R ₃ and R ₄ are each preferably a methyl group, an alkyl group or a propyl group, more preferably a methyl group.

<Porous polymer self-supporting structure>
The porous polymer self-supporting structure is a crosslinked polymer structure and is porous. The self-supporting structure means a structure that is not in the form of particles or the like but is itself a lump and can retain its shape. In this porous polymer self-supporting structure, a porogen (described later) is added to a monomer having two or more ethylenic double bonds (polyvinyl monomer) and a monomer having one ethylenic double bond (monovinyl monomer). Can be obtained by bulk polymerization. It becomes porous by removing the inert solvent. The porous pores have a uniform pore size distribution throughout. The pore diameter is in the range of 1 to 1000 nm. The shape is an integral structure such as a plate, a tube, or a cylinder.

As the monomer for constituting the porous polymer self-supporting structure, for example, a polyvinyl monomer and a monovinyl monomer can be used.

Examples of polyvinyl monomers include divinylbenzene, divinylnaphthalene, divinylpyridine, dimethacrylates, diacrylates, vinyl esters, vinyl ethers such as divinyl ether, alkylene bisacrylamides such as ethylene bisacrylamide and propylene bisacrylamide, and mixtures thereof. Can be used.
Examples of the dimethacrylates include alkylene dimethacrylates such as ethylene glycol dimethacrylate and propylene glycol dimethacrylate. Furthermore, pentaerythritol di-, tri- or tetramethacrylate, trimethylolpropane trimethacrylate or acrylate can be used.
Diacrylates include ethylene glycol diacrylates. Further, pentaerythritol di-, tri-, or tetraacrylate can be used.

Monovinyl monomers include styrene, substituted styrene (wherein the substituent is a chloromethyl group, an alkyl group having up to 18 carbon atoms, a hydroxyl group, a t-butyloxycarbonyl group, a halogen group, a nitro group, an amino group, a protected hydroxyl group or (Including amino groups), vinyl naphthalene, acrylic esters such as ethyl acrylate, methacrylic esters such as glycidyl methacrylate, methyl methacrylate, hydroxyethyl methacrylate, vinyl acetate and N-vinyl pyrrolidone, and mixtures thereof can do.

As the monovinyl monomer constituting the porous polymer self-supporting structure, a monomer having a functional group capable of holding an anion exchange group is preferable. Specific examples include glycidyl methacrylate and alkylene dimethacrylates, preferably glycidyl methacrylate and ethylene glycol dimethacrylate.

Specific examples of porous polymer self-supporting structures include CIM (registered trademark) QA DISK, QA-1 Tube Monolithic Column, CIM QA-8 Tube Monolithic Column, CIM QA-80 Tube Monolithic Column Q Tube Monolithic Column, CIM q, A-8000 Tube Monolithic Column, DEAE DISK, DEAE-1Tube Monolothic Column, CIM DEAE-8 Tube Monolithic Column, CIM QA-80 Tube Monolithic Column, CIM QA-800 Tube Monolithic Column, CIM DEAE- 8 00 Tube Monolithic Column and the like. These are all modified products of copolymers of glycidyl methacrylate and ethylene glycol dimethacrylate. This copolymer is produced from a mixture of glycidyl methacrylate and ethylene glycol dimethacrylate in the presence of a porogen and a polymerization initiator.
Porogen is an additive material for forming pores, such as aliphatic hydrocarbons, aromatic hydrocarbons, esters, alcohols, ketones, ethers, soluble polymers, and mixtures thereof. Different types of materials can be used. Normal hexane is preferred.

A free radical generating initiator can be used as the polymerization initiator. Specifically, azo compounds such as azobisisobutyronitrile and 2,2′-azobis (isobutylamide) dihydrate, and peroxides such as benzoyl peroxide and dipropyl dicarboxylic acid ester can be used. When different types of polymerization initiators are used, pore structures having different shapes can be formed. The amount of the polymerization initiator is preferably 0.5 to 4 parts by mass with respect to 100 parts by mass of the monomer.
When producing the copolymer, a soluble polymer may be added as a porogen. The soluble polymer is dissolved and removed by a washing solvent after polymerization. When a soluble polymer is added, more pore structures are formed. The amount of the soluble polymer is preferably 10 to 40 parts by mass with respect to 100 parts by mass of the whole monomer.
The mixture of glycidyl methacrylate and ethylene glycol dimethacrylate containing a porogen or polymerization initiator is preferably degassed using an inert gas such as nitrogen or argon before being placed in the mold. The mold is preferably sealed to prevent air contamination.
The polymerization can be performed, for example, by heating at a temperature of 50 ° C. to 90 ° C. for 40 to 50 hours.
After the polymerization, the tube is washed, and the solvent and soluble polymer used as the porogen are removed. As a solvent for washing, methanol, ethanol, benzene, toluene, acetone, tetrahydrofuran or the like can be used. The washing process may be repeated a plurality of times.
As an example of introducing a quaternary ammonium group as an anion exchange group, a quaternary trimethylamino group can be introduced using triethylamine hydrochloride.

This structure, when left standing, retains mechanical strength that can support its shape. That is, the physical structure is different from that using a bead-like porous polymer carrier or a porous membrane.

The thickness of the porous polymer self-supporting structure in the liquid passing direction is preferably 1 to 100 mm. The thickness is preferably 2 to 70 mm, more preferably 3 to 60 mm, and still more preferably 3 to 50 mm. By setting the thickness of the porous polymer self-supporting structure to 1 mm or more, it has sufficient mechanical strength. By setting the thickness of the porous polymer self-supporting structure to 100 mm or less, it is possible to maintain a pressure that does not cause the porous polymer self-supporting structure to be destroyed and to prevent an increase in pump pressure.

<Column>
The column includes a porous polymer self-supporting structure having an anion exchange group immobilized thereon. That is, the column may be composed of only a porous polymer self-supporting structure having a plate shape, a tubular shape, a cylindrical shape, etc., and a predetermined amount of the porous polymer self-supporting structure is formed into a cylindrical shape. It may be accommodated (held) in a container.
The column size (column volume) is not particularly limited, and is appropriately adjusted according to the amount of protein aggregate to be adsorbed.

<Protein>
The protein capable of removing protein aggregates by the method of the present invention is not particularly limited, but is preferably an aggregating protein that easily causes aggregation in an aqueous solution. The likelihood of aggregation of an aggregating protein depends on the composition of amino acids constituting the protein, its sequence, and steric configuration. Protein structures that are prone to aggregation include proteins in which amino acids with hydrophilic side chains are localized on a part of the surface of the molecule, and specific higher-order structures that induce association by hydrogen bonding force, such as β sheets. Examples thereof include a protein presented on the surface, a protein having a chelating site by binding of metal ions and the like. These are not limited to proteins having these structures as a result of the proper folding of the respective proteins, but the pH and hydrophobicity of the solution, presence of denaturing agents, denaturation caused by physical actions such as heating, shaking and stirring, and charge Proteins altered to have these structures as a result of neutralization and the like are also included. Proteins having these structures include IgG, IgA, IgM and other immunoglobulins, interleukins, chemokines, interferons, G-CSF, erythropoietin, EGF, FGF, TGF, BDNF, VEGF, GM-CSF, PDGF, EPO, Cytokines such as TPO, bFGF, HGF, TNF-α, TGF-β, PAI-1, HB-EGF, leptin, adiponeptin, NGF, protein hormones such as human growth hormone, insulin, glucagon, blood coagulation factors, albumin , Lysozyme, RNase A, cytochrome c and the like. Among them, proteins with highly hydrophobic amino acids oriented outside the molecule, or proteins in which such amino acids are localized on a part of the surface of the molecule, anions that tend to cause electrostatic association between molecules based on dipolarity More preferred are proteins in which amino acids having a cationic or cationic charge are localized and have a polarization tendency. Specific examples of such proteins include immunoglobulins such as IgG, IgA, and IgM, and albumin.

<Protein monomer>
A protein monomer means one protein molecule.

<Protein aggregate>
A protein aggregate is a protein complex in which protein monomers are adsorbed and aggregated reversibly or irreversibly by hydrophobic interaction, electrostatic interaction, and other interactions.

(First embodiment)
<Adsorption of protein aggregate to column and flow through of protein monomer>
As a first embodiment of the present invention, a protein monomer and a protein aggregate are dissolved in a salt-containing buffer to prepare a solution (hereinafter, this solution is referred to as “protein solution”), and this solution is used as a mobile phase. As a result, the protein aggregate can be adsorbed to the column and the protein monomer can be recovered by flow-through by passing the solution through a column holding the porous polymer self-supporting structure to which the anion exchange group is fixed. The salt-containing buffer used for the protein solution is preferably a salt-containing buffer in which an inorganic salt is dissolved in the buffer.

<Flow-through style>
The flow-through mode means that an object to be collected passes through without being adsorbed on the chromatography device. In the present invention, protein aggregates are adsorbed to a column and protein monomers are recovered in this manner.

Although it does not restrict | limit especially as a buffer solution, For example, a phosphate buffer solution, a citrate buffer solution, a tris (trishydroxymethylaminomethane) buffer solution, an acetate buffer solution, a borate buffer solution etc. can be used. Among these, a phosphate buffer solution, a citrate buffer solution, and a Tris buffer solution are preferable from the point of use pH range having buffer capacity.
The concentration of the buffer is not particularly limited, but is preferably 1 to 100 mM, more preferably 2 to 50 mM, and further preferably 5 to 30 mM.
The pH of the buffer solution is not particularly limited, but is preferably 2 to 9, more preferably 3 to 8, and still more preferably 4 to 7.5.

<Salt>
Examples of the salt used in the salt-containing buffer include, but are not limited to, sodium chloride, sodium sulfate, sodium acetate, ammonium sulfate, and metal salts of citric acid, phosphoric acid, or glycine. Sodium chloride, sodium sulfate, sodium acetate, ammonium sulfate are preferable, and sodium chloride is more preferable.

The salt concentration is preferably an amount sufficient to adsorb only protein aggregates without adsorbing protein monomers on the column. The amount is preferably small enough not to cause binding or precipitation of protein monomers and protein aggregates. Specifically, when sodium chloride is used as the salt, the concentration relative to the buffer is preferably 0.05 to 0.50M, more preferably 0.05 to 0.30M, and even more preferably 0.10 to 0.30M. It is. For each purification process, it is preferable to select the optimum amount and preferred type of salt by preliminary experiments and the like.

When the protein is dissolved in a salt-containing buffer, the concentration is preferably 0.01 to 10 mg / mL, more preferably 0.1 to 5 mg / mL, and still more preferably 0.2 to 3 mg / mL.
The protein loading per mL of column volume is preferably 250 μg to 60 mg, more preferably 500 μg to 50 mg, and even more preferably 750 μg to 40 mg.

Before passing the protein solution through the column, it is preferable to equilibrate the column with a buffer solution.
The kind, concentration, and pH of the buffer used for equilibration can be the same as the salt-containing buffer that dissolves the protein.
The amount of the buffer solution required for equilibration is not particularly limited, but is preferably 1 CV (column volume multiple) or more, more preferably 2 CV or more, and further preferably 4 CV or more.

When the protein solution is passed through the column, the temperature of the column and the protein solution is not particularly limited, but is preferably 2 to 50 ° C., more preferably 4 to 40 ° C., and still more preferably 8 to 30 ° C. By setting it within this range, freezing of protein solution and destruction of protein can be prevented.

The flow rate of the protein solution is not limited as long as the purpose can be achieved, but is preferably 2 to 12.5 CV / min, more preferably 2.5 to 5 CV / min, and further preferably 4 to 5 CV / min.

In the present invention, the pH range of the protein solution is not limited. Moreover, this invention may be performed independently and may be performed after the refinement | purification process by an ion exchange chromatography and affinity chromatography.
Therefore, the present invention can obtain a protein monomer in high yield and high purity under more free conditions.

If the conditions for selectively recovering protein monomers are examined using the above knowledge, the conditions can be shortened. In particular, in the present invention, even if the thickness of the column passage direction or the column diameter is changed, the ion exchange groups, constituent monomers, temperature, pressure, buffer solution, salt-containing buffer solution, mobile phase flow rate of the column are changed. Since it is possible to purify without changing the conditions such as (CV / min), it is possible to shorten the time required for studying the conditions.

<Elution of protein aggregates and column regeneration>
In addition, after eluting a protein monomer by the above-mentioned method, a protein aggregate can be eluted by letting the salt containing buffer solution which raised salt concentration pass to a column. After eluting the protein aggregate, the column can be regenerated by passing again the same buffer as that used for equilibration.

(Second embodiment)
In one embodiment of the present invention, after the column equilibration, the “loading of protein monomer and protein aggregate to the column” step and the “elution of protein monomer” step can be performed separately.

In the method for removing a protein aggregate according to the second embodiment, a buffer solution containing a protein monomer and a protein aggregate is supplied to a column holding a porous polymer self-supporting structure to which an anion exchange group is fixed. A step of loading the protein monomer and the protein aggregate onto the column, and adsorbing the protein aggregate by passing a salt-containing buffer through the column loaded with the protein monomer and the protein aggregate, And a step of eluting and recovering the protein monomer.

As the buffer solution and salt used in this step, the buffer solution and salt used in the first embodiment can be used.
The type, concentration, and pH of the buffer can be the same as the buffer used to equilibrate the column, and may be different, but are preferably the same.

When the salt-containing buffer is passed through the column as a mobile phase, the temperature of the column and the mobile phase is not particularly limited, but is preferably 2 to 50 ° C., more preferably 4 to 40 ° C., and still more preferably 8 to 30 ° C. . By setting it in this range, freezing of protein solution and destruction of protein can be prevented.

<Elution of protein aggregates and column regeneration>
In the second embodiment of the present invention, the protein aggregates can be eluted and the column regenerated as in the first embodiment of the present invention.

Hereinafter, the effects of the present invention will be made clearer by examples. In addition, this invention is not limited to a following example, In the range which does not change the summary, it can change suitably and can implement.
In the examples, recovery rate, purity, and aggregate content are defined as follows, using albumin as an example of protein.
<Recovery rate>
The recovery rate of the present invention is the albumin monomer recovered in the flow-through mode with respect to the total amount of albumin exiting the column in the flow-through mode and the total amount of albumin aggregate adsorbed on the column, ie, the sum of albumin and albumin aggregates. Calculated as the ratio of Specifically, the value obtained by dividing the area of the flow-through part on the chromatogram by the sum of the areas of the flow-through part and the protein aggregate elution part by the salt-containing buffer during column regeneration is defined as the recovery rate.
<Purity>
The purity of the present invention is calculated as the ratio of albumin monomer to the total amount of albumin in the collected sample, ie the sum of albumin monomer and albumin aggregate. Specifically, the amount recovered by flow-through is determined by measuring using size exclusion chromatography with Shodex (registered trademark) KW403-4F manufactured by Showa Denko KK. Purity is the value obtained by dividing the peak area of the protein monomer on the chromatogram by the area of the total amount of albumin. The total albumin area is the sum of the aggregate peak area and the monomer peak area. For preparation of the chromatogram, a UV-VIS detector SPD-M30A manufactured by Shimadzu Corporation was used. The unit is%.
<Aggregate content>
The aggregate content of the present invention is defined as 100-purity (%), where 100 is a state where no aggregate is contained. Purity (%) is calculated as an area ratio in the chromatogram.

<Example 1>
In the following steps (1) to (4), albumin monomer was recovered from the mixed solution containing albumin monomer and albumin aggregate, and the albumin aggregate was removed.
(1) Equilibration of column CIM (registered trademark) QA-1 Tube manufactured by BIA separations (thickness: 4.2 mm, outer diameter: 18.6 mm, inner diameter: 6.7 mm, porosity: 60 v / v%) Then, 20 mM phosphate buffer (pH 7.4) containing 0.10 M sodium chloride was allowed to pass through 5 CV or more for equilibration. CIM QA-1 Tube is a modified product of a copolymer of glycidyl methacrylate and ethylene glycol dimethacrylate, and is a porous polymer self-supporting structure in which quaternary ammonium groups are held in some glycidyl methacrylates. is there. Further, since the central hole of the column is blocked by the structure of the apparatus, the injected liquid does not pass through the central hole of the column as it is.
(2) Albumin monomer and albumin aggregate 10 mg of human serum albumin (manufactured by Sigma Aldrich) was dissolved in 10 mL of a pH 7.4 20 mM phosphoric acid solution containing 0.10 M sodium chloride. This solution is defined as an albumin solution. The aggregate content of this solution was approximately 85%. 10 mL of this solution was injected into the column equilibrated in (1) and purified. That is, the albumin load per 1 mL of column volume is 10 mg. The solution flow rate was 5 CV / min.
(3) Flow-through of albumin Albumin monomer when 10 mL of the albumin solution was passed was collected in a flow-through manner. The solution flow rate was 5 CV / min.
(4) Elution of albumin aggregate A 20 mM phosphate buffer (pH 7.4) containing 4 M sodium chloride was then passed through a 7.5 mL column to elute the albumin aggregate. The solution flow rate was 5 CV / min. In Example 1, the temperature of the column, protein solution, and mobile phase was 25 ° C., and the column pressure was 0.3 to 2 MPa.

<Example 2>
The same operation as in Example 1 was performed except that the sodium chloride concentration in Step (2) of Example 1 was 0.15M.
<Example 3>
The same procedure as in Example 1 was conducted except that the albumin load per mL of column volume in step (2) of Example 1 was changed to 30 mL (30 mg) and the sodium chloride concentration was changed to 0.07M.

<Example 4>
CIM (registered trademark) DEAE-1 Tube (thickness: 4.2 mm, outer diameter: 18.6 mm, inner diameter: 6.7 mm, porosity: 60 v / v%) was used as the column in step (1) of Example 1. The same operation as in Example 1 was carried out except that it was used. CIM DEAE-1 Tube is a porous polymer self-supporting structure in which diethylamino groups are held in a copolymer of glycidyl methacrylate and ethylene glycol dimethacrylate based on product information.
<Example 5>
The same procedure as in Example 1 was performed except that the pH of the buffer solution in step (2) of Example 1 was 7.0.
The results of Examples 1 to 5 are shown in Table 1.

<Comparative Example 1>
As the porous polymer beads constituting the column, strong anion exchange chromatography HiTrap Q FF (average particle size 90 μm) manufactured by GE Healthcare was used, and the flow rate of the protein solution in the column was 1 CV / min. In the same manner as in Example 1, albumin monomer was eluted.

<Comparative Example 2>
Example except that HiTrap Q FF (average particle size 90 μm) manufactured by GE Healthcare was used as the porous polymer beads constituting the column, and the flow rate of the protein solution in the column was 2 CV / min. In the same manner as in 1, the protein solution was injected into the column. As a result, the column pressure increased, exceeded the column allowable pressure, and could not be operated. The results of Comparative Examples 1 and 2 are shown in Table 1.

In Comparative Example 1 using porous polymer beads, the elution flow rate of the protein solution was set to about 1/5 of the example, and the recovery rate and purity comparable to those of the example were achieved. However, in Comparative Example 2 in which the elution flow rate was increased to about 2/5 of the example, the process could not be continued because the allowable pressure was exceeded.

Claims

From the mixture containing protein monomers and protein aggregates, the protein is passed through a column holding a porous polymer self-supporting structure having anion exchange groups immobilized, using a salt-containing buffer as a mobile phase. A method for removing a protein aggregate, comprising adsorbing the aggregate to a column and recovering the protein monomer.
The mixture is a salt-containing buffer containing protein monomers and protein aggregates,
The salt-containing buffer containing the protein monomer and the protein aggregate is used as a mobile phase to pass through the column, thereby adsorbing the protein aggregate to the column and recovering the protein monomer by flow-through. The method for removing a protein aggregate according to claim 1.
The mixture is a buffer containing the protein monomer and the protein aggregate,
Loading the buffer containing the protein monomer and the protein aggregate onto the column by supplying the buffer with the protein monomer and the protein aggregate;
Elution and recovery of the protein monomer while adsorbing the protein aggregate by passing the salt-containing buffer as a mobile phase through the column loaded with the protein monomer and the protein aggregate When,
The method for removing a protein aggregate according to claim 1, comprising:
The removal method according to any one of claims 1 to 3, wherein monomers for constituting the porous polymer self-supporting structure are glycidyl methacrylate and alkylene dimethacrylates.
The removal method according to claim 4, wherein the alkylene dimethacrylate is ethylene glycol dimethacrylate.
The removal method according to any one of claims 1 to 5, wherein the anion exchange group is a quaternary ammonium group.
The removal method according to any one of claims 1 to 6, wherein a thickness of the porous polymer self-supporting structure in a liquid passing direction is 1 to 100 mm.
The removal method according to any one of claims 1 to 7, wherein the flow rate of the mobile phase is 2 CV / min or more.
The removal method according to any one of claims 1 to 8, wherein the protein is albumin or immunoglobulin.