JP2014129319A - Purification method of antibody proteins - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a purification method for an antibody protein which can efficiently purify the antibody protein.SOLUTION: The method for purifying the antibody protein using a temperature-responsive cationic ion-exchange resin which adsorbs an antibody protein on its high temperature side and desorbs on the low temperature side. And the method comprises allowing the temperature-responsive cationic ion-exchange resin to adsorb a monomer component of the antibody protein and a component of the aggregate equal to or more than the dimer of the antibody protein comprised in a solution, in a first temperature region where the temperature-responsive cationic ion-exchange resin adsorbs the antibody protein, and allowing the temperature-responsive cationic ion-exchange resin to desorb the monomer component of the antibody protein using an eluate from the second temperature region where the temperature-responsive cationic ion-exchange resin desorbs the monomer component of the antibody protein.

Description

  The present invention relates to a biomolecule purification technique, and to a method for purifying antibody proteins.

  An immunoglobulin (antibody protein) is a physiologically active substance that controls an immune reaction. In recent years, the usefulness of antibody proteins has been increasing in applications such as pharmaceuticals, diagnostics, and corresponding antigen protein separation and purification materials. The antibody protein is obtained from the blood of an immunized animal or a cell culture medium of cells possessing antibody-producing ability or an ascites culture medium of an animal. However, blood and culture fluid containing antibody protein includes impurities other than antibody protein or impurity components such as complicated contaminant components derived from raw material fluid used for cell culture. Therefore, in order to separate and purify antibody proteins from impurity components, a complicated and time-consuming operation is usually necessary.

  Liquid chromatography is important for the separation and purification of antibody proteins. Chromatographic techniques for separating antibody proteins include gel filtration chromatography, affinity chromatography, ion exchange chromatography, reverse phase chromatography, and the like, and antibody proteins are separated and purified by combining these techniques.

  Ion exchange chromatography is a method in which separation is performed by reversibly adsorbing counter ions present in a mobile phase using an ion exchange group on the surface of a substrate as a stationary phase. As the shape of the substrate, beads, membranes such as flat membranes, hollow fibers, etc. are adopted, and those obtained by binding cation exchange groups or anion exchange groups to these substrates as solid adsorbents (stationary phase) It is commercially available. A solid adsorbent having a cation exchange group has a characteristic of mainly adsorbing antibody proteins and allowing most of other impurities to pass through, and is therefore used for concentration separation of antibody proteins.

  Cation exchange groups are roughly classified into weak cation exchange groups such as carboxyl groups and strong cation exchange groups such as sulfonic acid groups. The adsorbent having a weak cation exchange group has a drawback that the charge of the adsorbent surface changes when the pH of the mobile phase changes, and the binding capacity of the antibody protein varies. Therefore, when an adsorbent having a weak cation exchange group is used for separation and purification of antibody protein, the reproducibility of separation is poor, and the recovery rate of antibody protein may be lowered. On the other hand, the adsorbent having a strong cation exchange group does not change the binding capacity of the antibody protein because the charge on the adsorbent surface does not change even when the pH of the mobile phase varies. Although it is difficult to keep the pH of the mobile phase constant in an industrial antibody protein separation and purification process, on the other hand, since reproducibility of separation is strictly required, an adsorbent having a strong cation exchange group is required. It is used.

  In the conventional method for purifying antibody protein using an adsorbent having an ion exchange group, by increasing the salt concentration of the mobile phase, the electrostatic interaction between the antibody protein and the adsorbent is weakened, and the stationary phase It is a common practice to elute antibody proteins adsorbed on the gel. Here, the higher the salt concentration of the mobile phase, the easier the antibody protein elution tends to be. However, when the salt concentration of the mobile phase increases, the hydrophobic interaction between proteins increases, and protein aggregates and aggregates tend to occur. Thus, there is a trade-off relationship between the elution of the antibody protein and the likelihood of formation of aggregates. Therefore, it is difficult to set the salt concentration of the mobile phase that satisfies various conditions required for purification, such as the yield, concentration, and elution rate of the antibody protein.

  Therefore, in order to solve the problems of conventional adsorbents having ion exchange groups, when eluting physiologically active substances such as antibody proteins adsorbed on the stationary phase, the salt concentration of the mobile phase is not increased. A temperature-responsive adsorbent capable of changing the effective ion exchange group density depending on the temperature and eluting the physiologically active substance has been proposed.

  Patent Document 1 discloses a packing containing a charged copolymer that can change the effective charge density on the surface of the stationary phase according to temperature change, a production method, and a temperature-responsive chromatography method using the same. Has been. Patent Document 2 discloses a stationary phase of temperature-responsive chromatography in which a polymer whose hydration power changes within a temperature range of 0 to 80 ° C. is immobilized at a high density on the surface of the substrate by an atom transfer radical polymerization method. ing. Patent Document 3 discloses a temperature response characterized by causing a growth reaction of a polymer having charge and changing hydration power within a temperature range of 0 to 80 ° C. by an atom transfer radical method using isopropyl alcohol as a solvent. A method for producing a chromatographic chromatography carrier is disclosed. In Patent Document 4, a high molecular weight physiologically active substance useful in the fields of biology, medicine, pharmacy and the like can be separated under a specific condition including an aqueous mobile phase, within a temperature range of 0 to 80 ° C. A method for producing a carrier for liquid chromatography in which a charged polymer with varying hydration power is fixed is disclosed. Non-Patent Document 1 discloses a temperature-responsive chromatography carrier having a carboxyl group prepared by an atom transfer radical polymerization method and a method for producing the same. Among them, a monomer composition optimized for the separation of lysozyme in the monomer composition used in the atom transfer radical polymerization method is disclosed.

International Publication No. 99/061904 Pamphlet JP 2007-69193 A JP 2009-85933 A International Publication No. 01/074822 Pamphlet

Polymer Preprints, Japan Vol. 58, no. 2,3T1-13 (2009)

  However, even using the methods disclosed in the above-mentioned literature, the antibody protein cannot be purified efficiently. Then, an object of this invention is to provide the purification method of the antibody protein which can refine | purify an antibody protein efficiently.

  In order to solve the above problems, the present inventors have studied and developed from various angles. As a result, the present inventors adsorbed the antibody protein to the temperature-responsive cationic ion exchange resin on the high temperature side, and desorbed the antibody protein from the temperature-responsive cationic ion exchange resin on the low temperature side. Since the electrostatic interaction between the antibody protein and the temperature-responsive cationic ion exchange resin is weakened, the hydrophobic interaction between the proteins is also weakened. It was found that the antibody protein can be purified.

  The present invention based on the knowledge of the present inventors is a method for purifying an antibody protein using a temperature-responsive cationic ion exchange resin in which the antibody protein is adsorbed on the high temperature side and desorbed on the low temperature side. The antibody protein monomer component and the antibody protein dimer or higher aggregate component contained in the solution are in a first temperature range where the antibody protein is adsorbed to the temperature-responsive cationic ion exchange resin, Adsorbing to a temperature-responsive cationic ion exchange resin, and using an eluate in a second temperature region in which the monomer component of the antibody protein is desorbed from the temperature-responsive cationic ion exchange resin, the temperature-responsive cation The gist of the present invention is a method for purifying an antibody protein, which comprises desorbing a monomer component of an antibody protein from a cationic ion exchange resin.

  According to the method for purifying an antibody protein according to the present invention, it is possible to efficiently purify the antibody protein.

2 is a graph of absorbance when the fraction eluted from the protein A column according to Example 1 was subjected to size exclusion chromatography. 2 is a graph of absorbance of an eluate from a column packed with a temperature-responsive adsorbent according to Example 1. FIG. FIG. 3 is a graph of absorbance when the fraction eluted with “1” in FIG. 2 is subjected to size exclusion chromatography. FIG. 3 is a graph of absorbance when the elution fraction of the portion indicated by “2” in FIG. 2 is subjected to size exclusion chromatography. FIG. 3 is a graph of absorbance when the elution fraction of a portion indicated by “3” in FIG. 2 is subjected to size exclusion chromatography. FIG. 3 is a graph of absorbance when the elution fraction of the portion indicated by “4” in FIG. 2 is subjected to size exclusion chromatography. FIG. 3 is a graph of absorbance when the fraction eluted with “5” in FIG. 2 is subjected to size exclusion chromatography. Photo of the result of electrophoresis of the elution fraction from the column packed with the temperature-responsive adsorbent according to Example 1 and the elution fraction from the column packed with SP Sepharose (trademark) Fast Flow according to Comparative Example 1 It is. 4 is a graph of the absorbance of an eluate from a column packed with SP Sepharose (trademark) Fast Flow according to Comparative Example 1. FIG. 10 is a graph of absorbance when the elution fraction of the portion indicated by “1” in FIG. 9 is subjected to size exclusion chromatography. FIG. 10 is a graph of absorbance when the elution fraction of the portion indicated by “2” in FIG. 9 is subjected to size exclusion chromatography. FIG. 10 is a graph of absorbance when the elution fraction of the portion indicated by “3” in FIG. 9 is subjected to size exclusion chromatography. FIG. 10 is a graph of absorbance when the elution fraction of the portion indicated by “4” in FIG. 9 is subjected to size exclusion chromatography. FIG. 10 is a graph of absorbance when the elution fraction of the portion indicated by “5” in FIG. 9 is subjected to size exclusion chromatography. FIG. 10 is a graph of absorbance when the elution fraction of the portion indicated by “6” in FIG. 9 is subjected to size exclusion chromatography. 3 is a graph of absorbance when an elution fraction from a protein A column according to Example 2 was subjected to size exclusion chromatography. It is a graph of the light absorbency when the elution fraction of Example 2 is applied to size exclusion chromatography.

  Hereinafter, preferred embodiments of the present invention (hereinafter referred to as “embodiments”) will be described in detail. The following embodiments exemplify apparatuses and methods for embodying the technical idea of the present invention, and the technical idea of the present invention specifies combinations of components and the like as follows. Not what you want. The technical idea of the present invention can be variously modified within the scope of the claims.

  In the method for purifying antibody protein according to the embodiment, a temperature-responsive cationic ion exchange resin that adsorbs antibody protein on the high temperature side and desorbs antibody protein on the low temperature side is used as a solid adsorbent (fixed) for ion exchange chromatography. Phase).

  An antibody protein purification method according to an embodiment includes an antibody protein solution containing a monomer component of an antibody protein and an aggregate component of a dimer or more of the antibody protein, wherein the antibody protein is a temperature-responsive cationic ion exchange resin. Adsorbing the antibody protein to the temperature-responsive cationic ion exchange resin in the first temperature region adsorbed on the second phase, and the second step in which the monomer component of the antibody protein is desorbed from the temperature-responsive cationic ion exchange resin. Desorbing the monomer component of the antibody protein from the temperature-responsive cationic ion exchange resin using an eluate in the temperature range. Here, in the second temperature region, the monomer component of the antibody protein is desorbed, and the monomer component of the antibody protein can be isolated.

  The first temperature region (hereinafter also referred to as “high temperature region”) where the antibody protein is adsorbed to the temperature-responsive cationic ion exchange resin is, for example, 30 ° C. or more and less than 60 ° C., preferably 30 ° C. or more and less than 50 ° C. Most preferably, it is 30 ° C. or higher and lower than 45 ° C. The second temperature region (hereinafter also referred to as “low temperature region”) where the antibody protein is desorbed from the temperature-responsive cationic ion exchange resin is, for example, 0 ° C. or higher and lower than 30 ° C., preferably 10 ° C. or higher. It is less than 30 ° C, more preferably 15 ° C or more and less than 30 ° C, and still more preferably 20 ° C or more and less than 27 ° C.

  The antibody protein to be purified by the method according to the embodiment is a glycoprotein molecule (also referred to as gamma globulin or immunoglobulin) produced by B lymphocytes as a vertebrate infection prevention mechanism, as is generally defined in biochemistry. It is. For example, an antibody protein purified by the method according to the embodiment is used as a human pharmaceutical and has substantially the same structure as an antibody protein in a human body to be administered.

  The antibody protein may be a human antibody protein, or may be an antibody protein derived from mammals such as non-human bovines and mice. 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 region is substituted with a human-derived immunoglobulin. In addition, the humanized antibody protein has a complementarity-determining region (CDR) in a variable region derived from an organism other than a human, but other framework regions (FR) are derived from a human. It is an antibody protein. Humanized antibody proteins are further reduced in immunogenicity than chimeric antibody proteins.

  The class (isotype) and subclass of the antibody protein to be purified by the method according to the embodiment is not particularly limited. For example, antibody proteins are classified into five classes, IgG, IgA, IgM, IgD, and IgE, depending on the structure of the constant region. However, the antibody protein to be purified by the method according to the embodiment may be any of the five classes. In human antibody proteins, IgG has four subclasses IgG1 to IgG4, and IgA has two subclasses IgA1 and IgA2. However, any subclass of the antibody protein to be purified by the method according to the embodiment may be used. Note that antibody-related proteins such as Fc fusion proteins in which a protein is bound to the Fc region can also be included in the antibody protein to be purified by the method according to the embodiment.

  Furthermore, antibody proteins can also be classified by origin. However, the antibody protein to be purified by the method according to the embodiment is any of a natural human antibody protein, a recombinant human antibody protein produced by gene recombination technology, a monoclonal antibody protein, and a polyclonal antibody protein. Also good. Among these antibody proteins, human IgG is preferable from the viewpoint of demand and importance as an antibody drug.

  The solid adsorbent including the temperature-responsive cationic ion exchange resin used in the purification method according to the embodiment includes, for example, a base material and a copolymer containing N-isopropylacrylamide and the like fixed on the base material surface. Including. The copolymer has at least a cationic ion exchange group. For example, the temperature-responsive cationic ion exchange resin according to the embodiment is a monomer mixture comprising a monomer having a cationic ion exchange group and / or a precursor monomer for introducing a cationic ion exchange group, and an N-isopropylacrylamide monomer. Is polymerized on the surface of the substrate by a surface graft polymerization method.

  The shape of the substrate used in the embodiment is not particularly limited, but may be, for example, a bead shape, a flat plate shape, or a tubular shape. When the base material has a bead shape, beads having various particle sizes are available. The particle size of the bead-shaped substrate is not particularly limited, but is, for example, 1 to 300 μm, preferably 10 to 200 μm, and more preferably 20 to 150 μm. When the particle size is 1 μm or less, since consolidation of beads tends to occur in the column filled with the temperature-responsive cationic ion exchange resin, it tends to be difficult to pass the solution through the column at high speed. On the other hand, when the particle size is 300 μm or more, the gap between the beads becomes large, and when the antibody protein is adsorbed on the temperature-responsive cationic ion exchange resin, the solution tends to leak.

  The base material used in the embodiment has, for example, a plurality of pores. Although a pore diameter is not specifically limited, For example, it is 5-1000 nm, Preferably it is 10-700 nm, More preferably, it is 20-500 nm. If the pore diameter is 5 nm or less, the molecular weight of the separable antibody protein tends to be low. Further, when the pore diameter is 1000 nm or more, the surface area of the substrate is decreased, and the binding capacity of the antibody protein tends to be decreased.

  The material of the substrate is not particularly limited, but when the substrate is in the form of beads, glass, silica, polystyrene resin, methacrylic resin, crosslinked agarose, crosslinked dextran, crosslinked polyvinyl alcohol, crosslinked cellulose, and the like can be used.

  In the embodiment, a temperature-responsive polymer having a cation exchange group is fixed to the substrate. As the fixing method, an atom transfer radical polymerization initiator is fixed on the surface of the substrate, and a temperature responsive polymer is grown from the initiator in the presence of a catalyst. There is a “radiation graft polymerization method” in which a radical is generated by irradiation and a temperature-responsive polymer is grown from the generated radical as a starting point, but is not particularly limited. As another fixing method, there is an “atom transfer radical polymerization method” which is a surface living radical polymerization method. The “atom transfer radical polymerization method” is preferably used because the polymer can be fixed at a high density on the surface of the substrate.

When the temperature-responsive polymer is fixed to the substrate surface by the “atom transfer radical polymerization method”, the initiator used at that time is not particularly limited, but when the substrate has a hydroxyl group, For example, 1-trichlorosilyl-2- (m, p-chloromethylphenyl) ethane, 2- (4-chlorosulfonylphenyl) ethyltrimethoxysilane, (3- (2-bromoisobutyryl) propyl) dimethylethoxysilane , And 2-bromoisobutyric acid bromide. Polymer chains are grown from this initiator. The catalyst at that time is not particularly limited, and examples of the copper halide (CuIX) include CuICl and CuIBr. The ligand complex for the copper halide is not particularly limited, but tris (2- (dimethylamino) ethyl) amine (Me ₆ TREN), N, N, N ″, N ″ -pentamethyl. Diethylenetriamine (PMDETA), 1,1,4,7,10,10-hexamethyltriethylenetetraamine (HMTETA), 1,4,8,11-tetramethyl 1,4,8,11-azacyclotetradecane (Me) ₄ Cyclam), bipyridine and the like.

  When the temperature-responsive polymer is fixed to the substrate surface by the “radiation graft polymerization method”, any means can be adopted to generate radicals on the substrate, but when the substrate is irradiated with ionizing radiation, This is preferable because uniform radicals are generated throughout the material. As types of ionizing radiation, γ rays, electron beams, β rays, neutron rays and the like can be used. However, electron beams or γ rays are preferable for implementation on an industrial scale. The ionizing radiation is obtained from radioactive isotopes such as cobalt 60, strontium 90, and cesium 137, or by an X-ray imaging apparatus, an electron beam accelerator, an ultraviolet irradiation apparatus, or 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 500 kGy or less, and further preferably 5 kGy or more and 200 kGy or less. If the irradiation dose is less than 1 kGy, radicals tend not to be generated uniformly. Further, when the irradiation dose exceeds 1000 kGy, the physical strength of the substrate tends to be lowered.

  In the graft polymerization method by irradiation with ionizing radiation, generally, after generating radicals on the base material, the pre-irradiation method in which the radicals are then contacted with the reactive compound, and the film in contact with the reactive compound on the base material. It is roughly divided into a simultaneous irradiation method for generating radicals. In the embodiment, any method can be applied, but a pre-irradiation method with less oligomer formation is preferable.

  In the embodiment, the solvent used in the polymerization is not particularly limited as long as the reactive compound can be uniformly dissolved. Examples of such a solvent include alcohols such as ethanol, isopropanol, and t-butyl alcohol, ethers such as diethyl ether and tetrahydrofuran, ketones such as acetone and 2-butanone, water, and a mixture thereof. .

  For example, the polymer fixed to the substrate surface has N-isopropylacrylamide. Poly (N-isopropylacrylamide) is known to have a lower critical temperature at 32 degrees. The solid adsorbent in which a polymer having N-isopropylacrylamide is introduced onto the substrate surface greatly changes the hydrophilic / hydrophobic surface properties at a critical temperature. Therefore, by grafting or coating a polymer having N-isopropylacrylamide on the surface of a chromatographic packing material to form a solid adsorbent, it becomes possible to change the force with which the solid adsorbent retains antibody protein depending on the temperature. . As a result, the retention behavior of the solid adsorbent can be controlled by temperature without changing the composition of the eluate.

  In order to set the lower critical temperature to 32 ° C. or higher, acrylamide, methacrylic acid, acrylic acid, dimethylacrylamide, vinylpyrrolidone, etc., which are more hydrophilic monomers than isopropylacrylamide, are combined with N-isopropylacrylamide as a hydrophilic comonomer. It can be adjusted by copolymerization.

  When the lower critical temperature is desired to be 32 ° C. or lower, it can be adjusted by copolymerizing styrene, alkyl methacrylate, alkyl acrylate, etc., which are hydrophobic monomers, with N-isopropylacrylamide as a hydrophobic comonomer. is there.

  For example, the polymer fixed on the substrate surface has strong cation exchange groups such as sulfonic acid groups as cation exchange groups. A method for providing a strong cation exchange group is not particularly limited, but as a first method, when synthesizing a temperature-responsive polymer chain fixed to the substrate surface, a method including copolymerization including a monomer having a strong cation exchange group Is mentioned. Examples of the monomer unit having a sulfonic acid group include (meth) acrylamide alkyl sulfonic acid, vinyl sulfonic acid, acrylamide t-butyl sulfonic acid, and styrene sulfonic acid, which are constituent units of a polymer having sulfonic acid.

For example, when at least a part of the monomer unit of the copolymer is derived from a vinyl monomer having a sulfonic acid group such as vinyl sulfonic acid, the sulfonic acid group is bonded to the main chain without a linker. Therefore, since the hydrophobic interaction between the linker and the antibody protein does not occur, it may be possible to increase the temperature elution amount when the antibody protein is eluted from the surface of the solid adsorbent due to a temperature change. In addition, at least a part of the monomer unit of the copolymer having a strong cation exchange group can also be represented by the following chemical formula (1), where each of R ₁ , R ₂ , and R ₃ is H or Me.
—CR ₁ R ₂ —CR ₃ (—SO ₃ H) — (1)

  In the embodiment, as a second method for giving a strong cation exchange group to a polymer fixed on the substrate surface, after copolymerization including a monomer having a “strong cation exchange group introduction precursor”, the precursor is converted to sulfone. The method of converting into an acid group is mentioned. The “strong cation exchange group-introduced precursor” may include “a strong cation exchange group precursor”. The “precursor of a strong cation exchange group” is, for example, a strong cation exchange group with a protective group. Examples of the monomer having a sulfonic acid group precursor include phenyl vinyl sulfonate, but are not limited thereto.

  In the embodiment, as a third method for giving a strong cation exchange group to a polymer fixed on the surface of a substrate, a monomer having a functional group capable of imparting a strong cation exchange group is used as a strong cation exchange group introduction precursor monomer. A method of converting a functional group capable of providing a strong cation exchange group into a sulfonic acid group after copolymerization is included. Examples of the monomer having a functional group that can impart a strong cation exchange group include styrene and glycidyl methacrylate. When a monomer having a strong cation exchange group is polymerized by the surface living radical polymerization method, a sufficient polymerization rate is often not obtained, but a strong cation in which at least a part of glycidyl methacrylate or the like is a methacrylic acid derivative or an acrylic acid derivative A sufficient polymerization rate can be obtained by using the exchange group-introduced precursor monomer.

  In addition, since at least a part of the monomer unit of the copolymer having a strong cation exchange group is a methacrylic acid derivative or an acrylic acid derivative, the hydrophobicity of the antibody protein and the other part of the substrate or the copolymer It may be possible to suppress the interaction and increase the temperature elution amount when the antibody protein is eluted from the surface of the solid adsorbent by a temperature change.

Furthermore, when at least a part of the monomer unit of the copolymer having a strong cation exchange group is a methacrylic acid derivative or an acrylic acid derivative, at least a part of the monomer unit of the copolymer having a strong cation exchange group is: It has a group represented by chemical formula (2) or (3).
_{-CH (-OH) -CH 2 -SO 3} H ··· (2)
—CH (—SO ₃ H) —CH ₂ —OH (3)
Sulfonic acid groups of the monomer units of Formula (2) is at least -CH (-OH) -CH ₂ - via a linker comprising, bonded to the main chain. In addition, the sulfonic acid group of the monomer unit represented by the chemical formula (3) is bonded to the main chain via a linker containing at least -CH-. Because the linker reduces steric hindrance, the antibody protein may be able to quickly bind to the sulfonic acid group. Furthermore, the monomer units of the chemical formulas (2) and (3) have a hydroxyl group in the vicinity of the sulfonic acid group. Therefore, the hydroxyl group suppresses the hydrophobic interaction between the antibody protein and the other part of the substrate or copolymer, and the temperature elution amount when the antibody protein is eluted from the surface of the solid adsorbent by temperature change. It may be possible to increase.

  In the embodiment, a monomer composition in which the ratio of the monomer having a strong cation exchange group and / or the strong cation exchange group-introduced precursor monomer to N-isopropylacrylamide is 0.01 to 5 mol% is obtained by surface grafting. Polymerize by polymerization method. As said ratio, Preferably it is 0.1-4 mol%, More preferably, it is 0.2-3 mol%, More preferably, it is 0.3-2 mol%, Most preferably, it is 0.5-1.5 mol%. If the ratio exceeds 5 mol%, the amount of strong cation exchange groups for N-isopropylacrylamide in the copolymer becomes excessive, and the amount of antibody protein adsorbed to the temperature-responsive solid adsorbent increases. However, most of the adsorbed antibody protein tends to be difficult to elute due to temperature change. On the other hand, when the ratio is less than 0.01 mol%, the amount of strong cation exchange group introduced is too small, and the amount of antibody protein adsorbed to the solid adsorbent tends to decrease.

  In the embodiment, the copolymerization ratio (composition) of the monomer unit having a strong cation exchange group with respect to N-isopropylacrylamide can be quantified by analyzing the copolymer immobilized on the substrate surface. is there. Various analysis techniques such as elemental analysis and NMR can be used for the analysis of the copolymerization ratio. Analyzing the copolymerization ratio after isolating the copolymer from the substrate is preferable from the viewpoint of analysis accuracy because the influence of the substrate on the analysis can be eliminated. When the copolymer cannot be isolated from the substrate, the copolymer used for analysis of the copolymerization ratio can be obtained by polymerizing the copolymer in the solution without using the substrate.

  The polymer fixed on the surface of the substrate causes hydration and dehydration by changing the temperature. The temperature range is 0 ° C. to 80 ° C., preferably 5 ° C. to 50 ° C., more preferably 10 ° C. to 45 ° C. If the temperature exceeds 80 ° C., the mobile phase is water, and thus evaporation occurs and the workability tends to deteriorate. On the other hand, if it is lower than 0 ° C., the mobile phase tends to freeze.

  The temperature-responsive solid adsorbent obtained by the embodiment is packed in a column of a normal liquid chromatography apparatus and used as a liquid chromatography system. At that time, the temperature loading method to the temperature-responsive solid adsorbent is not particularly limited. For example, the temperature-responsive solid adsorbent is brought into contact with an aluminum block, a water bath, and an air layer having a predetermined temperature, or For example, attaching a solid adsorbent to a jacket or the like.

  When the antibody protein is purified using the temperature-responsive solid adsorbent according to the embodiment, the monomer component of the target antibody protein in the first adsorbent in the first temperature region, the aggregate component, Is then adsorbed once, then the temperature is changed to a second temperature range lower than the first temperature range to change the characteristics of the surface of the solid adsorbent, and the solid adsorbent remains adsorbed on the solid adsorbent. A bind-and-elut (B & E) method is used in which only the monomer component of the antibody protein adsorbed to the agent is substantially eluted. The amount of antibody protein given to the solid adsorbent may or may not exceed the amount that the solid adsorbent can adsorb. Since antibody protein aggregates have a higher charge amount than monomers, the binding to ion exchange resins tends to be stronger than monomers. Furthermore, since the aggregate is more hydrophobic than the monomer, the aggregate tends to interact with the hydrophobic portion of the ion exchange resin (hydrophobic interaction) and bond with the ion exchange resin.

  Since the surface characteristics of the solid adsorbent according to the embodiment can vary greatly depending only on the temperature change, the temperature at which the surface characteristics of the solid adsorbent changes beforehand is confirmed, and the confirmed temperature is sandwiched between them. Preferably, the temperature is lowered from the first temperature range to the second temperature range, and the antibody protein adsorbed on the solid adsorbent is eluted with the aggregate adsorbed on the solid adsorbent.

 When the column temperature is changed from the first temperature range to the second temperature range, a gradient elution method for continuously changing the temperature, and a step of changing stepwise from the first temperature range to the second temperature range There is a width elution method. However, in the stepwise elution method, the temperature may be changed from the first temperature region to the second temperature region in one step and then eluted at a constant temperature.

 Here, the higher the temperature in the second temperature region, the stronger the adsorptive power of the solid adsorbent, the aggregate component and the monomer component, and thus the aggregate removal ability of the antibody protein tends to increase. However, the higher the temperature in the second temperature region, the less likely the monomer component of the antibody protein is eluted, and the recovery rate tends to decrease.

 In the embodiment of the present invention, any of a gradient elution method and a stepwise elution method can be used. However, in the gradient elution method and the stepwise elution method, the adsorptive powers of the aggregate component and monomer component differ from the solid adsorbent, which may affect the antibody recovery rate, aggregate removal ability, and the like.

 In order to improve the antibody recovery rate, a stepwise elution method is preferably used.

 Further, when the suitable conditions for the second temperature range are known, it is preferable to use the stepwise elution method because the purified purity of the antibody can be maintained and the separation time can be shortened. When changing the temperature stepwise, prepare an aluminum block, water bath, air layer, etc. set in advance in the first temperature range and the second temperature range, and use temperature-responsive antibody adsorbent and / or adsorption. This is achieved by contacting the mobile phase flowing into the agent.

 For the unknown antibody protein, a gradient elution method is preferably used to determine the most suitable elution temperature in order to improve the antibody recovery amount and aggregate removal ability.

  As the mobile phase of the chromatography according to the embodiment, a neutral buffer may be used, and an organic solvent is not required. Here, the buffer solution is an aqueous solution containing inorganic salts, and specifically includes a phosphate buffer solution, a Tris buffer solution, an acetate buffer solution, and the like. It is not limited. The concentration of the inorganic salt is 1 to 50 mmol / L, preferably 3 to 40 mmol / L, and more preferably 5 to 30 mmol / L.

  When the concentration of inorganic salts in the buffer solution is lower than 1 mmol / L, the activity of antibody protein as a solute tends to be impaired. Furthermore, when the concentration of inorganic salts in the buffer solution is lower than 1 mmol / L, the degree of dissociation of ion exchange groups on the surface of the temperature-responsive adsorbent increases, and the antibody protein is strongly adsorbed on the surface of the temperature-responsive adsorbent. In subsequent operations, it tends to be difficult to elute the antibody protein from the adsorbent surface.

  In addition, when the concentration of inorganic salts in the buffer solution is higher than 50 mmol / L, the dissociation degree of the ion exchange groups on the temperature-responsive adsorbent surface tends to be low, so that it is difficult for the adsorbent surface to hold the antibody protein. It is in. Therefore, it tends to be difficult to efficiently separate the antibody protein from the aggregate component.

  The hydrogen ion concentration of the buffer is, for example, pH 4.0 to 7.5, preferably pH 4.5 to 7.0, and more preferably pH 5.0 to 6.5. When the pH of the buffer solution is higher than 7.5, the antibody protein (isoelectric point 7.5 to 10) is negatively charged. Therefore, charge repulsion with the strong cation exchange group of the temperature-responsive adsorbent of the embodiment is performed. The adsorption capacity tends to decrease. On the other hand, when the pH is lower than 4.0, the antibody protein is denatured, which tends to cause a decrease in activity and quality such as formation of aggregates.

  The temperature-responsive cation exchange resin according to the embodiment described above is adsorbed with antibody protein monomers and aggregates on the high temperature side, and the antibody protein monomer is temperature-responsive cation exchange resin on the low temperature side. Since the electrostatic interaction between the antibody protein monomer and the temperature-responsive cation exchange resin weakens at the same time, the hydrophobic interaction between the antibody proteins also weakens at low temperatures. It is possible to efficiently purify antibody protein monomers while suppressing the formation of protein aggregates. Therefore, according to the method for purifying an antibody protein according to the embodiment, it is possible to efficiently purify a very useful antibody protein that can be used for pharmaceuticals and the like.

Hereinafter, the embodiments will be described in more detail based on examples, but these do not limit the embodiments.
In Example 1, a bead-like temperature-responsive adsorbent having a sulfonic acid group was synthesized by an atom transfer radical polymerization method.

1) Fixation of initiator 1 g of crosslinked polyvinyl alcohol beads (particle size 100 μm) was wetted with pure water and placed in a 300 mL glass Erlenmeyer flask. In an Erlenmeyer flask, 200 ml of tetrahydrafuran (without stabilizer, manufactured by Kanto Chemical Co., Inc.), 1.23 ml of 2-bromoisobutyric acid bromide (manufactured by Tokyo Chemical Industry Co., Ltd.), and triethylamine (Wako Pure Chemical Industries, Ltd.) 1) mL was added and shaken at room temperature for 16 hours. After the reaction, the mixture was filtered, washed with 200 mL ethanol three times, and stored in dehydrated isopropanol. Thereby, 2-bromoisobutyric acid bromide which is an atom transfer radical polymerization (ATRP) initiator was introduced on the surface of the crosslinked polyvinyl alcohol beads.

2) Surface Graft Polymerization A monomer composition containing glycidyl methacrylate (GMA, manufactured by Tokyo Chemical Industry Co., Ltd.), which is a sulfonic acid group precursor monomer, at a ratio of 1 mol% with respect to N-isopropylacrylamide was prepared. Specifically, N-isopropylacrylamide (IPAAm, manufactured by Wako Pure Chemical Industries, Ltd.) 18.40 g, GMA 0.231 g, butyl methacrylate (BMA, manufactured by Tokyo Chemical Industry Co., Ltd.) 1.217 g, copper chloride I ( 0.085 g of CuCl, manufactured by Wako Pure Chemical Industries, Ltd.) and 0.012 g of copper chloride II (CuCl2, manufactured by Wako Pure Chemical Industries, Ltd.) were dissolved in 42.8 mL of 90% by volume isopropanol (IPA) aqueous solution. Nitrogen bubbling was performed for 30 minutes. Thereafter, 0.221 g of tris (2-dimethylaminoethyl) amine (Me6TREN) (Alfa Aesar) was added to the solution under a nitrogen atmosphere and stirred for 5 minutes to form a catalyst of CuCl / CuCl2 / Me6TREN. This reaction solution was reacted with initiator-introduced crosslinked polyvinyl alcohol beads in a nitrogen atmosphere, and ATRP was performed at room temperature for 16 hours. After the reaction, ethanol, 50 mmol / L-EDTA aqueous solution, and pure water were washed in this order to wash the monomer, polymer, and copper catalyst.

3) Introduction of sulfonic acid group Beads introduced with graft chains by the atom transfer radical polymerization method are added to 200 g of a mixed aqueous solution of sodium sulfite and IPA (sodium sulfite / IPA / pure water = 10/15/75 wt%). Then, the reaction was carried out at 80 ° C. for 24 hours to convert the epoxy group in the graft chain into a sulfonic acid group. After the reaction, the beads were washed with pure water. Thereafter, the beads were put into 0.5 mol / L sulfuric acid and reacted at 80 ° C. for 2 hours to convert the epoxy group remaining in the graft chain into a diol group. After the reaction, the beads were washed with pure water to obtain a temperature-responsive adsorbent according to Example 1. The temperature-responsive adsorbent was packed in the column.

4) Measurement of copolymerization ratio Using a monomer composition containing glycidyl methacrylate (GMA, manufactured by Tokyo Chemical Industry Co., Ltd.), which is a sulfonic acid group precursor monomer, in a proportion of 1 mol% with respect to N-isopropylacrylamide. The copolymer was polymerized without using a substrate. Specifically, the reaction solution described in 2) above was reacted with ethyl 2-bromoisobutyrate under a nitrogen atmosphere, and ATRP was performed at room temperature for 16 hours. After the reaction, the monomer solution and the copper catalyst are removed by immersing the reaction solution in a dialysis membrane (Spectra / po Dialyzation Membrane, MWCO1000, Spectrum Laboratories) and immersing in the order ethanol, 50 mmol / L-EDTA aqueous solution, and pure water. did. Next, the copolymer obtained by freeze-drying the reaction solution was added to 200 g of a mixed aqueous solution of sodium sulfite and IPA (sodium sulfite / IPA / pure water = 10/15/75 wt%), and 80 The reaction was carried out at 0 ° C. for 24 hours to convert the epoxy group in the graft chain into a sulfonic acid group. After the reaction, the reaction solution was placed in a dialysis membrane and immersed in pure water to remove sodium sulfite and IPA, and the reaction solution was freeze-dried to obtain a copolymer.

  30 mg of the above copolymer was dissolved in 670 mg of heavy water, and 1H-NMR was measured using a nuclear magnetic resonance apparatus (Bruker Avenve-600). Thereafter, the copolymerization ratio (composition) of the monomer unit having a strong cation exchange group with respect to N-isopropylacrylamide was calculated from the signal integrated value derived from the N-isopropylacrylamide unit and the signal integrated value derived from the sulfonic acid group. As a result, the copolymerization ratio (composition) of the monomer unit having a strong cation exchange group with respect to N-isopropylacrylamide was 0.72 mol%.

(Adjustment of cell culture medium)
As an antibody protein, a culture supernatant containing 0.115 mg / L of AE6F4 antibody (human monoclonal antibody) was prepared. AE6F4-producing cells were provided by Associate Professor Yoshinori Katakura, Graduate School of Agriculture, Kyushu University. The AE6F4 antibody-producing cells were cultured with reference to literature (Abstracts of the Japanese Society for Biotechnology, 1994, Volume 65, page 65). The culture solution containing AE6F4 antibody-producing cells was filtered using a filtration membrane (trade name BioOptimal (registered trademark) MF-SL, manufactured by Asahi Kasei Medical Co., Ltd.) to obtain a solution (culture supernatant) containing the antibody. Filtration was performed with reference to the instruction manual of the provider.

(Purification of antibody protein by protein A column)
10 ml of the filtered culture solution was added to a protein A column (made by GE Healthcare Biosciences, packed with MabSelect) equilibrated with 10 ml of phosphate buffer (50 mM sodium phosphate + 150 mM NaCl (pH 8.0)), Antibody protein was adsorbed to protein A. Next, 20 mL of phosphate buffer (50 mM sodium phosphate + 150 mM NaCl (pH 8.0)) was passed through the column for washing, and then the elution buffer (100 mM sodium citrate + 300 mM (pH 3.0)) was applied to the column. 10 mL was passed through, and the antibody protein was eluted from the protein A column and recovered. When the recovered elution fraction was subjected to size exclusion chromatography (SEC), as shown in FIG. 1, the rise of the absorption peak was not steep, and not only the antibody protein monomer but also the antibody protein It was shown to also contain an agglomerate component.

(Virus inactivation treatment)
1 mol / LTris-HCl (pH 8.0) of 0.5% of the volume of the eluted fraction was added to the obtained eluted fraction to adjust the hydrogen ion index of the eluted fraction to pH 3.8. Furthermore, citric acid was added dropwise to the elution fraction, the hydrogen ion index of the elution fraction was adjusted to pH 3.5, and the elution fraction was allowed to stand for 1 hour for virus inactivation treatment. Thereafter, the hydrogen ion index of the eluted fraction was adjusted to pH 5.0 using Tris buffer, and buffer exchange was performed with an acetate buffer (15 mmol / L acetate buffer (pH 6.0)).

(Removal of aggregate components)
To a column packed with a temperature-responsive adsorbent (1.7 ml), an elution fraction from a protein A column that has been subjected to virus inactivation treatment is added. The aggregate was adsorbed. The amount of the eluted fraction added was 20 mL (0.57 mg / mL), the flow rate was 0.4 mL / min, and the temperature was 40 ° C. Thereafter, the column packed with the temperature-responsive adsorbent was washed with a 40 ° C. acetate buffer (15 mmol / L acetate buffer (pH 6.0)) flowing at a flow rate of 0.4 mL / min. Next, as shown in FIG. 2, the temperature gradient of the elution buffer (15 mmol / L acetate buffer (pH 6.0)) is set from 40 ° C. to 10 ° C., and the temperature response of the elution buffer at a flow rate of 0.4 mL / min. It flowed through the column packed with the adsorbent.

In FIG. 2, when the elution fractions numbered “1” to “5” were subjected to size exclusion chromatography, as shown in FIG. 3 to FIG. The protein monomer was shown to be purified. In addition, when non-reducing SDS-PAGE was performed on each of the fractions eluted with numbers “1” to “5” in FIG. 2, as shown in FIG. It was not confirmed.
Further, when the amount of antibody (mg) input to the temperature-responsive chromatography is A, and the amount of antibody eluted in the second temperature region in FIG. 2 (mg) is B, the recovery rate (= B / A) is 54.3. %Met.

[Comparative Example 1]
The antibody protein was purified from the cell culture using a protein A column in the same manner as in Example 1 to inactivate the virus. Thereafter, the fraction eluted from the protein A column subjected to virus inactivation treatment was added to a column packed with SP Sepharose (trademark) Fast Flow (GE Healthcare) (1.7 mL). The amount of the eluted fraction added was 20 mL (0.57 mg / mL), the flow rate was 0.4 mL / min, and the temperature was room temperature. Thereafter, as in Example 1, the column packed with SP Sepharose beads was washed. Next, as shown in FIG. 9, while increasing the salt concentration (conductivity) of the elution buffer (15 mmol / L acetate buffer (pH 6.0)), the elution buffer was changed to SP Sepharose (trademark) at a flow rate of 0.4 mL / min. ) It flowed through a column filled with Fast Flow.

  In FIG. 9, the elution fractions assigned with the numbers “1” to “6” were subjected to size exclusion chromatography. As shown in FIGS. 10 and 11, the numbers “1” and “2” were assigned. The rise of the absorption peak of the eluted fraction of the shaken portion was steep, but as shown in FIGS. 12 to 15, the absorption peak of the eluted fraction of the portion assigned the numbers “3” to “6” was shown. The rise was disturbed, indicating that the protein aggregate component was not removed. This seems to be because the hydrophobic interaction between proteins became stronger as the salt concentration increased. In addition, when non-reducing SDS-PAGE was performed on each of the fractions eluted with the numbers “1” to “6” in FIG. 9, the number “1” was assigned as shown in FIG. In the eluted fraction of the portion, a low molecular impurity component was confirmed.

[Example 2]
The antibody protein was purified from the cell culture using a protein A column in the same manner as in Example 1 to inactivate the virus. When the recovered elution fraction was subjected to size exclusion chromatography (SEC), as shown in FIG. 16, the rise of the absorption peak was not steep, and not only the antibody protein monomer but also the antibody protein It was shown to also contain an agglomerate component. Further, the amount of the aggregate component was larger than that in Example 1 and Comparative Example 1.

  Thereafter, the elution fraction from the protein A column subjected to virus inactivation treatment was added to the column filled with the bead temperature-responsive adsorbent (2.0 mL) prepared in the same manner as in Example 1, and the temperature-responsive adsorption was performed. The antibody protein monomer and aggregates were adsorbed on the agent. The amount of the eluted fraction added was 4 mL (4.97 mg / mL), the flow rate was 0.4 mL / min, and the temperature was 37 ° C. Thereafter, the column packed with the temperature-responsive adsorbent was washed with an acetic acid buffer (15 mmol / L acetic acid buffer (pH 6.0)) at 37 ° C. flowing at a flow rate of 0.4 mL / min. Next, 20 ml of an elution buffer (15 mmol / L acetate buffer (pH 6.0)) set at a temperature of 5 ° C. was passed through a column packed with a temperature-responsive adsorbent at a flow rate of 0.4 mL / min.

When 20 ml of the eluted fraction collected after purification by temperature-responsive chromatography was subjected to size exclusion chromatography, as shown in FIG. 17, the rise of the absorption peak was steep and the antibody protein monomer was purified. It showed that.
The recovery rate (= B / A) was 82.6%, where A is the amount of antibody (mg) input into the temperature-responsive chromatography and B is the amount of eluted antibody (mg).
Thus, the recovery rate was greatly improved by changing from the first temperature range (37 ° C.) to the second temperature range (5 ° C.) by the stepwise elution method.

  According to the method according to the embodiment, the monomer of the antibody protein can be purified on an industrial scale by changing the temperature.

Claims (17)

A method for purifying antibody protein using a temperature-responsive cation exchange resin in which antibody protein is adsorbed on a high temperature side and desorbed on the low temperature side,
In the first temperature region where the antibody protein is adsorbed to the temperature-responsive cation exchange resin, the monomer component of the antibody protein and the aggregate component of the antibody protein dimer or more contained in the solution, Adsorbing to a temperature-responsive cation exchange resin;
The antibody protein monomer component is desorbed from the temperature-responsive cation exchange resin by using an eluate in a second temperature region in which the antibody protein monomer component is desorbed from the temperature-responsive cation exchange resin. A method for purifying an antibody protein, wherein   The method for purifying an antibody protein according to claim 1, wherein the antibody protein is a monoclonal antibody.   The method for purifying an antibody protein according to claim 1 or 2, wherein the first temperature region is 30 ° C or higher and lower than 60 ° C, and the second temperature region is 0 ° C or higher and lower than 30 ° C. The temperature-responsive cation exchange resin comprises a base material and a copolymer containing at least N-isopropylacrylamide fixed on the base material surface,
The copolymer has at least a strong cation exchange group;
The copolymer contains 0.01 to 5 mol% of the strong cation exchange group in terms of monomer with respect to the N-isopropylacrylamide.
The method for purifying the antibody protein according to any one of claims 1 to 3. The at least one part of monomer unit of the copolymer which has the said strong cation exchange group is an acrylic acid derivative or a methacrylic acid derivative, and has group shown by following Chemical formula (1) or (2). The antibody protein purification method.
_{-CH (-OH) -CH 2 -SO 3} H ··· (1)
—CH (—SO ₃ H) —CH ₂ —OH (2)   The method for purifying an antibody protein according to claim 4, wherein at least a part of the monomer unit of the copolymer having a strong cation exchange group is derived from a vinyl monomer having a sulfonic acid group. The antibody protein purification according to claim 6, wherein at least a part of the monomer unit of the copolymer having a strong cation exchange group is represented by the following chemical formula (3), wherein each of R1, R2, and R3 is H or Me. Method.
—CR ₁ R ₂ —CR ₃ (—SO ₃ H) — (3) The temperature-responsive cationic ion exchange resin comprises a base material and a copolymer containing at least N-isopropylacrylamide fixed on the base material surface,
The copolymer has at least a strong cation exchange group;
A monomer composition in which the copolymer contains the monomer having a strong cation exchange group and / or a strong cation exchange group-introduced precursor monomer in a proportion of 0.01 to 5 mol% with respect to the N-isopropylacrylamide. , Formed by polymerization by surface graft polymerization method,
A method for purifying the antibody protein according to claim 1 or 2. The at least one part of the monomer unit of the copolymer which has the said strong cation exchange group is an acrylic acid derivative or a methacrylic acid derivative, and has group shown by following Chemical formula (4) or (5). The antibody protein purification method.
—CH (—OH) —CH ₂ —SO ₃ H (4)
—CH (—SO ₃ H) —CH ₂ —OH (5) At least a part of the strong cation exchange group-introduced precursor monomer is an acrylic acid derivative or a methacrylic acid derivative, and the copolymer has a side chain represented by the following chemical formula (6) or (7). 10. A method for purifying the antibody protein according to 9.
—CH (—OH) —CH ₂ —SO ₃ H (6)
—CH (—SO ₃ H) —CH ₂ —OH (7)   The method for purifying an antibody protein according to claim 10, wherein at least a part of the monomer unit of the copolymer having a strong cation exchange group is derived from a vinyl monomer having a sulfonic acid group.   The method for purifying an antibody protein according to claim 8 or 11, wherein at least a part of the monomer having a strong cation exchange group is a vinyl monomer having a sulfonic acid group. The monomer unit of the copolymer having the strong cation exchange group is represented by the following chemical formula (8), wherein each of R1, R2, and R3 is H or Me. 2. A method for purifying the antibody protein according to item 1.
—CR ₁ R ₂ —CR ₃ (—SO ₃ H) — (8)   The method for purifying an antibody protein according to any one of claims 4 to 13, wherein the strong cation exchange group is a sulfonic acid group.   The method for purifying an antibody protein according to any one of claims 8 to 13, wherein the surface graft polymerization method is a surface living radical polymerization method.   The method for purifying an antibody protein according to any one of claims 8 to 13, wherein the surface graft polymerization method is a radiation radical polymerization method.  The antibody protein according to any one of claims 1 to 16, wherein a stepwise elution method is used when the column temperature is changed from the first temperature range to the second temperature range. Purification method.