JP2014161833A - Affinity separating agent for protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an affinity separating agent for proteins, which can selectively adsorb or separate proteins.SOLUTION: The affinity separating agent for proteins comprises: ligand particles each of which comprises a copolymer having a structural unit derived from a compound shown by formula (1) (in which Ris a hydrogen atom or a 1-3C optionally-branched alkyl group; Ris a 1-3C optionally-branched alkyl group) and which have 20-500 nm average particle size; and an organic polymer particle carrier.

Description

  The present invention relates to a separation agent that is chemically stable, can selectively adsorb and separate proteins, and can be produced at low cost.

  In the research and development of biopolymers such as proteins, chromatography is often used for their adsorption, separation, and purification. In particular, affinity chromatography using a fixed bed in which a ligand having an affinity for these proteins and the like is bound on a carrier has high selectivity for the target molecule and enables high-speed, high-purity separation / purification with high yield. Therefore, it is preferably used.

  In recent years, antibody therapy has been shown to be effective as a specific drug for cancer, rheumatism and the like. In antibody therapy, for example, targeting cancer cells, drugs that resemble antibodies that attack only the cancer cells are relied on by using markers that the cancer cells do not have in normal cells. The therapy to use. However, antibody drugs are expensive, and there is a demand for inexpensive and stable production methods.

  As an effective method for purifying such antibody drugs, an affinity separation agent using protein A as a ligand has been reported (for example, Patent Document 1).

JP2012-018135A

  Protein A used as a ligand in the affinity separation agent described in Patent Document 1 has a problem that it is very expensive and is an unstable substance. There is a problem to use.

  The present invention has been made for the purpose of solving such problems of the prior art. That is, since the present invention is chemically stable and can selectively adsorb and separate proteins, it is useful in the field of affinity purification used in the purification of antibody drugs and the like, and is produced at low cost. It is an object of the present invention to provide an affinity separation agent for proteins.

  As a result of intensive studies in view of the above problems, the present inventors have found that a separating agent comprising a copolymer having a specific chemical structure supported on a carrier has excellent performance and is inexpensive. The present inventors have found that it is possible to produce the present invention and have arrived at the present invention. That is, the gist of the present invention resides in the following [1] to [6].

[1] From a copolymer containing as a main component a structural unit derived from each of a compound represented by the following formula (1), a compound represented by the formula (2), and a compound represented by the formula (3) A protein affinity separation agent comprising a ligand particle having an average particle size of 20 nm to 500 nm and an organic polymer particle carrier.
(In the above formula (1), R ¹ is a hydrogen atom or an optionally branched alkyl group having 1 to 3 carbon atoms, and R ² is an optionally branched alkyl group having 1 to 3 carbon atoms. In the above formula (2), R ³ is a hydrogen atom or an optionally branched alkyl group having 1 to 3 carbon atoms, and R ⁴ is an optionally branched alkyl group having 4 to 8 carbon atoms. In the above formula (3), R ⁵ is a hydrogen atom or an optionally branched alkyl group having 1 to 3 carbon atoms.)

[2] All vinyls in which the structural unit derived from each of the compound represented by formula (1), the compound represented by formula (2), and the compound represented by formula (3) constitutes the copolymer. The protein affinity separation agent according to [1], which has the following ratio with respect to the total weight of the system compound.
Structural unit derived from the compound represented by the formula (1): 5% by weight or more and 90% by weight or less Structural unit derived from the compound represented by the formula (2): 5% by weight or more and 90% by weight or less Formula (3) Structural unit derived from a compound represented by: 3 wt% or more and 50 wt% or less

[3] The compound represented by the formula (1) is N-isopropylacrylamide, the compound represented by the formula (2) is tert-butylacrylamide, and the compound represented by the formula (3) is The protein affinity separation agent according to [1] or [2], which is acrylic acid.

[4] The organic polymer particle carrier is a porous cross-linked organic polymer particle carrier having an average particle diameter of 1 μm or more and 1000 μm or less and a pore diameter of 10 mm or more and 10,000 mm or less by a mercury intrusion method. [3] The protein affinity separation agent according to any one of [3].

[5] The above [1] to [1], wherein the organic polymer particle carrier is a porous crosslinked organic polymer particle carrier made of a crosslinked copolymer having a structural unit derived from a (meth) acrylate monomer. 4] The protein affinity separation agent according to any one of [4].

[6] The organic polymer particle carrier includes an epoxy group, a carboxyl group, a sulfonic acid group, a phosphoric acid group, an amino group, a hydroxyl group, an imino group, an imidazole group, a carbamate group, a cyano group, a thiol group, and an aldehyde group. The protein affinity separation agent according to any one of [1] to [5], which has at least one functional group in the group.

  INDUSTRIAL APPLICABILITY According to the present invention, since it is chemically stable and can selectively adsorb and separate proteins, it is useful in the field of affinity purification used in the purification of antibody drugs and the like, and is produced at low cost. A protein affinity separation agent is provided.

  DESCRIPTION OF EMBODIMENTS Embodiments of the present invention will be described in detail below. However, the description of the constituent elements described below is an example of the embodiments of the present invention, and the present invention is described below unless the gist of the present invention is exceeded. It is not limited to. In addition, when using the expression “to” in the present specification, it is used as an expression including numerical values or physical property values before and after the expression.

[Affinity separation agent for protein]
The protein affinity separation agent of the present invention is mainly composed of a structural unit derived from each of the compound represented by the following formula (1), the compound represented by the formula (2) and the compound represented by the formula (3). And comprising an organic polymer particle carrier and a ligand particle having an average particle diameter of 20 nm to 500 nm. Hereinafter, the protein affinity separation agent of the present invention may be abbreviated as “the separation agent of the present invention”.

In the above formula (1), R ¹ is a hydrogen atom or an optionally branched alkyl group having 1 to 3 carbon atoms, R ² is an optionally branched alkyl group having 1 to 3 carbon atoms, In the above formula (2), R ³ is a hydrogen atom or an optionally branched alkyl group having 1 to 3 carbon atoms, and R ⁴ is an optionally branched alkyl group having 4 to 8 carbon atoms. In the above formula (3), R ⁵ is a hydrogen atom or an optionally branched alkyl group having 1 to 3 carbon atoms.

<Ligand particles>
The ligand particle used in the present invention contains as a main component a structural unit derived from each of the compound represented by formula (1), the compound represented by formula (2), and the compound represented by formula (3). It consists of a copolymer, and an average particle diameter is 20 nm or more and 500 nm or less. Here, the main component is represented by the compound represented by the formula (1), the compound represented by the formula (2), and the formula (3) with respect to the total weight of all vinyl compounds constituting the ligand particles. It means that the total weight of the compound is 50% by weight or more.

The copolymer constituting the ligand particles expresses a function as a basic skeleton of a polymer having intermediate properties of hydrophilicity and hydrophobicity by a structural unit derived from the compound represented by the formula (1). The structural unit derived from the compound represented by (2) mainly expresses a hydrophobic interaction for adsorbing with the target molecule, and the structural unit derived from the compound represented by formula (3) Thus, the hydrophilic interaction for adsorbing to the target molecule and the electrostatic interaction are mainly expressed, and therefore, it has an excellent affinity adsorption property for proteins, particularly antibodies.
Here, “affinity adsorptivity” means an adsorptivity based on a mechanism in which hydrophobic interaction, hydrophilic interaction, electrostatic interaction, intermolecular force, hydrogen bond, coordination bond, etc. are involved in a complex manner. That is.

In the formula (1), R ¹ is a hydrogen atom or an optionally branched alkyl group having 1 to 3 carbon atoms, preferably a hydrogen atom or a methyl group. R ² is an optionally branched alkyl group having 1 to 3 carbon atoms, preferably 3 carbon atoms, and particularly preferably an isopropyl group. Examples of the compound represented by the formula (1) include N-methyl (meth) acrylamide, N-ethyl (meth) acrylamide, N-propyl (meth) acrylamide, N-isopropyl (meth) acrylamide, and the like. N-isopropyl (meth) acrylamide is preferable, and N-isopropylacrylamide is particularly preferable. In the present invention, the expression “(meth) acryl” means “acryl or methacryl”. The copolymer constituting the ligand particle may contain only one type or two or more types of structural units derived from the compound represented by the formula (1).

In the formula (2), R ³ is a hydrogen atom or an alkyl group having 1 to 3 carbon atoms which may be branched, preferably a hydrogen atom or a methyl group. R ⁴ is an optionally branched alkyl group having 4 to 8 carbon atoms, preferably 4 to 6 carbon atoms, and particularly preferably R ⁴ is a tert-butyl group. Examples of the compound represented by the formula (2) include butyl (meth) acrylamide, tert-butyl (meth) acrylamide, pentyl (meth) acrylamide, hexyl (meth) acrylamide, octyl (meth) acrylamide, and the like. Tert-butyl (meth) acrylamide is preferable, and tert-butylacrylamide is particularly preferable. The copolymer constituting the ligand particle may contain only one type or two or more types of structural units derived from the compound represented by the formula (2).

In the formula (3), R ⁵ is a hydrogen atom or an optionally branched alkyl group having 1 to 3 carbon atoms, preferably a hydrogen atom or a methyl group, and acrylic acid in which R ⁵ is a hydrogen atom. It is preferable that The copolymer constituting the ligand particle may contain only one type or two or more types of structural units derived from the compound represented by the formula (3).

  The compound represented by the formula (1) is particularly suitable as the copolymer constituting the ligand particle in terms of the stability of the copolymer constituting the ligand particle and the affinity adsorption property of the ligand particle to the protein. A copolymer of isopropylacrylamide (NIPAm), the compound represented by formula (2) is tert-butylacrylamide (TBAm), and the compound represented by formula (3) is acrylic acid (AAc). is there.

In addition, the copolymer may be referred to as a vinyl compound other than the compounds represented by the above formulas (1) to (3) (hereinafter referred to as “other vinyl compound”) within a range not impairing the effects of the present invention. It may contain a structural unit derived from.
Other vinyl compounds include a polyfunctional vinyl compound that functions as a cross-linking agent described later, a vinyl compound that contains a covalently-bonded functional group (with an organic polymer particle carrier), a vinyl compound that functions as a quantitative marker, etc. Is mentioned.

A suitable ratio of the structural units derived from the compounds represented by the formulas (1) to (3) in the copolymer is:
The structural unit derived from each of the compound represented by the formula (1), the compound represented by the formula (2) and the compound represented by the formula (3) is a total of all vinyl compounds constituting the copolymer. For weight, preferably
Structural units derived from the compound represented by formula (1): 5% by weight or more and 90% by weight or less,
Structural units derived from the compound represented by formula (2): 5% by weight or more and 90% by weight or less,
Structural units derived from the compound represented by formula (3): 3% by weight or more and 50% by weight or less,
More preferably, the structural unit derived from the compound represented by the formula (1): 10 wt% or more and 80 wt% or less,
Structural units derived from the compound represented by the formula (2): 10 wt% or more and 80 wt% or less,
Structural units derived from the compound represented by formula (3): 5% by weight or more and 40% by weight or less,
And particularly preferably
Structural units derived from the compound represented by the formula (1): 20% by weight or more and 60% by weight or less,
Structural units derived from the compound represented by the formula (2): 20% by weight or more and 60% by weight or less,
Structural units derived from the compound represented by formula (3): 10% by weight or more and 30% by weight or less,
It is.
The “total weight of all vinyl compounds constituting the copolymer” is a structure derived from other vinyl compounds in addition to the structural unit derived from the compound represented by the formula (1) to (3). When a unit is included, it is the total weight of the compound represented by Formula (1)-(3) and another vinyl compound. That is, “the total weight of all vinyl compounds composing the copolymer” means the total weight of all vinyl compounds composing the copolymer. The weight proportion of each structural unit is determined based on the weight proportion of each vinyl compound used as a raw material.

  The copolymer preferably has a particulate steric structure, particularly for function expression as a ligand particle. The average particle size is 20 to 500 nm, and preferably the average particle size is 25 to 200 nm. It is. Ligand particles need to have a three-dimensional structure in order to have adsorption selectivity with respect to the target molecule (protein). In order to adopt such a structure, it is preferable that the average particle diameter is not less than the above lower limit value. . Moreover, it is preferable that an average particle diameter is below the said upper limit from a viewpoint of the adsorption amount with respect to the target molecule | numerator (protein) of a ligand particle.

The copolymer can be produced by a conventionally known synthesis method such as a solution polymerization method, a bulk polymerization method, a suspension polymerization method, an emulsion polymerization method, or a precipitation polymerization method.
In particular, in order to obtain a particulate copolymer having an average particle size of 20 to 500 nm (preferably, an average particle size of 25 to 200 nm), it is preferable to produce by a precipitation polymerization method.
The precipitation polymerization method is preferably produced by a method according to the methods described in the documents Journal of the American Chemical Society, 134, 15765-15772, 2012 and Polymer Preprints, 52, 502-503, 2011.
The average particle size of the synthesized particulate copolymer can be measured by a dynamic light scattering method.

The precipitation polymerization method is a method in which the polymerization is performed using a solvent in which the raw material monomer is soluble and the produced copolymer is difficult to dissolve.
The particulate copolymer according to the present invention contains a compound represented by the above formula (1), a compound represented by (2) and a compound represented by (3) as raw material monomers in a solvent. When the polymerization reaction is allowed to proceed in the presence of a cross-linking agent and a polymerization initiator as necessary, the polymer that is produced precipitates as the reaction proceeds. It can be obtained by collecting and drying. The obtained particulate copolymer is preferably purified by a generally known method such as gel filtration, reprecipitation, liquid separation extraction, or dialysis.

The solvent used for the polymerization is not particularly limited as long as the raw material monomer and the polymerization initiator are soluble at room temperature or by heating, and the produced copolymer is not dissolved.
For example, water, dimethylformamide (DMF), dimethyl sulfoxide, methanol, ethanol, butanol, tetrahydrofuran, dioxane, acetone and the like can be mentioned. These may be used alone or in admixture of two or more.

A suitable ratio of the compounds represented by the above formulas (1) to (3), which are raw material monomers for the copolymer, is a compound represented by the formula (1): 5 to 90% by weight (more preferably 10 to 10%). 80% by weight, particularly preferably 20-60% by weight), compound represented by formula (2): 5-90% by weight (preferably 10-80% by weight, particularly preferably 20-60% by weight) Compound represented by formula (3): 3 to 50% by weight (preferably 5 to 40% by weight, particularly preferably 10 to 30% by weight).
This ratio (% by weight) is a value when the total weight of all vinyl compounds constituting the copolymer is 100% by weight.

The particulate copolymer preferably has a cross-linked structure from the viewpoint of increasing mechanical strength. As a method for imparting a crosslinked structure to the copolymer, it is possible to carry out copolymerization with a raw material monomer in the presence of a crosslinking agent when polymerizing the raw material monomer.
Examples of the crosslinking agent include polyfunctional vinyl compounds, and examples thereof include bifunctional monomers having a plurality of vinyl groups such as N, N-methylenebisacrylamide, hydroxyethyl dimethacrylate, and divinylbenzene.

The ratio of the crosslinking agent is preferably 0.1 to 5% by weight, more preferably 0.5 to 2% by weight, based on the total weight of all vinyl compounds constituting the copolymer.
When the ratio of the crosslinking agent is not less than the above lower limit value, it is preferable from the viewpoint of mechanical strength, and when it is not more than the above upper limit value, it is preferable from the viewpoint of obtaining the function as a ligand particle.

Moreover, it is preferable that the copolymer which comprises ligand particle | grains contains the structural unit which consists of a vinyl compound containing a support | carrier and a covalent bond functional group.
Examples of such a vinyl compound include vinyl compounds having an amino group such as N- (3-aminopropyl) methacrylamide when the carrier has an epoxy group as a reactive functional group.
When the carrier has a quaternary amino group as a reactive functional group, examples thereof include anionic groups such as a sulfonic acid group and a carboxylic acid group.

  The concentration of the whole raw material monomer (the whole vinyl compound constituting the copolymer) in the reaction solution is preferably 1 to 50% by mass from the viewpoints of production efficiency, stirring properties and adhesion prevention.

  There are no particular restrictions on the method and order of addition of raw material monomers (including other vinyl compounds such as crosslinking agents) and other components (polymerization initiators, etc.) to the solvent in the precipitation polymerization method. Then, a method of reacting after mixing may be used, or a method of reacting after mixing any two components or three or more components in advance and then mixing the remaining components may be used.

Optimum conditions for the polymerization reaction vary depending on the polymerization initiator, monomer, type of solvent, concentration, etc., but the temperature is usually 10 to 80 ° C. and the polymerization time is usually about 1 to 20 hours.
Moreover, in order to suppress inactivation of the polymerization initiator by oxygen, it is preferable to make it react in inert gas atmosphere.

<Organic polymer particle carrier>
By supporting the ligand particles according to the present invention on an organic polymer particle carrier, the separating agent of the present invention can be prepared. Regarding the organic polymer particle carrier, polymer-based, cellulose-based, agarose, etc. Any organic particle carrier such as a polymer can be used, and a polymer organic particle carrier is particularly preferable. From the viewpoint of having an internal surface area for supporting the ligand particles, the organic particle carrier is particularly preferably a porous crosslinked organic polymer particle carrier having porosity.

  The average particle size of such an organic polymer particle carrier is 1 μm or more, preferably 5 μm or more, and more preferably 10 μm or more. On the other hand, it is 1000 μm or less, preferably 700 μm or less, and more preferably 500 μm or less. When the average particle size of the organic polymer particle carrier is equal to or greater than the above lower limit, the decrease in the flow rate due to pressure loss when the separation agent of the present invention is packed in the column and passed through the column is suppressed, and the productivity is improved. On the other hand, the amount of protein adsorbed and the separation performance tend to be improved when it passes through the column.

  The average particle diameter of the organic polymer particle carrier can be measured by a known method. For example, an average particle diameter can be obtained by measuring 100 or more particle diameters with an optical microscope and calculating a volume median diameter from the distribution.

  When the organic polymer particle carrier is porous, the pore diameter is measured by a mercury intrusion method, and is preferably 10 mm or more, more preferably 20 mm or more from the viewpoint of obtaining a surface area for supporting the ligand particles. More preferably, it is 50 mm or more. On the other hand, from the viewpoint of obtaining the physical strength of the organic polymer particle carrier itself, it is preferably 10,000 kg or less, more preferably 5,000 kg or less, and still more preferably 2,000 kg or less.

  The mercury intrusion method is a method of calculating the diameter of a pore assumed to be cylindrical from the equation of Washburn using a pressure value and a corresponding intruding mercury volume by injecting mercury into the opening by applying pressure. Therefore, JIS R1655 defined for ceramic molded bodies can be applied mutatis mutandis.

Among the above carriers, an organic material composed of porous crosslinked polymer particles described in JP 2012-18135 A is capable of sufficiently supporting the copolymer constituting the ligand particles and having excellent chemical stability. A polymer particle carrier or an organic polymer particle carrier into which a spacer is introduced is preferable.
Hereinafter, a porous crosslinked organic polymer particle carrier suitable as an organic polymer particle carrier in the separating agent of the present invention will be described.

  The porous crosslinked organic polymer particle carrier suitably used as the carrier in the separating agent of the present invention has a crosslinked structure and has pores suitable for supporting the copolymer constituting the ligand particle. It is a particulate carrier.

(1) Method for Producing Porous Crosslinked Organic Polymer Particle Carrier A method for obtaining a particulate porous crosslinked organic polymer particle carrier includes conventionally known suspension polymerization and emulsion polymerization. For example, using a method as disclosed in Japanese Patent Publication No. 58-058026, a polymerizable monomer such as a styrene monomer or a (meth) acrylic monomer, and a reactive functional group as necessary. It can be carried out by subjecting a polymerizable monomer having group-providing properties to suspension polymerization or emulsion polymerization.
In particular, it is preferably produced by a suspension polymerization method as described, for example, in JP-A-1-54004, in that a porous crosslinked organic polymer particle carrier having an average particle size of 1 μm or more and 1000 μm or less can be formed.

Porous cross-linked organic polymer particle carrier has a high affinity with aqueous medium at the time of separation and a structure derived from (meth) acrylic monomer, especially (meth) acrylic acid ester monomer, which is effective for improving selectivity It is preferable to have a unit. In particular, the structural unit derived from the (meth) acrylic acid ester monomer is preferably 50% by weight or more of the total structural units of the polymer.
In the present invention, the expression “(meth) acryl” means “acryl or methacryl” as described above.

Examples of the styrene monomer include monomers such as styrene, ethylstyrene, methylstyrene, hydroxystyrene, and chlorostyrene.
(Meth) acrylic monomers include methyl (meth) acrylate, ethyl (meth) acrylate, hydroxyethyl (meth) acrylate, hydroxypropyl (meth) acrylate, butyl (meth) acrylate, (meth ) (Meth) acrylic acid ester monomers such as 2-ethylhexyl acrylate, cyclohexyl (meth) acrylate, glycerin mono (meth) acrylate, (meth) acrylamide, dimethyl (meth) acrylamide, hydroxyethyl (meth) acrylamide, etc. Epoxy group content bodies such as (meth) acrylamides, nitriles such as (meth) acrylonitrile, glycidyl (meth) acrylate, 4,5-epoxybutyl (meth) acrylate, 9,10-epoxystearyl (meth) acrylate, etc. Etc. can be exemplified.

  The average particle size of the porous crosslinked organic polymer particle carrier is determined by the operating conditions of suspension polymerization, for example, selection of the types and amounts of the above various raw material monomers, and selection of the types and amounts of the emulsifier and / or protective colloid agent. , And the intensity of stirring (such as the number of rotations of stirring) and the like can be appropriately controlled.

  The pore diameter and the total volume of the pores of the porous crosslinked organic polymer particle carrier are the type and amount of the polymerizable monomer used, the amount ratio of water and monomer during polymerization, or polymerization during polymerization. It can be adjusted by allowing a predetermined amount of an organic solvent inert to the reaction system to coexist in the reaction system and controlling the type and amount thereof. Furthermore, it can be adjusted by the kind and amount of the polymerization initiator.

The supported amount (immobilization amount) of the ligand particles is preferably 10 to 50 mg / mL (resin), and more preferably 20 to 40 mg / mL (resin).
The amount of ligand particles supported is determined by polymerizing a vinyl compound that functions as a quantitative marker, such as DNS methacrylate, shown in Examples below, into a copolymer that is a ligand particle, The fluorescence spectrum of the solution before and after adsorption can be measured, and the amount of ligand particles immobilized on the organic polymer particle carrier can be determined from the difference in fluorescence intensity at each maximum wavelength.

  Ligand particles may be physically adsorbed on the organic polymer particle carrier as a method for supporting the ligand particles on the organic polymer particle carrier. However, in order to more firmly fix the ligand particles, the ligand particles are supported by an ion exchange reaction. A method or a method in which the reactive functional group of the copolymer constituting the ligand particle and the reactive functional group of the organic polymer particle carrier are bonded via a covalent bond by a chemical reaction is preferable.

  As a method for supporting ligand particles on an organic polymer particle carrier by ion exchange reaction, for example, a copolymer and a solution constituting the ligand particles are combined with a quaternary ammonium group such as a trimethylammonium group or a dimethylethanolammonium group. For example, a method of supplying ions onto an organic polymer particle carrier into which various ion exchange groups have been introduced and bringing the copolymer constituting the ligand particles into contact with a solution for ion exchange can be mentioned.

  On the other hand, a method for supporting (immobilizing) ligand particles on an organic polymer particle carrier through a covalent bond by chemical reaction is to introduce a structural unit having a reactive functional group into a copolymer constituting the ligand particles. In addition, by introducing a reactive functional group capable of binding to the reactive functional group of the copolymer constituting the ligand particle into the organic polymer particle carrier, the ligand particle and the organic polymer particle carrier are chemically bonded. It is a method of immobilization.

The structural unit having a reactive functional group to be introduced into the ligand particle may be selected according to the type of the reactive functional group on the organic polymer particle carrier side.
A preferred example is a method in which a structural unit having an amino group is introduced into the copolymer constituting the ligand particle, and a functional group capable of covalently bonding to the amino group is introduced into the organic polymer particle carrier.
As functional groups that can be covalently bonded to amino groups, they consist of epoxy groups, carboxyl groups, sulfonic acid groups, phosphoric acid groups, amino groups, hydroxyl groups, imino groups, imidazole groups, carbamate groups, cyano groups, thiol groups, and aldehyde groups. There may be mentioned at least one functional group of the group. Among these, an epoxy group and a carboxyl group are preferable, and among these, an epoxy group is preferable from the viewpoint of reactivity.

  Examples of the polymerizable monomer having an epoxy group that is preferably used for introducing a functional group capable of covalently bonding with an amino group into an organic polymer particle carrier include an epoxy group-containing monomer such as glycidyl (meth) acrylate. Body, allyl glycidyl ether, vinyl glycidyl ether, 4-epoxy-1-butene, and the like.

  Hereinafter, a method for supporting ligand particles via a covalent bond to a porous crosslinked organic polymer particle carrier which is a suitable organic polymer particle carrier in the separation agent of the present invention will be described in more detail.

  As a method of covalently immobilizing a copolymer as a ligand particle to a porous crosslinked organic polymer particle carrier, a polymerizable monomer having a reactive functional group-providing property on the porous crosslinked organic polymer particle carrier. In the form of copolymerization or the like, and the reactive functional group directly reacts with the functional group introduced into the ligand particle, or the component of the porous crosslinked organic polymer particle carrier. A low molecular or high molecular compound (hereinafter, these compounds are collectively referred to as “spacers”) each having one or more functional groups each capable of reacting with a functional group and a functional group introduced into the ligand particle. The method of making it couple | bond through is mentioned.

When immobilizing ligand particles into which amino groups are introduced, the former method is to include a functional group that forms a covalent bond with an amino group such as an epoxy group or a carboxyl group in the porous crosslinked organic polymer particle carrier. An example is a method of directly reacting this with a copolymer constituting the ligand particle and immobilizing it.
In the latter method, an amino acid (amine carboxylic acid) is used as a spacer, the amino group site is reacted with the epoxy group contained in the porous crosslinked organic polymer carrier, and then the other terminal. Porous cross-linking organic system by reacting with the amino group of the copolymer constituting the ligand particle by carboxyl group, or by sequentially using diamine or diol and diglycidyl compound such as (poly) ethylene glycol diglycidyl ether as spacer A method in which an epoxy group of a polymer carrier is bonded to one end of a diamine or diol, one epoxy group of a diglycidyl compound is bonded to the other end, and the remaining epoxy group is bonded to a copolymer which is a ligand particle. Etc.

Examples of the diamine used as a part of the spacer in the above method include aliphatic diamines such as tetramethylene diamine and hexamethylene diamine. Examples of the diol include propylene glycol, butane diol, diethylene glycol, and triethylene glycol. And aliphatic diols such as polyethylene glycols.
In consideration of the reactivity with the ligand particles and the steric hindrance relationship with the porous crosslinked organic polymer carrier at the time of immobilization, the spacer has a branched structure even if it has a linear structure. However, it preferably has a linear structure. When a spacer having a linear structure is used, the amount of adsorption tends to be improved because steric hindrance is reduced and an affinity bond between the ligand particle and the antibody is easily formed.

A preferable specific method is exemplified by using the particulate copolymer constituting the ligand particle having the reactive functional group as an aqueous solution on the porous crosslinked organic polymer particle carrier having the reactive functional group such as the epoxy group. And the reaction is carried out.
In this case, the temperature of the immobilization reaction is preferably from room temperature (20 ° C.) to about 30 ° C. If the temperature is too high, the ligand particles and the like may be inactivated, while if the temperature is too low, the reaction may take a long time.

After the immobilization reaction as described above, the reactive functional group remaining on the porous crosslinked organic polymer particle carrier side is preferably inactivated by post-treatment.
As such post-treatment, for example, when an epoxy group is taken as an example of a reactive functional group, a method of reacting with an aqueous solution of amines such as ethanolamine to inactivate it can be exemplified.

  The separation agent after the ligand particle immobilization reaction or the separation agent subjected to post-treatment in addition to the immobilization reaction is preferably washed with water in order to remove unreacted substances.

The obtained separating agent is temporarily stored except when used as it is. As a medium for storage, an ethanol aqueous solution having a concentration of 1 to 50% by weight is preferably used.
When the separating agent using the porous crosslinked organic polymer particle carrier is stored in the storage medium, the swelling degree of the separating agent is appropriate, and the affinity for the separating medium for the separating agent is good. .

<Application>
The separation agent of the present invention is preferably used for separation of proteins, particularly antibodies, as an affinity separation agent because the copolymer constituting the ligand particles has affinity adsorption to proteins, particularly antibodies. can do.
Particularly preferred target molecules include immunoglobulins, fusion proteins containing at least part of the Fc region of immunoglobulins, or chemically modified products thereof. Among these, those in which the immunoglobulin is a monoclonal antibody or a polyclonal antibody are preferred.

The target molecule separation treatment is preferably performed so as to include the following steps (a) and (b). In such a separation treatment, it is preferable to use a liquid chromatography column which is provided with at least one container and is filled with the separation agent of the present invention.
(A) The process which makes the solution containing a target molecule contact said separation agent, and makes a target molecule adsorb | suck to a separation agent.
(B) A step of eluting the target molecule from the separating agent that has adsorbed the target molecule.

By such a method, it is possible to separate various proteins as described above with high selectivity. In such a separation process, a liquid chromatography column including the above separating agent and including at least one container is preferably used.

  The antibody adsorption amount of the separating agent of the present invention is preferably 1 mg / mL (resin) or more. In addition, mL (resin) shows the volume of wet resin.

The method for measuring the amount of adsorbed antibody was that the separating agent was wetted with water and 1 volume part was weighed into a tube, and 50 volume parts of a mouse polyclonal IgG aqueous solution (reagent manufactured by Wako Pure Chemical Industries, Ltd. was added at a concentration of 2.5 mg / and diluted with mL) and stirred at room temperature for 5 hours to adsorb IgG. The absorbance of the supernatant before and after adsorption is measured, and the amount of antibody adsorbed is determined from a separately prepared calibration curve.
In addition, it is thought that when the mouse polyclonal antibody adsorption amount is small, the adsorption amount of the target human antibody is also reduced.

  EXAMPLES Hereinafter, although this invention is demonstrated more concretely based on an Example, this invention is not limited at all by the following example. In addition, the value of various manufacturing conditions and evaluation results in the following examples has a meaning as a preferable value of the upper limit or the lower limit in the embodiment of the present invention, and the preferable range is the above-described upper limit or lower limit value. A range defined by a combination of values of the following examples or values of the examples may be used.

[Examples 1 to 6]
1) Synthesis of DNS methacrylate DNS methacrylate as a marker for quantitative determination of the copolymer constituting the ligand particle was synthesized by the following method.
First, 0.94 g of dansyl chloride was dissolved in 75 ml of anhydrous tetrahydrofuran (hereinafter referred to as THF), and slowly added to 2.32 ml of ethylenediamine dissolved in 150 ml of anhydrous THF in an ice bath. The reaction was shielded from light with aluminum and reacted at 0 ° C. for 3 hours. Thereafter, 10 ml of 1 mol% KOH was added to make the solution basic. THF was removed with an evaporator, and the aqueous phase was separated with methylene chloride. The organic phase was dried over MgSO ₄ and the methylene chloride was blown off with an evaporator. Recrystallization from benzene and filtration gave a yellow-green solid.

  In 33 ml of anhydrous THF, 0.333 g of this solid was dissolved, 0.112 ml of methacryloyl chloride and 0.16 ml of triethylamine were added, and the mixture was stirred at room temperature for 16 hours. Thereafter, 0.056 ml of methacryloyl chloride and 0.08 ml of triethylamine were added and stirred for 3 hours. The salt was removed by filtration, washed with THF, and THF was removed by an evaporator. This was column-purified with a developing solvent (ethyl acetate / hexane) to obtain a light green solid (DNS methacrylate).

2) Synthesis of Ligand Particles The copolymer constituting the ligand particles is prepared by converting vinyl compounds (copolymerizable monomers) as various raw materials into a precipitation weight as described in Journal of the American Chemical Society, 134, 15765-15772,2012. Synthesized according to law.
As raw material monomers for ligand particles, N-isopropylacrylamide (NIPAm), tert-butylacrylamide (TBAm), acrylic acid (AAc), methylenebisacrylamide (Bis) as a cross-linking agent, and N-as a vinyl compound having a reactive functional group (3-Aminopropyl) methacrylamide (Am), and 1) for DNS methacrylate as a marker for quantification were used. Each chemical structure is as shown below.
As a vinyl compound (raw material monomer) as a raw material, 400 ml of NIPAm, TBAm, AAc, Bis, Am, and DNS methacrylate are mixed at a ratio shown in Table 1 below so that the total concentration of raw material monomers in the solution is 6.5 mM. Dissolved in water. At that time, TBAm was dissolved in 4 ml of ethanol and DNS methacrylate was dissolved in 5 ml of acetone and then charged into the solution. Also 40 mg of sodium dodecyl sulfate was added. The solution was placed in an eggplant flask, sealed with a septum, and degassed with nitrogen bubbling for 120 minutes while stirring. The solution was then stirred at room temperature for 17 hours. After completion of the reaction, the solution was put into a dialysis membrane and dialyzed with water for 4 days to obtain a copolymer solution (ligand particle solution).

  Table 1 shows the ratio of the monomer of each ligand particle, and the average particle diameter of each obtained ligand particle. In Table 1, the average particle size of the ligand particles (particulate copolymer) was measured by a dynamic light scattering method (Zeta Sizer Nano manufactured by Malvern).

3) Synthesis of organic polymer particle carrier Organic polymer particle carrier S1 for binding to ligand particles by covalent bond as an organic polymer particle carrier for binding to ligand particles by chemical bond, ion exchange Two types of organic polymer particle carrier S2 for binding with ligand particles by the method were used.

  The organic polymer particle carrier S1 for binding to the ligand particles by covalent bonding was synthesized by the following method.

  First, in water obtained by dissolving 0.5 parts by weight of polyvinyl alcohol in 100 parts by weight of water, 80 parts by weight of glycidyl methacrylate, 20 parts by weight of ethylene glycol dimethacrylate, 2,2′-azobis (2,4-dimethylvaleronitrile) 1 A mixture of parts by weight and 100 parts by weight of dichloropropane was added and stirred to form a suspended state. At this time, the stirring speed was adjusted so that the average diameter of the droplets was about 100 μm. The suspension was heated to 70 ° C. and reacted for 6 hours. After cooling, the obtained porous particles having a crosslinked structure were washed with water, washed with methanol, and dried to obtain porous particles of a crosslinked copolymer.

  Next, the porous particles were wetted with water, and 100 parts by weight thereof was collected in a flask. 200 parts by weight of ethanol / water (50/50) solvent was added thereto, 100 parts by weight of 1,4-butanediol was further added and mixed, and then 2 parts by weight of sulfuric acid (98%) was added as a catalyst. The reaction was carried out at 40 ° C. for 5 hours to obtain an adduct in which 1,4-butanediol reacted with the epoxy groups of the porous particles.

  Next, 50 parts by weight of ethylene glycol diglycidyl ether is added per 100 parts by weight of 1,4-butanediol-added particles (water wet state), an alkali catalyst is added, and 1,4-butanediol is added to the hydroxyl terminal. And a porous cross-linked polymer carrier having a spacer having an epoxy group as a terminal group was prepared. The epoxy group content of this particulate porous crosslinked polymer carrier was 1.5 μmol / g (resin).

  Further, MCI (registered trademark) gel CQA35P manufactured by Mitsubishi Chemical Corporation was used as the organic polymer particle carrier S2 for binding to the ligand particles by the ion exchange method.

The average particle diameter and pore diameter of the organic polymer particle carriers S1 and S2 were evaluated by the following methods.
The average particle diameter of the organic polymer particle carrier is an average value obtained by taking a micrograph using an optical microscope and measuring the diameter of 100 particles per field of view. In addition, the pore diameter (Å) of the organic polymer particle carrier is determined by a histogram showing the pore distribution measured with a mercury porosimeter manufactured by Shimadzu Corporation and the pore diameter as the vertical axis and horizontal axis, respectively. The pore diameter of the portion having the largest total pore volume was taken as the average pore diameter.
Table 2 summarizes the physical properties of each organic polymer particle carrier.

4) Loading of ligand particles on organic polymer particle carrier (production of separating agent)
1 part by weight of the organic polymer particle carrier (S1, S2) of 3) above is collected in a wet state, and 5 to 50 parts by weight of the aqueous ligand particle solution of 2) is added thereto and reacted at room temperature for 3 hours or more. Ligand particles were supported on an organic polymer particle carrier. The organic polymer particle carrier carrying the ligand particles was sufficiently washed with water to obtain protein affinity separation agents (R1 to R6).

5) Evaluation of ligand particle loading For the separation agent using ligand particles with DNS introduced, the fluorescence spectrum of the solution before and after adsorption is measured, and the amount of ligand particle loading is determined from the difference in fluorescence intensity at each maximum wavelength. evaluated. In addition, regarding ligand particles into which DNS was not introduced, SEM observation of the separating agent after loading was performed to confirm that the ligand particles were supported.

6) IgG adsorption amount evaluation 1 volume part of the protein affinity separation agents (R1 to R6) was collected in a tube in a wet state. 50 volume parts of mouse polyclonal IgG aqueous solution (concentration 2.5 mg / ml) was added thereto and stirred at room temperature for 5 hours. The amount of adsorbed IgG was quantified by measuring the absorbance of the supernatant before and after the adsorption of IgG.

7) IgG elution rate evaluation The IgG affinity elution is performed by contacting the protein affinity separation agent adsorbed with IgG with pH 2.3 glycine / hydrochloric acid buffer solution for 3 hours, and measuring the absorbance of the eluted IgG. The amount was evaluated and the elution rate was determined.

  The above results are summarized in Table-3.

[Evaluation of results]
As shown in Table 3, it was shown that all of the protein affinity separation agents of Examples 1 to 6 adsorb IgG well. Moreover, from the results of Examples 3 to 6 in Table 3, the affinity for protein also showed the ability to favorably elute IgG after adsorbing IgG. From these results, it can be seen that the protein affinity separation agent of the present invention is effective.

  The separation agent of the present invention exhibits separation / adsorption performance with a high selectivity for proteins, particularly antibodies, and is excellent in strength and durability, and thus has a high practical value particularly in the fields of medicine and diagnosis.

Claims (6)

It consists of a copolymer containing, as a main component, a structural unit derived from each of the compound represented by the following formula (1), the compound represented by the formula (2) and the compound represented by the formula (3). A protein affinity separation agent comprising a ligand particle having a particle diameter of 20 nm to 500 nm and an organic polymer particle carrier.
(In the above formula (1), R ¹ is a hydrogen atom or an optionally branched alkyl group having 1 to 3 carbon atoms, and R ² is an optionally branched alkyl group having 1 to 3 carbon atoms. In the above formula (2), R ³ is a hydrogen atom or an optionally branched alkyl group having 1 to 3 carbon atoms, and R ⁴ is an optionally branched alkyl group having 4 to 8 carbon atoms. In the above formula (3), R ⁵ is a hydrogen atom or an optionally branched alkyl group having 1 to 3 carbon atoms.) The structural unit derived from each of the compound represented by the formula (1), the compound represented by the formula (2), and the compound represented by the formula (3) is an all vinyl compound constituting the copolymer. The protein affinity separation agent according to claim 1, which has the following ratio with respect to the total weight.
Structural unit derived from the compound represented by formula (1): 5% by weight or more and 90% by weight or less Structural unit derived from the compound represented by formula (2): 5% by weight or more and 90% by weight or less Formula (3) Structural unit derived from a compound represented by: 3 wt% or more and 50 wt% or less   The compound represented by the formula (1) is N-isopropylacrylamide, the compound represented by the formula (2) is tert-butylacrylamide, and the compound represented by the formula (3) is acrylic acid. The protein affinity separation agent according to claim 1 or 2.   4. The organic polymer particle carrier according to claim 1, wherein the organic polymer particle carrier is a porous crosslinked organic polymer particle carrier having an average particle size of 1 μm to 1000 μm and a pore diameter of 10 to 10,000 μm by a mercury intrusion method. 2. The protein affinity separation agent according to item 1.   The organic polymer particle carrier is a porous crosslinked organic polymer particle carrier made of a crosslinked copolymer having a structural unit derived from a (meth) acrylic acid ester monomer. The affinity separation agent for protein according to Item.   The organic polymer particle carrier is an epoxy group, carboxyl group, sulfonic acid group, phosphoric acid group, amino group, hydroxyl group, imino group, imidazole group, carbamate group, cyano group, thiol group, or aldehyde group. The protein affinity separation agent according to any one of claims 1 to 5, which has at least one functional group.