JP2004201752A - Protein adsorption suppressing material and column using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a material which can suppress an adsorption of protein or the other useful substances and is particularly useful as the material for a blood purification column. <P>SOLUTION: The protein adsorption material is made to have a positively charged cationic substance with an amount of functionality of 2.0mmol/g or more and 10.0mmol/g or less immobilized on a base material and a molecular weight of the cationic substance is 600 or more. Thereby the material which is suppressed to adsorb protein or the other useful substances in a liquid and blood can be obtained. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a protein adsorption-suppressing material, particularly to a material in which protein or useful substance present in liquid or blood is suppressed from adsorbing, and a column using the same.
[0002]
[Prior art]
In a material used for a blood purification column, particularly a material made of a hydrophobic material, it is necessary to suppress the adsorption of a substance that causes thrombus formation or a useful substance that is not an object to be removed. For this purpose, various attempts have been made to develop materials in which adsorption of proteins and the like in blood is suppressed by grafting a nonionic hydrophilic polymer (for example, see Patent Documents 1 and 2). However, the function of immobilizing a physiologically active substance or the like using a nonionic hydrophilic polymer immobilized on a material cannot be modified.
[0003]
In addition, when a useful substance such as a cell is captured using a protein-immobilized material such as an antibody, and the useful substance is recovered by an external stimulus such as reduction, the protein detached from the material is re-adsorbed to the material, Since there is a possibility that the useful substance cannot be recovered, it is necessary to suppress the adsorption in such a case.
[0004]
[Patent Document 1]
JP 2000-237557 A
[Patent Document 2]
JP-A-6-212078
[Problems to be solved by the invention]
An object of the present invention is to solve the above-mentioned drawbacks of the prior art and to provide a material capable of suppressing adsorption of a protein or a useful substance.
[0007]
[Means for Solving the Problems]
In order to achieve the above object, the present invention has the following configuration.
(1) A protein adsorption-inhibiting material, characterized in that a cationic substance having a positive charge amount of 2.0 mmol / g or more and 10.0 mmol / g or less is immobilized on a substrate.
(2) The protein adsorption suppressing material according to (1), wherein the cationic substance has a molecular weight of 600 or more.
(3) The material for suppressing protein adsorption according to (1) or (2), wherein the cationic substance is a substance having an amino group.
(4) The protein adsorption suppressing material according to any one of (1) to (3), wherein the physiologically active substance is immobilized via an amino group.
(5) The protein adsorption suppressing material according to (4), wherein the physiologically active substance is a protein.
(6) The protein adsorption-suppressing material according to (5), wherein the protein is an antibody.
(7) The protein adsorption suppressing material according to (3), wherein the substance having an amino group is polyethyleneimine or a derivative thereof.
(8) The protein adsorption suppressing material according to any one of (1) to (7), wherein the substrate is a fiber.
(9) The protein adsorption suppressing material according to (8), wherein the fiber is a sea-island type fiber.
(10) The protein adsorption-inhibiting material according to (8) or (9), wherein the substrate shape is any of a knitted fabric, a woven fabric, and a nonwoven fabric.
(11) The material according to any one of (1) to (10), wherein the base material is made of one or more substances selected from the group consisting of polystyrene, polyester, polypropylene, polyethylene, nylon, and derivatives thereof. The material for suppressing protein adsorption described in the above.
(12) A column using the protein adsorption suppressing material according to any one of (1) to (11), which is used for extracorporeal circulation.
(13) A column using the material for suppressing protein adsorption according to (12), which is used for any of cell separation, cell concentration, and blood purification.
[0008]
BEST MODE FOR CARRYING OUT THE INVENTION
The present invention is a protein adsorption suppressing material, characterized in that a cationic substance having a positively charged functional group content of 2.0 mmol / g or more and 10.0 mol / g or less is immobilized on a substrate.
[0009]
The cationic substance used in the present invention is not particularly limited as long as it is a substance having a cationic property (a substance having a functional group having a positive charge). Among them, a substance containing a nitrogen atom, that is, a primary, secondary, Amino group-containing substances, imino group-containing substances, amide group-containing substances and the like having at least one of a tertiary amino group and a quaternary ammonium salt are preferably used.
[0010]
As the cationic substance used in the present invention, a polymer having a molecular weight of 600 or more is preferably used from the viewpoint of the effect on protein non-adsorption. Among them, polymers containing an amino group are easily available and are preferably used.
[0011]
Examples of the amino group-containing polymer include polyalkyleneimine, polyallylamine, polyvinylamine, dialkylaminoalkyldextran, chitosan, polyornithine, polylysine, and those having a substituent introduced therein, and monomer units constituting these. A copolymer, a nonionic, a copolymer with an anionic compound, and the like can be given. Here, the polymer may be linear, branched, or cyclic. The molecular weight is preferably 600 or more and 10,000,000 or less. More preferably, it is not less than 1000 and not more than 100,000.
[0012]
Among the amino group-containing polymers, branched polyethylene imine and diethylaminoethyl dextran are particularly preferably used because of their low toxicity, availability, and ease of handling.
[0013]
As the polyethyleneimine, a linear or branched one having a molecular weight of 600 to 10,000,000 is preferably used. Examples of the polyethyleneimine derivative include those obtained by derivatizing polyethyleneimine at a desired ratio such as alkylation, carboxylation, phenylation, phosphorylation, and sulfonation.
[0014]
Although the substrate used in the present invention is not particularly limited, an organic polymer compound is preferable in consideration of chemical resistance, heat resistance, radiation resistance and low cost. For example, polyvinyl chloride, polystyrene, polyester, polypropylene, polyethylene, polyurethane, nylon and derivatives thereof can be mentioned. When used as an extracorporeal circulation column, the above organic polymer compound is preferably used in the form of a fiber, a hollow fiber, a net, a knitted fabric, a woven fabric, a cut fiber, a non-woven fabric, etc. Considering the low flow resistance without clogging in the case, fibers, hollow fibers, knitted fabrics, woven fabrics and nonwoven fabrics are more preferably used.
[0015]
The sea-island type conjugate fiber can be used as a base material even when a material having low strength is used, since a certain strength can be imparted by using a material having strength for the core material (island portion). . For example, when the ligand is immobilized on a material such as polystyrene, it can be used as a base material by reinforcing the core with polypropylene or polyethylene.
[0016]
In the present invention, as a method for immobilizing a cationic substance on a substrate, an active group that reacts with the cationic substance is introduced into the surface of the substrate, followed by immersion or adhesion in a solution containing the cationic substance. Can be mentioned. It is also possible to introduce an active group after processing the fiber into a knitted fabric or a nonwoven fabric, and immerse or adhere the solution in a solution containing a cationic substance to fix the fiber. Further, in the case of a polyamide-based material such as nylon, the amino group-containing substance can be immobilized by heating with the amino group-containing substance and performing a transamidation reaction. Further, the material can be grafted by light or radiation together with a vinyl monomer or polymer having an amino group.
[0017]
In the present invention, the amount of the cationic substance immobilized on the substrate needs to be 2.0 mmol / g or more in terms of the amount of the functional group having a positive charge. When the amount of the cationic substance immobilized is 2.0 mmol / g or more in terms of the amount of the functional group having a positive charge, the adsorption of the protein is suppressed to a level that does not cause any problem when used as a medical material, particularly as a blood purification column. . If the amount of the cationic substance immobilized is less than 2.0 mmol / g, protein adsorption to the base material increases, and the adsorption can be suppressed to the extent necessary when used as a medical material, particularly as a blood purification column. Absent. In addition, if the amount of the cationic substance immobilized is too large, the material itself is largely positively charged, and there is a concern that biological materials, particularly cells, may be adversely affected or the material may be denatured. The amount is desirably 10.0 mmol / g or less.
[0018]
Here, “adsorption is suppressed” refers to a state in which the amount of protein adsorbed per 1 g of the base material is less than 0.5 mg. When the amount of protein adsorbed per 1 g of the base material is 0.5 mg or more, the loss of useful substances in the blood increases, and when the protein is desorbed from the material by external stimulation, the protein cannot be desorbed from the immobilized material. .
[0019]
The amount of protein adsorbed on the substrate is determined by immersing the substrate in a protein solution having a predetermined concentration for 30 minutes or more, and adjusting the concentration of the protein solution before and after the immersion of the substrate by TSK manufactured by Tosoh filled with hydrophilic silica. -It can be measured by using high-performance liquid chromatography with a buffer having a stable pH as an eluent using a biopolymer column such as GEL SW series.
[0020]
The amount of the functional group having a positive charge is calculated based on the type of the cationic substance to be immobilized and the amount of the cationic substance immobilized on the substrate.
[0021]
The amount of the cationic substance immobilized on the base material is measured by measuring the weight before and after the reaction, obtaining the amount of immobilization from the weight increase, or using an acid dye such as Acid Orange 7 having a carboxylic acid or sulfonic acid in the molecule. And dyeing the substrate, immersing the substrate in dilute hydrochloric acid, binding HCl in dilute hydrochloric acid to the substrate, taking out the substrate, and neutralizing and titrating the consumed HCl with an aqueous sodium hydroxide solution. Can be obtained by doing
[0022]
According to the material of the present invention, that is, a material in which a cationic substance, in particular, an amino group-containing substance is immobilized, various ligands can be immobilized using an amino group and used for a blood purification column or a cell separation column. I can do it.
[0023]
When used in a cell separation column, in order to recover the cells captured by the ligand, it is preferred that the bond between the ligand and the material be cut off by an external stimulus, and the ligand in which the cells are captured be detached. Reactions that break the bond using a chemical reaction include hydrolysis, oxidation, reduction, and enzymatic reactions, but they require stability in water and maintain cell viability. A reduction reaction is preferred. In particular, a reaction that cleaves a disulfide bond by a reduction reaction with a reducing agent is a reaction often performed in vivo such as refolding of a protein, and can be suitably used as a reaction for removing a ligand. Examples of a reducing agent that causes such a reduction reaction include, but are not limited to, dithiothreitol, mercaptoethanol, cysteine, and reduced glutathione.
[0024]
There are various proteins that can suppress adsorption by the material of the present invention. For example, proteins that cause thrombus formation include fibrinogen, collagen, von Wille blind factor, fibronectin, platelet-derived growth factor, and the like. Often, albumin and antibodies should not be removed. When an antibody is immobilized on a material and used for cell separation, the antibody desorbed by external stimulus during collection must be prevented from being re-adsorbed.
[0025]
Examples of the form of the column of the present invention include a laminated type, a spiral type, a pleated type, a tubular type, a plate and plate type, and the like.For example, a knitted fabric or a nonwoven fabric punched into a circle having the same diameter as a cylindrical module inner diameter is laminated. A fabric having an opening through which cell groups can flow at both ends, or having an entrance through which cell groups can flow on one side, and wrapping a knitted fabric or a nonwoven fabric around a tube having an opening at the side serving as an outlet for cell groups. Incorporate in the module and attach a header to the module to make a column.
[0026]
The column using the material obtained in the present invention is used for an artificial kidney, a blood adsorbent and the like. A column using a material on which a ligand such as an antibody that can be detached by an external stimulus is immobilized is used for cell separation. It is used for concentration use or cell activation.
[0027]
The method for producing the extracorporeal circulation column material using the base material of the present invention will be described below with reference to an example in which polyethyleneimine is immobilized on polystyrene fibers.
[0028]
【Example】
Hereinafter, the present invention will be described in detail with reference to examples, but the content of the invention is not limited to the examples.
[0029]
The immobilized amount of the cationic substance and the protein concentration in the examples were determined by the following neutralization titration and high performance liquid chromatography, respectively.
[0030]
(Neutralization titration)
0.5 g of the material thoroughly washed with pure water was immersed in 20 ml of 0.1N diluted hydrochloric acid overnight, and then 10 ml was taken out and titrated with 10 ml of a 0.1N aqueous sodium hydroxide solution using phenolphthalein as an indicator.
[0031]
(High performance liquid chromatography)
The measurement conditions are as follows. Eluent: 0.5 M acetate buffer, pH 4.5, Column: G3000SWXL manufactured by Tosoh, apparatus: Tosoh Corporation, HPLC system, flow rate: 0.5 ml / min.
[0032]
Production Example 1. Preparation of Chloroacetamidomethylated Crosslinked Polystyrene Knitted Fabric 1 Sea-island composite fiber (50 weight ratio of a sea component (a mixture of 46 weight ratio of polystyrene and 4 weight ratio of polypropylene)) and 50 weight ratio of an island component (polypropylene) ( Thickness: 2.6 denier, number of islands 16) was obtained by mixing 35 g of N-methylol-α-chloroacetamide, 232.5 g of nitrobenzene, 232.5 g of 98% sulfuric acid, and 0.5 g of paraformaldehyde. The reaction was performed at 10 ° C. for 2 hours. The fibers were washed with nitrobenzene and quenched with methanol. As a result, chloroacetamidomethylated crosslinked polystyrene fibers (hereinafter abbreviated as AMPSt fibers) were obtained. The obtained AMPSt fiber was used as a knitted fabric (hereinafter referred to as AMPSt knitted fabric 1).
[0033]
Production Example 2. Preparation of chloroacetamidomethylated crosslinked polystyrene knitted fabric 2 10.0 g of polyethyleneimine (molecular weight: 10,000, manufactured by Wako Pure Chemical Industries) was dissolved in 0.95 g of triethylamine and 39.0 g of dimethyl sulfoxide, and prepared in this solution in Preparation Example 1. 1.0 g of the AMPSt knitted fabric 1 was added and stirred. The reaction was performed at 30 ° C. for 3 hours. Thereafter, the resultant was washed with dimethyl sulfoxide, methanol and pure water to obtain AMPSt knitted fabric 2 containing an amino group. When the amount of amino groups introduced was determined by titration, it was found to be 3.0 mmol / g.
[0034]
Production Example 3 Preparation of chloroacetamidomethylated cross-linked polystyrene knitted fabric 3 2.0 g of polyethyleneimine (molecular weight: 10,000, manufactured by Wako Pure Chemical Industries) was dissolved in 0.95 g of triethylamine and 47.0 g of dimethyl sulfoxide, and prepared in this solution in Preparation Example 1. 1.0 g of the AMPSt knitted fabric 1 was added and stirred. The reaction was performed at 30 ° C. for 3 hours. Thereafter, the resultant was washed with dimethyl sulfoxide, methanol and pure water to obtain AMPSt knitted fabric 3 containing an amino group. When the amount of amino groups introduced was determined by titration, it was found to be 2.0 mmol / g.
[0035]
Production Example 4. Preparation of chloroacetamidomethylated cross-linked polystyrene knitted fabric 4 0.5 g of polyethyleneimine (molecular weight 10,000, manufactured by Wako Pure Chemical Industries) was dissolved in 0.95 g of triethylamine and 48.5 g of dimethyl sulfoxide, and prepared in this solution in Preparation Example 1. The AMPSt knitted fabric 1 thus obtained was added in an amount of 1.0 g (corresponding to a chloro content of 2.6 mmol) and stirred. The reaction was performed at 30 ° C. for 3 hours. Thereafter, the resultant was washed with dimethyl sulfoxide, methanol and pure water to obtain AMPSt knitted fabric 4 containing an amino group. When the amount of amino group introduced was determined by titration, it was found to be 1.5 mmol / g.
[0036]
(Examples 1-2, Comparative Examples 1-2)
Purified human immunoglobulin G (manufactured by Sigma) obtained by dissolving 0.05 g of the knitted fabrics 1 to 4 obtained in Preparation Examples 1 to 4 in a phosphate buffer solution (manufactured by Nissui, "Dulbecco") at 0.1 mg / ml. (Hereinafter abbreviated as IgG). Immersed in 1 ml, adsorbed for 2 hours at room temperature, and the change in concentration before and after adsorption was examined by high performance liquid chromatography. The knitted fabric 2 did not adsorb IgG at all (Example 1). ), Only 0.4 mg was adsorbed on the knitted fabric 3 (Example 2). On the other hand, the knitted fabric 1 adsorbed all the antibodies (Comparative Example 1), and the knitted fabric 4 adsorbed 1.2 mg of IgG (Comparative Example 2).
[0037]
Production Example 5 Preparation of Polystyrene Knitted Fabrics 5 and 6 Having Antibodies Removable by Reduction 0.05 g of the above AMPSt knitted fabric 1 and AMPSt knitted fabric 2 were added to 20 mM phosphate buffer (pH 7.5) containing 0.15 mM NaCl, respectively. Was immersed in 1 ml of a dimethyl sulfoxide solution in which N-succinimidyl-3- (2-pyridyldithio) -propionate (Pierce, SPDP) was dissolved in 27 mM for 0.5 hour (room temperature). After washing the reacted knitted fabric with the above-mentioned phosphate buffer, it is reduced with the above-mentioned phosphate buffer containing 100 mM dithiothreitol, and then washed with 100 ml of the above-mentioned phosphate buffer, and mercapto-coated on the surface. A polystyrene knitted fabric having a group was obtained.
[0038]
Further, a solution prepared by dissolving an anti-human CD4 monoclonal antibody (Sigma) at a concentration of 1.1 mg / ml in a 20 mM phosphate buffer (pH 7.5) containing 0.15 mM NaCl was added to a solution of 27 mM dissolved in dimethyl sulfoxide. SPDP was added so that SPDP was 10 mol per 1 mol of the antibody, and reacted at room temperature for 30 minutes. Thereafter, gel filtration was performed on a PD-10 column (manufactured by Pharmacia), and only the high molecular weight fraction was fractionated to purify the SPDP-conjugated anti-human CD4 monoclonal antibody.
[0039]
The AMPSt knitted fabric into which the mercapto group was introduced as described above was immersed in a phosphate buffer solution (pH 7.5) containing an SPDP-modified anti-human CD4 monoclonal antibody at room temperature for 18 hours. The SPDP group of the antibody was subjected to a disulfide exchange reaction to prepare a knitted fabric (antibody-immobilized knitted fabrics 5 and 6) in which the antibody was immobilized via a disulfide bond on the AMPSt knitted fabric.
[0040]
(Example 3, Comparative Example 3)
0.05 g of each of the antibody-immobilized knitted fabrics 5 and 6 was immersed in the above-mentioned phosphate buffer containing 1 mM dithiothreitol at room temperature for 30 minutes, and the amount of the CD4 antibody detached in the reducing solution was measured by high performance liquid chromatography. As measured by chromatography, no antibody was detached from the antibody-immobilized knitted fabric 5 (Comparative Example 3), but 10 μg of the antibody was detached from the antibody-immobilized knitted fabric 6 (Example 3).
[0041]
【The invention's effect】
According to the present invention, it is possible to provide a material in which adsorption of proteins or useful substances in liquid and blood is suppressed.

Claims (13)

A protein adsorption suppressing material, characterized in that a cationic substance having a positively charged functional group content of 2.0 mmol / g to 10.0 mmol / g is immobilized on a substrate. 2. The protein adsorption suppressing material according to claim 1, wherein the molecular weight of the cationic substance is 600 or more. The protein adsorption inhibiting material according to claim 1 or 2, wherein the cationic substance is a substance having an amino group. The protein adsorption suppressing material according to any one of claims 1 to 3, wherein the physiologically active substance is immobilized via the amino group. The protein adsorption inhibiting material according to claim 4, wherein the physiologically active substance is a protein. The protein adsorption suppressing material according to claim 5, wherein the protein is an antibody. The protein adsorption inhibiting material according to claim 3, wherein the substance having an amino group is polyethyleneimine or a derivative thereof. The protein adsorption suppressing material according to any one of claims 1 to 7, wherein the substrate is a fiber. The protein adsorption suppressing material according to claim 8, wherein the fiber is a sea-island type fiber. The protein adsorption suppressing material according to claim 8 or 9, wherein the base material is any of a knitted fabric, a woven fabric, and a nonwoven fabric. The protein adsorption suppression according to any one of claims 1 to 10, wherein the base material is made of one or more substances selected from the group consisting of polystyrene, polyester, polypropylene, polyethylene, nylon, and derivatives thereof. material. A column using the protein adsorption-suppressing material according to any one of claims 1 to 11, which is used for extracorporeal circulation. 13. The column using the protein adsorption-suppressing material according to claim 12, wherein the column is used for cell separation, cell concentration, or blood purification.