JPH0622628B2 - Carrier for immobilization of physiologically active substance and method for producing the same - Google Patents

Description

TECHNICAL FIELD The present invention relates to a carrier for immobilizing a physiologically active substance and a method for producing the same.

(Prior Art) A substance in which a physiologically active substance is immobilized on an insoluble carrier is widely used as an adsorbent for affinity chromatography, immobilized enzyme, therapeutic blood purification agent, therapeutic immunomodulator, antibacterial agent and other analytical reagents. It is an important substance that is expected to expand further in the future.

The requirements to be provided as an immobilization carrier are (1) under the conditions of use, be mechanically, chemically and thermally stable,
(2) It is possible to immobilize a physiologically active substance at a high density, (3) it is easy to manufacture, and (4) it is not decomposed by a hydrolase in a short time.

As examples of the carrier for immobilization, sepharose, those obtained by bromocyanating polysaccharide gel such as agarose, those obtained by bromocyanating porous silica gel beads, crosslinked polyacrylamide beads, etc. are known, but the polysaccharide gel is The water content is too high and the liquid permeability is poor, and it is especially difficult to use with highly viscous liquids such as blood. Furthermore, it is chemically unstable. Porous silica gel beads and cross-linked polyacrylamide beads have the drawbacks of low immobilization density and low chemical stability.

In addition, as a modification of these drawbacks, a fibrous carrier provided by the present inventors (ethylenediaminoacetamidomethylated polystyrene fiber; JP-A-59-6405).
There is 3). It is chemically and thermally stable,
Although it has the advantage of high immobilization density compared to the above carrier,
There is a drawback that the volume change is large with respect to the pH change. For example, when the pH is changed from 7.4 to 4.0, the water content (the number of grams of water contained in 1 g of the carrier) becomes 6 times (Example 4, Comparative Fiber 5). If the molded product has a large volume change, it tends to cause self-destruction, and when it is packed in a column and used, a drift tends to occur due to repeated reuse.

As the bond between the immobilizing carrier and the substance to be immobilized, an amide bond, an alkyl bond, an ether bond, an ester bond, a ureido bond, a urea bond and the like are known. Among these, the amide bond and the alkyl bond are known. High chemical stability.
Especially, the amide bond is preferably used because the range of applicable substances to be immobilized is wide and the immobilization condition is mild. Therefore, an aliphatic primary amino group or a carboxyl group capable of forming an amide bond is most preferably used as the functional group to be retained on the carrier. Aliphatic secondary amino groups and aromatic primary amino groups can also be used, but they are generally not widely used because of the low reaction rate.

(Problems to be Solved by the Invention) The present invention has reached the present invention as a result of extensive studies to solve the problems of the conventional art.

(Means for Solving Problems) The present invention provides (1) immobilization of a physiologically active substance composed of a vinyl polymer having an aromatic nucleus into which a functional group represented by the following general formula (I) is introduced as a side chain. Chemical carrier.

(In the above formula, R ₁ and R ₂ represent a hydrogen atom or a lower alkyl group, R ₃ represents an NH ₂ or NH—R ₄ —NH ₂ group, R 3
₄ represents an alkylene group having 4 to 16 carbon atoms. (2) A vinyl polymer having an aromatic nucleus in which a functional group represented by the following general formula (II) is introduced as a side chain and a molded product thereof are ammonia, (In the above formula, R ₁ and R ₂ are hydrogen atoms or lower alkyl groups, and X is a halogen atom.) When reacting with a diamine represented by the following general formula (III), an NH ₂ —R ₄ —NH ₂ group (III) (R ₄ represents an alkylene group having 4 to 16 carbon atoms) An aprotic polar organic solvent is used to produce a carrier for immobilizing a physiologically active substance.

Is provided.

The vinyl polymer having an aromatic nucleus in the present invention means a homopolymer of a vinyl monomer having an aromatic nucleus represented by styrene, α-methylstyrene, vinyltoluene or the like or a copolymer containing these as the main components. However, it is more preferable that these polymers are crosslinked. Further, if the polymer is reinforced with poly α-olefin represented by crystalline polypropylene, polyethylene, etc., the mechanical properties are improved, which is more preferable. Examples thereof include a copolymer with a polyvinyl compound represented by divinylbenzene or methylenebisacrylamide, and a product obtained by subjecting the molded product of the above-mentioned monovinyl compound to a cross-linking treatment with formaldehyde, chlorosulfonic acid or the like. Since the crosslinked polymer has no fluidity and is difficult to mold, when the polymer molded product is a fiber, a film or the like, a method of crosslinking after molding is preferably adopted.

Molded product in the present invention means fiber, membrane, hollow fiber, diameter 10μ
It means granules of m or more and their higher-order processed products. In particular, it is preferable that fibers and hollow fibers can be used in such a manner that a flow path can be secured. This property is important when the immobilizate is used as a treatment agent for highly viscous liquids such as blood.

If the surface area of the molded article of the present invention is too small, it will not be possible to immobilize the physiologically active substance at a high density, but if it is too large, the liquid permeability of the column packed with the molded article of the present invention will deteriorate. Surface area of 0.01 or more 50m ² / g
Hereafter, it is more preferably 0.05 or more and 10 m ² / g or less.

The swelling properties of the polymer and the molded product of the present invention are important factors that affect the ability to immobilize physiologically active substances. If the swelling property is too large, the immobilization density will increase, but the mechanical properties will deteriorate, and if it is too small, the immobilization density will decrease, so the water content will be 0.6 to 3. 0 g, especially 1.0 to 2.0 g is preferable.

R ₁ and R ₂ in the functional groups (I) and (II) in the present invention are not particularly limited as long as they are hydrogen atoms or lower alkyl groups, but the functional groups are functional groups having a small number of carbon atoms. In particular, a hydrogen atom has less steric hindrance, which is advantageous in that it is easy to prepare and the immobilization density can be increased.

In the general formula (I) for the functional group in the present invention, R ₃ is NH ₂
When it is, there is an advantage that it can be easily manufactured at low cost, and since there are few extra amino groups remaining after immobilization, it has good biocompatibility when the immobilized product is used for a living body, and It is preferable because it has little ionic effect when used for applications. In addition, R ₃ in the functional group general formula (I) is N
In the case of H—R ₄ —NH ₂ , if the carbon number of R ₄ is too small, the ratio of amino groups to alkyl groups becomes high, and the ionic properties of the carrier become too strong, or coloring easily occurs. Not preferable. On the other hand, if the carbon number of R ₄ is too large, there is a drawback that the carrier becomes too hydrophobic and the immobilization density becomes small. Therefore, the carbon number of R ₄ is preferably 4 or more and 16 or less. Specific examples of R ₄ include a hexamethylene group, a dodecamethylene group, and an undecamethylene group, which are particularly inexpensive and easily available. In addition, a branched one such as a 2-methylhexamethylene group can also be used.

The aprotic polar organic solvent referred to in the present invention has a dielectric constant of 1
Means 5 or more aprotic polar organic solvents. Specific examples thereof include dimethyl sulfoxide, N, N-dimethylformamide, N, N-dimethylacetamide, hexamethylphosphoramide, tetramethylurea and the like. Further, it can be used in combination with a solvent other than the aprotic polar organic solvent. The larger the amount of the aprotic polar organic solvent used, the higher the swelling property of the molded product of the present invention is, but usually 1 g or more, and more preferably 100 g or more per 1 g of the raw material polymer and the molded product.

The molded product of the present invention is produced by a vinyl polymer having an aromatic nucleus having a functional group represented by the general formula (II) described in claim 1 and a molded product thereof, that is, an α-haloacetamidomethylated aromatic compound. The vinyl polymer and its molded product (Japanese Patent Laid-Open No. 57-12008) are immersed in a solution of an aprotic polar solvent containing ammonia or a diamine represented by the general formula (III), or this raw material molded product is previously treated with an aprotic compound. It can be achieved by swelling with a polar organic solvent and then contacting with ammonia or the diamine represented by the general formula (III).

The amount of ammonia and the diamine represented by the general formula (III) used in the present invention, when the amount is too small, the crosslinking reaction occurs preferentially, and the swelling property of the obtained carrier becomes too low,
It is preferable to use an equimolar amount or more, especially 5 times or more moles, with respect to the α-haloacetamidomethyl group of the α-haloacetamidomethylated aromatic vinyl polymer.

The higher the concentration of ammonia in the solution, the more swellable the carrier can be made. If the concentration of ammonia in the solution is too low, only a carrier having a small swelling property can be produced, so 0.5% or more, particularly 2.0% or more is preferable. On the other hand, if the concentration of the diamine in the solution is too high, the swelling power of the solution with respect to the vinyl polymer having a functional group-introduced aromatic nucleus represented by the general formula (II) and the molded article thereof will decrease. 0.5% or more and 50% or less, especially 2.0% or more and 20% or less are preferable.

The reaction temperature is usually 0 to 100 ° C., especially 0 to
30 ° C. is preferably used. When the reaction temperature is low, a carrier having a large swelling property can be obtained.

The polymer of the present invention and its molded product are used as a carrier for immobilizing a physiologically active substance.

(Effect of the invention) Since the polymer of the present invention and the molded product thereof have a functional group that is immobilized on the tip of a long chain, a physiologically active substance can be immobilized at a high density. In addition, regarding swellability, pH
It is easy to use and durable because it has little dependence.

The functional group (I) of the polymer of the present invention has an advantage that after the immobilization treatment, the remaining amino groups can be acylated and eliminated, and as a result, the physiologically active substance-immobilized product can be converted to neutral.

Further, the functional group (I) of the polymer of the present invention has an advantage that it can be easily converted into a carrier having a carboxyl group by ring-opening acylation with a dicarboxylic acid anhydride such as succinic anhydride.

The reaction for obtaining the polymer of the present invention is conceptually represented by the general formula (II)
A vinyl polymer having an aromatic nucleus having a functional group introduced therein or a molded product thereof is treated with ammonia or the general formula (II
It should be allowed to react with the diamine represented by I), but since these amino compounds are polyvalent reactive, a crosslinking reaction occurs and only a low swelling polymer or molded product is obtained. In order to prevent the crosslinking reaction, increasing the concentration of the amino compound is one of the improvement measures, but it is not preferable because the solubility is limited or the cost is high.
However, the use of the method of the present invention is economical because a carrier having a high swelling property can be prepared even when the concentration of the amino compound is low.

(Example) An example is shown below.

Example 1 50 parts of polypropylene (Mitsui "Noblen" J3HG) as an island component and polystyrene ("Stylon" 666)
46 parts, polypropylene (Sumitomo "Noblen" WF-7
27-F) Sea-island type composite fiber containing 4 parts of the mixture as a sea component (16 islands, single yarn fineness 2.6 denier, tensile strength 2.9).
g / d, elongation 50%, filament number 42) 50g, N
-Methylol-α-chloroacetamide 50 g, nitrobenzene 400 g, and paraformaldehyde 0.8
It was immersed in a mixed solution of 5 g and reacted at 20 ° C. for 1 hour. The fiber was taken out of the reaction solution, poured into ice water 5 at 0 ° C. to stop the reaction, washed with water, and then nitrobenzene attached to the fiber was extracted and removed with methanol. This fiber (fiber A) is vacuum dried at 50 ° C., and 71 g of chloracetamide methylated fiber which is a raw material molded product
(Fiber A) was obtained.

20 g of fiber A was added to 300 ml of a 1.8 M ammonia-dimethyl sulfoxide solution obtained by blowing ammonia gas into dimethyl sulfoxide while cooling,
The reaction was carried out at 20 ° C for 72 hours. The fiber was taken out and washed thoroughly with dilute hydrochloric acid and water to obtain the carrier of the present invention (fiber B). The exchange capacity of the fiber B is 1.15 meq / g, and the water content is 1.76 g at pH 4 and 1.74 at pH 7.4 per 1 g of fiber.
It was 45. The amount of primary amino groups in the fiber was 0.96 mmol / g as a result of amino acid analysis.

(Preparation of immobilized lipopolysaccharide) After swelling 32 g of the above fiber B in 300 ml of water overnight, 0.4 mg / ml of Escherichia coli 055: B5 lipopolysaccharide (trichloroacetic acid extraction method, manufactured by Difco Laboratories) Mix with 500 ml of aqueous solution, then 1
The pH was adjusted to 4.5 with N-hydrochloric acid and 1N-sodium hydroxide.
While maintaining at -6.0, 5.0 g of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide was added little by little and dissolved. After shaking this mixture at room temperature for 2 days, the fiber was taken out and the operation of immersing the fiber in boiling water for 30 minutes was repeated three times. Next, add 0.0% of this fiber.
The lipopolysaccharide-immobilized fiber (immobilized lipopolysaccharide) was obtained by washing with 7 mol of phosphate buffer (pH 7.4) until the pH of the washing solution reached 7.4. The concentration of lipopolysaccharide in the above-mentioned immobilized mother liquor and washing solution was determined by the phenol / sulfuric acid method (sample solution 1 ml + 5% phenol water ml + concentrated sulfuric acid 5 ml; 485 mμ).
I asked for. The amount of immobilized lipopolysaccharide in the immobilized lipopolysaccharide was 5.0 mg / g.

Example 2 100 ml of 17N-ammonia water cooled to 0 ° C. and 0 to 900 ml of N, N-dimethylformamide cooled to 0 ° C. were mixed to prepare an ammonia / DMF solution. This solution was cooled to 10 ° C., 3 g of fiber A was added, and 10-20
The reaction was carried out at 24 ° C. for 24 hours (10 days when Comparative Fiber 1 was prepared). The fibers were taken out and washed thoroughly with dilute hydrochloric acid and water to obtain the carriers of the present invention (fibers C to H) and comparative fiber 1.

From the table, it can be seen that the reaction is difficult to proceed without the aprotic polar organic solvent.

Example 3 Freshly distilled hexamethylenediamine was dissolved in dimethylsulfoxide to prepare hexamethylenediamine / DMSO solutions having concentrations of 5, 10 and 20%. 15 g of fiber 9.3 g in these solutions (140-987 g)
Then, the mixture was reacted at 15 to 25 ° C for 5 days.
The fibers were taken out and thoroughly washed with dilute hydrochloric acid and water to obtain the carriers of the present invention (fibers I to M).

For comparison, Comparative Fiber 2 was obtained by the same treatment using 10% hexamethylenediamine / water solution instead of the hexamethylenediamine / DMSO solution. The measurement results of the exchange capacity and water content of these fibers are shown in the table.

From the table, it can be seen that the functional groups are often introduced in the examples of the present invention, while the functional groups are hardly introduced in the comparative examples. Further, it can be seen that in the examples of the present invention, the pH change of the water content is small.

Example 4 20 g of dodecamethylenediamine and 240 g of a mixed solvent of dimethyl sulfoxide / ethanol (weight mixing ratio 1: 1)
And was cooled to 5 ° C., and 6.8 g of the fiber A was immersed therein. After allowing the mixture to stand at room temperature of 15 to 25 ° C. for 4 days, the fibers were taken out, washed thoroughly with dilute hydrochloric acid and water,
The carrier of the present invention (fiber N) was obtained. The exchange capacity was 1.62 meq / g, and the water content was 1.06 at pH 4 and 0.72 at pH 7.4. From the exchange capacity, the amount of dodecamethylenediamine introduced is estimated to be 0.81 mmol / g.

As a comparison, when ethanol or n-butanol was used in place of the mixed solvent of dimethyl sulfoxide / ethanol and the same reaction was performed, the exchange capacity was 0.24 meq / g and the water content was 0. 59
(PH 4) and n-butanol solvent had an exchange capacity of 0.
Only fibers having a low functional group introduction ratio of 03 meq / g and a water content of 0.43 (pH 4) were obtained.

Example 5 10 g of undecamethylenediamine was dissolved in 190 g of dimethylsulfoxide, and at 24 ° C., fibers A6.
8 g was immersed. After allowing the mixture to stand at room temperature of 10 to 20 ° C. for 9 days, the fibers were taken out and washed thoroughly with diluted hydrochloric acid and water to obtain the carrier of the present invention (fiber M). Exchange capacity is 1.
93 meq / g, water content 1.0 at pH 4, pH 7.
It was 0.86 at 4. From the exchange capacity, the amount of undecamethylenediamine introduced was estimated to be 0.965 mmol / g.

For comparison, 40 g of the fiber A was dipped in ethylenediamine at 15 ° C. and reacted at a temperature of 15 to 20 ° C. for 24 hours to obtain an ethylenediaminoacetamide methylated fiber (comparative fiber 5). The exchange capacity of this fiber is as high as 3.60 meq / g, but the water content is 6.2 at pH 4,
It can be seen that the value is 7.4 at 1.0, which is a very large change with respect to the change in pH.

Claims (2)

[Claims] 1. A carrier for immobilizing a physiologically active substance, which comprises a vinyl polymer having an aromatic nucleus into which a functional group represented by the following general formula (I) is introduced as a side chain. (In the above formula, R ₁ and R ₂ represent a hydrogen atom or a lower alkyl group, R ₃ represents an NH ₂ or NH—R ₄ —NH ₂ group, R 3
₄ represents an alkylene group having 4 to 16 carbon atoms) 2. A vinyl polymer having an aromatic nucleus in which a functional group represented by the following general formula (II) is introduced as a side chain and a molded product thereof are ammonia, (In the above formula, R ₁ and R ₂ are hydrogen atoms or lower alkyl groups, and X is a halogen atom) or NH ₂ —R ₄ —NH ₂ when reacted with a diamine represented by the following general formula (III) Group (III) (R ₄ represents an alkylene group having 4 to 16 carbon atoms) An aprotic polar organic solvent is used to produce a carrier for immobilizing a physiologically active substance.