JPS5915401A - Immobilized physiologically active substance, its production, and carrier used in its production - Google Patents

Abstract

PURPOSE:To obtain a stable immobilized physiologically active substance, by reacting a reaction product between an epoxy group-containing insoluble polymer and a hydrazine with a carbonyl group-containing physiologically active substance in the presence of sodium cyanoborohydride. CONSTITUTION:An insoluble polymer having epoxy-terminated side chains or an insoluble polymer activated with epoxy groups is reacted with a hydrazine and/or a dihydrazide compound to obtain a compound of formula I (wherein formula II is a residue of the insoluble polymer or the epoxy group-activated insoluble polymer, R<1>, R<2> and R<3> are each H or a 1-5C alkyl, formula III is a residue of the dihydrazide compound, and X is 0 or 1). Then, this compound is reacted with a carbonyl group-containing physiologically active substance (e.g., enzyme or protein) in the presence of sodium cyanoborohydride to obtain an immobilized physiologically active substance excellent in stability, etc., in good yields.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an immobilized physiologically active substance, a method for producing the same, and a carrier used in the production method, and more particularly, the present invention relates to an immobilized physiologically active substance that has a high ligand concentration and is stable. The present invention relates to an active substance, a method for producing the same, and a carrier used in the method.

There are various immobilization methods for immobilizing physiologically active substances for industrial use, but considering the stability of the immobilized substance, non-specific reactivity between the immobilized substance and the reactant, etc. A method in which a ligand (physiologically active substance) is immobilized on an insoluble carrier by a covalent bond is desirable.

Such methods include the cyanogen bromide method and the boxy activation method [Organic Synthetic Chemistry, Vol. 38, pp. 128-138 (
[1980]], but in the former case, non-specific adsorption often occurs, and once immobilized, Gant binding is unstable, so the ligand.

However, it has the disadvantage that it often leaks out, and the amount of ligand bound is also not satisfactory. On the other hand, specific adsorbents (immobilized physiologically active substances) prepared by the latter method have the advantage of having excellent stability and less nonspecific adsorption, but they do not allow binding of ligands. However, since the process must be carried out under strong alkaline conditions at high temperatures, it is unstable and has been converted into an amino rust conductor.
IJ Gant has a disadvantage in that it requires a long time to react, especially with a compound having a carbonyl group with low reactivity such as sugar.

! ! There is also a method that utilizes a coupling reaction between an amine group and a carboxyl group using carbodiimide.
In this method, there is a possibility that side reactions such as the formation of isourea derivatives and Chapman rearrangement may occur, and even at the optimum acidity of -14.5, there is a possibility that the ligand is A side reaction of polymerization is likely to occur, and the yield of the immobilized product is not satisfactory, so a support having a more reactive functional group is desired.

As a result of extensive research to solve the above-mentioned problems, the present inventors have discovered that hydrazine obtained by reacting an insoluble polymer having an epoxy group or an epoxy-activated insoluble polymer with hydrazine and/or a dihydrazide compound. By using derivatives and/or dihydrazide rib conductors as immobilization carriers, physiologically active substances with low reactivity in sugar syrup can also be immobilized under mild external conditions, and the resulting stone immobilization The inventors discovered that the substance has a high ligand concentration and is stable, leading to the completion of the present invention.

That is, the immobilized physiologically active substance and insoluble polymer (
The immobilization carrier) is expressed by the following formula (1)! 1Oat combined insoluble polymer, residue or component i1? =*Si? 11, R2 and t are the aging insoluble polymer residues;
Sx is a hydrogen atom or an alkyl group having 1 to 5 carbon atoms, and Sx is a hydrogen atom, a η tri-active substance residue bonded via its carbonyl group or carboxyl group, or the following formula: X, -NIEI-(CH , ), - (wherein, X and -Nu are physiologically active substance residues bonded via their amino groups, and Sy is an integer from 2 to 10; each represents a residue represented by: - N) INH-C-y-c-NI
nax- represents a dihydrazide compound residue bonded via the two hydrazino groups; X represents 0 or l;

] This is shown in .

The insoluble polymer (immobilization carrier) of the present invention represented by the above formula (1) in which X is a hydrogen atom can be obtained as follows.

That is, by reacting an insoluble polymer having a side chain with an epoxy group at the end or an epoxy-activated insoluble polymer with a hydrazine and/or a dihydrazide compound, the following formula (n)! The dihydrazide compound bonded through the group has the same meaning as above. ) An immobilization carrier can be obtained.

Insoluble polymers and epoxy-activated insoluble polymers that have side chains with epoxy groups at their ends include insoluble polymers that have hydroxyl groups such as agarose, cellulose, chitin, mannan, polyacrylamide resins, and polyvinyl alcohol resins. , known methods [Organic Synthetic Chemistry, Vol. 38,
128-138 (19'io)), shrimp halohydrin such as epichlorohydrin, shrimp bromohydrin; or ethylene glycol diglycidyl ether, 1
．． 3-propanediol diglycidyl ether, 1,4
-Butanediol diglycidyl ether, 1,5-bentanediol diglycidyl ether, 1,6-hexanediol diglycidyl ether, and other bisoxirane compounds.

The insoluble polymer used as a carrier is used in a mild form depending on the purpose. For example, when used as an affinity adsorbent or immobilized enzyme, a porous granular material is preferable in order to increase the adsorption capacity and contact area, and when used for processing dilute physiologically active substance solutions. for,
Porous membrane-like hydrazines include hydrazine hydrate, hydrazine hydrate salt, hydrazine sulfate, etc.
h form is used.

Examples of dihydrazide compounds include oxalic acid dihydrazide,
Straight-chain saturated compounds with 2 to 12 carbon atoms such as malonic acid dihydrazide, succinic acid dihydrazide 9 sites, glutaric acid dihydrazide, adipic acid dihydrazide, pimelic acid dihydrazide, speric acid dihydrazide, azelaic acid dihydrazide, and nanano(sinic acid dihydrazide) Examples include unsaturated dicarboxylic acid dihydrazides such as dicarboxylic acid dihydrazide, maleic acid dihydrazide, heptamalic acid dihydrazide, and aromatic dicarboxylic acid dihydrazides such as phthalic acid dihydrazide and terephthalic acid dihydrazide.

These hydrazines and dihydrazide compounds may be used alone or as a mixture of two or more.

The m of the human 2 diZ and dihydrazide compounds used is 0.001 to 0.0 for the epoxy group-containing insoluble polymer or epoxy-activated insoluble polymer (dry type ■). 5F, for dihydrazide compounds, it varies depending on the species M, but generally 0.005
~1f. Generally, A water is used as the solvent, and the amount of water is the same as that of the insoluble polymer 1f (dry type i).
If necessary, sodium carbonate, hydrochloric acid, etc. may be appropriately added to the solvent. Under the above conditions, at 0 to 100°C, preferably 4 to 50°C,
By reacting for 0.5 to 72 hours, preferably 1 to 48 hours, the immobilization carrier represented by the formula (If) can be obtained.

The method for immobilizing the physiologically active substance on the immobilization body thus obtained is selected as appropriate depending on the IF of the physiologically active substance.

Physiologically active substances include lactose, dextran sulfate, heparin,
N-acetylgelcosaone, N-acetylmuramic acid,
Sugars such as tri-N-acetylchitotriose, chitin, glucose, maltose; mucin, acidic monoglycoprotein,
Glycoproteins such as prothrombin, no-butoglobin, and gonadotropins; 7-glycolipids such as kerasin, nervone, oxynervone, and ganglioside; 8-glucosides; created glycosides such as galactoside, mannoside, and fructocyto; as well as carbonyl groups and aldehydes. group, carboxyl group, alkoxycarbonyl group, amine carbonyl group,
In the case of a compound having a carbonyl group in a broad sense such as a haloformyl group, it can be immobilized as follows.

That is, the desired immobilized physiologically active substance can be obtained by reacting the physiologically active substance with the immobilization carrier represented by the formula (It) in the presence of sodium cyanoborohydride.

The concentration of sodium cyanoborohydride used may be 10 or more per the immobilization carrier xr (dry weight), but it is particularly preferably 100 to 500 per. The amount of physiologically active substances varies depending on the f1 class, but
Generally, the immobilization carrier 1F (dry 'M'R) is 50~! 10 (If7.

As a solvent, dipotassium hydrogen phosphate, aqueous solution, phosphate buffer, acetate buffer, borate buffer, carbonate buffer, bicarbonate buffer, etc. are generally used.
It is preferable that the amount is the same as that of the immobilization carrier IF.
(Dry weight) υ is preferably 3 to 20 ml. Under the above conditions, 0-100℃, preferably 4-8℃
By reacting at 0° C. for 1 to 72 hours, preferably 2 to 48 hours, the desired immobilized physiologically active substance can be obtained. The immobilized physiologically active substance thus obtained may be treated with acetic anhydride to acetylate unreacted sites, if necessary.

Next, the physiologically active substance is concanavalin A (hereinafter, [c
It's called onA,j. ), bovine Jfi + serum albumin (hereinafter referred to as [B8AJ), γ ~ globulin (antibody),
Enzymes, proteins such as phytohemagglutinin (PHA), fibronectin, and protein A; peptides such as kinin, corticotropin-releasing factor (C-F), calcitonin, and glucagon; nuclear proteins, chromoproteins, glycoproteins, and phosphorus Protein complex proteins: various amino acids; histamine, dopamine, glucose: ftM, tributamine, etc. When the compound has an amino group such as VCIri, it can be immobilized as follows.

That is, in the presence of sodium cyanoborohydride, the immobilization carrier represented by the formula (II) is converted into the immobilization carrier represented by the following formula (1).
)1 0HC-(CH,),! -CHO(1) (in the formula,
7 represents an integer from 2 to 1o. ) to form an aldehyde-activated insoluble polymer represented by the following formula 1.

The nail of sodium cyanoborohydride to be used differs depending on the water content XK of the carrier, but if the IP (dry weight) of the immobilization carrier is 50 cm or more, the desired purpose can be achieved, but especially 80 to 1,000 Preferably it is a cape. The linear dialdehyde compound generally has 2 to 1 carbon atoms.
For example, glyoxal, propane dial, buta, dial, glutaraldehyde (pentatosite), decanedial, etc. can be used alone or as a mixture of two or more. Water is used as the solvent, and the amount thereof is based on the immobilization carrier 12 (dry weight).
It is preferable that it is 5-20 tnl. Under the above conditions,
At 0-80°C, preferably 20-40°C, 0.5
The aldehyde-activated insoluble polymer can be obtained by reacting for 10 hours, preferably 1 to 4 hours.

Next, the desired immobilized physiologically active substance can be obtained by reacting this aldehyde-activated insoluble polymer with the physiologically active substance in the presence of sodium cyanoborohydride.

Examples of solvents used at this time include phosphate buffer, acetate buffer, borate buffer, carbonate buffer, etc.
is preferably 5 to 8, and the amount is 5 to 4 per aldehyde-activated insoluble polymer xr' (dry weight).
(preferably 0 m1).

The amount of sodium anopouride hydride used is 100 kg xr (dry weight) of the aldehyde-activated insoluble polymer.
It may be at least 200 km, but preferably 200 to 400 km. The amount of physiologically active substances varies depending on the type.1. Generally, the amount is 100 to 1,000 kg per aldehyde activated insoluble polymer xr (dry heavy duty). Under the above conditions, 0-80℃, preferably 4-40℃
By reacting for 5 to 100 hours, preferably 12 to 72 hours at 100°C, a mixture is prepared to obtain the desired immobilized physiologically active substance.

On the other hand, the physiologically active substance is a compound having a carboxyl group, such as the aforementioned proteins, peptides, complex proteins, amino acids, heparin, N-acetylmusionic acid 1 and fatty acids such as valmitic acid and oleic acid; penicillin, prostaglandin; If so, it can be immobilized as follows.

That is, by reacting the physiologically active substance with the immobilization carrier represented by the formula (II) in the presence of a carbodiimide compound, the desired immobilization carrier and substance can be obtained. . The carbodiimide compound used is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (hereinafter, jli3DcJ
That's what it means. ), 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide meso-P-toluenesulfonate, dicyclohexylcarbodiimide, etc. are used alone or as a combination of two or more types, and the amount thereof is The immobilization carrier 11 (dry type jfi') or 10 to 200
I like that it's a cape. The nature of the physiologically active substance varies depending on its moderation, but in general, the above-mentioned immobilization carrier xp
(Dry weight) In addition, it is 30 to 100 capes. As a solvent, water is generally used, and its weight, the above-mentioned immobilization carrier 11 (dry weight), υ, 2-10-ash,
Sodium chloride, potassium chloride, etc. may be appropriately added to the solvent as necessary. Under the above conditions, 0 to 80
By reacting at a temperature of preferably 4 to 40°C for 10 to 100 hours, preferably 24 to 72 hours, the objective immobilized physiologically active substance can be isolated.

According to the production method of the present invention, it is possible to achieve the desired immobilization physiology in a shorter reaction time than in the conventional method, under mild conditions-F and without performing any finishing or adjustment procedures during the reaction. When the active substance is obtained, it is made. Furthermore, the obtained immobilized physiologically active substance has a higher concentration of ligand than conventional substances.

Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example 1 (1) Preparation of hydrazine agarose Sepharose 4'B manufactured by Pharmacia was used as agarose.

After thoroughly washing with water on a glass filter, put the aspirated filtered agarose 20F into a flask, add 30 ml of water,
13 mt of 2 M Na0)1 aqueous solution and 3 ml of epichlorohydrin were added sequentially. The suspension was shaken in an incubator at 40° C. for 2 hours, and then thoroughly washed with water on a Glasnoyl plate to obtain epoxy-activated agarose. When the epoxy groups introduced into this epoxy-activated agarose were measured by neutralization titration using Nat St Os, it was found that approximately 500 μmole of epoxy groups had been introduced per 1 f of dry weight.

80c in 1 volume (20r) of this epoxy activated agarose
1.5 volumes (30 m/liter) of an aqueous solution of X hydrazine hydrate were added, and the mixture was shaken for 1.5 hours in an incubator at 40°C. After the reaction, it was thoroughly washed with water on a glass filter to obtain hydrazine agarose.

(2) Preparation of immobilized lactose To hydrazine agarose 2P [dry weight (wet weight) 1zoP] obtained in (1): I, lactose 520 Misaki and sodium cyanoborohydride 256 ■ were added at 0.2 MK, 1 (P
O.

A solution dissolved in 15 mg of an aqueous solution was added, and the mixture was shaken at room temperature for 3 days. After the reaction, pour 10 rn of water on a glass filter!
, and o, 2 M C)I3'CC00N aqueous solution 10
Washed sequentially with m1. Next, 5 ml of acetic anhydride was added, and after reacting at 0°C for 30 minutes, 5 ml of acetic anhydride was added.
1! was added and treated at room temperature for 30 minutes to acetylate unreacted (lactose is not immobilized) hydrazine residues.
7. After the reaction, it was washed once with water, 0.1 M Na0)I aqueous solution, water, and phosphate buffered saline (PBE) to obtain lactose-bound agarose.

Example 2 (1) Preparation of adipic acid dihydrazide agarose Sepharose CL manufactured by Pharmacia was used as agarose.
6B was used. Adipic acid dihydrazide 1152 was added to +1.1 MNa I C to epoxy-activated agarose lt obtained by treatment in the same manner as in Example 1 (1).
A solution of Os dissolved in aqueous solution and adjusted to rJ+ of 9 with hydrochloric acid was added, and the mixture was shaken at 40°C overnight. After the reaction, the glass filter was sufficiently washed with a 0.2 M NACt aqueous solution to obtain adipic acid dihydrazide agarose.

(2) Preparation of immobilized dextran sulfate Adipic acid dihydrazide agarose xoo obtained in (1)
r (dry weight (wet volume) t)], dextran sulfate (
Pharmacia) 26v and sodium cyanoborohydride 12.8 F at 0.2 M IC7) LPO,
A solution dissolved in 750 mg of an aqueous solution was added, and the mixture was shaken at room temperature for 3 days. After the reaction, water, 0.2
It was washed sequentially with MCI and C00NR aqueous solutions.

Next, add 250 ml of acetic anhydride and shake for 30 minutes at 0°C, then add 250 ml of acetic anhydride and shake for 30 minutes at room temperature to remove unreacted (dextran sulfate is not immobilized) ) Adipic acid dihydrazide residues were acetylated. After the reaction, water, 0.1M NaOH aqueous solution, water,
Dextran sulfate-bound agarose was obtained by sequential washing with PI35. The dextran sulfate content before reaction is 0.2
Dextran sulfate in MK, 1IPO, aqueous solution and supernatant after reaction was measured according to the phenol sulfuric acid method, and the resulting dextran sulfate-bonded agarose had
It was found that 25% of the dextran sulfate used was bound.

Example 3 (1) Preparation of aldehyde-activated agarose Example 2 (1)
), 25% gluta-4-aldehyde aqueous solution W was added to adipic acid dihydrazide agarose obtained by the same treatment as
After adding 1 ml of L1 ml and 82 caps of sodium cyanoborohydride and shaking in an incubator at 40° C. for 2 hours, the mixture was thoroughly washed with water on a glass filter to obtain aldehyde-activated agarose.

(2) Preparation of immobilized Can A [aldehyde-activated agarose obtained in (1)]7, C
OnA100+nIi and methyl-α-D-mannoside 110q were dissolved in 0.2 M phosphate buffer (pH 7).
.0) After adding 24 g of sodium cyanoborohydride, the mixture was shaken in an incubator at 4° C. for 2 days. After the reaction, the supernatant was removed and washed thoroughly with 0.2M phosphate buffer to obtain ConA-bound agarose.

(3) Measurement of Ligand Concentration: Calculate Con AQL1 bound to agarose by subtracting the amount of unbound Con A in the two supernatants and wash solution from the measured absorbance in 280 dishes. did. As a control, an enzyme bound to 117 agarose was used in the CNBr method [Author: Kazuo Chibata, "Affinity Chromatography", p. 40].

Tokyo, Kodansha (197 (1st year)) (Contrast 1), Kayo i
BSA bound to each weight polysaccharide by acid oxidation method (Imm
unology, Volume 20, 1061-1065
(1971)] (Control 2) and Bloe temltry bound to hydrazide derivatives, Vol. 8, pp. 4074-4082 (1969)] (Control 3).
) was used. The results are shown in Table 1.

From Table 1, it can be concluded that the immobilized physiologically active substance of the present invention has a higher ligand concentration than the conventional substance.

Example 4 Preparation of immobilized 13SA 80% aqueous hydrazine hydrate solution 15 was added to epoxy-activated agarose 102 obtained by the same treatment as in Example 1 (1), and the mixture was incubated in an incubator at 40°C. 1
Shake for half an hour. After the reaction, the mixture was thoroughly washed with water through a glass filter, and it was confirmed that the reaction mixture was colored by the phenolsuclein and had lost its strength.

Next, 0.5M N in which B5A30ff9 was dissolved
After adding 2- of the net aqueous solution, 33 g of EDC was added.

After adjusting −1 to 5 and shaking overnight at room temperature, F!
DC33 cape was added and shaken overnight. After the reaction, the glass filter was washed with a 0.5 M NaCl aqueous solution at 8 mP and water at 00 mA to obtain BSA-bonded agarose.

The amount of unbound residual BS in the washing solution was determined by
By calculating from the measured value of absorption at ffl, it was found that the obtained BsA-bound agarose contained 947 kg of l3SA per wet weight lf.

Example 5 (1) Preparation of adipic acid dihydrazide agarose q9-Epoxy activated agarose 201 (wet weight) VC obtained by the same treatment as in Example 1 (1), adipic acid dihydrazide 2.32 VC. A solution containing 1 M NA, CO, and water and adjusted from -1 to 9 with hydrochloric acid was added, and the mixture was shaken overnight at room temperature. After the reaction, wash 711 with trinitrobenzene sulfonic acid (TNBS) on a glass filter.
Approximately 500ml of 0.2M NaCl aqueous solution is reached when no reaction occurs! Adipic acid dihydrazide agarose was obtained by washing with water.

(2) Preparation of immobilized BSA (adipate dihydrazide agarose IV obtained by
BAA 5 (IP was dissolved (1.5 M Nnet aqueous solution 2 rne was added, then 1DD033 was added.-
After adjusting 1 to 5 and shaking at room temperature overnight, further EDC
33η was added and shaken overnight. After the reaction, 8 ml of 0.5 M NaCL aqueous solution was added on a glass filter.

-)3SA-bound agarose was obtained by sequential washing with -.

The amount of unbound residual Be in the washing solution was measured at 280 nm.
The obtained BS binding agarose has a wet weight of 12 cm and 38 cm.
．． It was found that 6■ BSA was bound.

Claims (1)

[Claims] 1. The following formula: 1Bl represents the combined insoluble polymer residue or epoxy-activated insoluble polymer residue, R2 and R3 each represent a hydrogen atom or an alkyl group having 1 to 5 carbon atoms. OX is a hydrogen atom, a physiologically active substance residue bonded via its carbonyl group or carboxyl group, or the following formula t-X, -(cHl), - (wherein, X and -NH are the amine group) y represents an integer from 2 to 10, respectively.
Ito is 8 A dihydrazide compound residue bonded via the two hydrazino groups is O or 1; respectively. ] An immobilized physiologically active substance and an insoluble polymer represented by 2. Reacting an insoluble polymer having a side chain with an epoxy group at the end or an epoxy-activated insoluble polymer with a hydrazine and/or a dihydrazide compound,
゛Following formula; The combined insoluble polymer residues or epoxy-activated insoluble polymer residues are bonded to S R', 11 and R3 through hydrogen atoms or alkyl groups having 1 to 5 carbon atoms, respectively. outside of Dividra, do compound residue; Su is 0 or 1;
Represent each. ) An immobilized physiologically active substance characterized by obtaining a hydrazine derivative and/or dihydrazide derivative represented by ゛, and then reacting it with a physiologically active substance having a carbonyl group in the presence of cyanoborohydride.) Method of manufacturing a substance. 3. An insoluble polymer having a side chain with an epoxy group at the end or an epoxy-activated insoluble polymer is reacted with a hydrazine and/or a dihydrazide compound to form an insoluble polymer residue having the following formula: kl is a group or an epoxy-activated insoluble polymer residue; R2 and R'' are each a dihydrazide compound residue bonded via a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; gx is 0 or 1; In the presence of hydrogenated cyanopurine), OHC-(CH,), -8-CHO (in the formula ,
y represents the number of grains from 2 to 1O. ) is reacted with a linear dialdehyde compound represented by the following formula! (In the formula, =NIIN)1-C-1'-C-N)INII
- is the two hi 8 Blue 3 5 1 X and y have the same meanings as above. ) A method for producing an immobilized physiologically active substance, which comprises obtaining an aldehyde-activated insoluble polymer represented by the formula and then reacting it with a physiologically active substance having an amino group in the presence of sodium cyanoborohydride. . An insoluble polymer having a side chain having an epoxy group at the end or an epoxy-activated insoluble polymer is reacted with hydrazine, etc. and/or a dihydrazide compound, and the insoluble polymer residue is combined with the following formula: or an epoxy-activated insoluble polymer residue; R1, -11, and R3 are each a dihydrazide compound residue bonded via a hydrogen atom or an alkyl group having 1 to 5 carbon atoms; gx is 0 or 1t; −
t respectively. ) A method for producing an immobilized physiologically active substance, which comprises obtaining a hydrazine derivative and/or dihydrazide derivative °, and then reacting this with a physiologically active substance having a carboxyl group in the presence of a carbodiimide compound.