JPH09210981A - Filler for separation, refining, and concentration of gelatine-affinity substance - Google Patents

Abstract

PROBLEM TO BE SOLVED: To make separation, refining, and concentration of a gelatine-affinity substance by using a filler of such a structure that a hyaluronic acid is fixed to a insoluble carrier for fixation, or a column containing such filler. SOLUTION: A carrier for fixation should be of insoluble type in an aqueous organic solvent. A hyaluronic acid is fixed to this carrier by the covalent bonding method. an activation reagent may be used for fixation reactions of the hyaluronic acid to the carrier. The favorable shape of the carrier is spherical, and it should preferably by subjected to amino activation treatment. A gelatine-affinity substance can be separated, refined, and concentrated by performing a column treatment or batch treatment using this filler.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for separating, purifying and concentrating a filler having immobilized hyaluronic acid and a gelatin affinity substance by a chromatographic technique.

[0002]

BACKGROUND OF THE INVENTION Hyaluronic acid is widely distributed in animal tissues, especially mesenchymal tissues, and it is vitreous, aqueous, umbilical cord, nuchal ligament, synovial fluid, costal fluid, skin, aorta, tendon, valvular heart, brain, cartilage,
It is a mucopolysaccharide obtained from serum, poultry, silkworm gastrointestinal membrane, streptococcal capsule, etc., β-D-N-acetylglucosamine and β-D
-It is known as a linear polymeric polysaccharide in which glucuronic acid is alternately bonded.

Columns containing a packing material in which gelatin is already immobilized include fibronectin, chondronectin,
Glycoproteins such as vitronectin, laminin and polynectin, proteoglycans such as proteoheparin, proteoheparan sulfate, proteochondroitin sulfate, growth factors such as acidic fibroblast growth factor and basic fibroblast growth factor, chondroitinase, etc. Enzymes, C
It is known to be effective in separating, purifying and concentrating complement factors such as 1q and the like, and gelatin affinity substances such as platelets and smooth muscle cells.

[0004]

However, gelatin is a denatured collagen and is a protein that has lost the triple helix structure, and is therefore easily decomposed by proteases. Furthermore, in the structure, NH ₂ of the amide group of glutamine and asparagine does not exist. In some cases, or because the covalent bond may be broken, there is a problem that it is difficult to obtain a product having a uniform structure with good reproducibility because the structure is not definite.

From the above, it has sufficient usability and versatility as a packing material for chromatographs, and further has a function of selectively separating, purifying and concentrating or quantifying a gelatin affinity substance, It is effective in that it is stable to proteases and has better structural homogeneity, and it is more advantageous than gelatin. A new and highly effective chromatographic method using a column containing a is desired.

Therefore, a filler having such a functional characteristic is prepared, and a gelatin affinity substance is obtained by batch mode chromatography using the filler itself or column chromatography using a column containing the filler. Has been intensively studied for the purpose of providing a method for separating, purifying, and concentrating.

[0007]

Means for Solving the Problems As a result of intensive studies to achieve this object, the present inventors have found that a filler containing hyaluronic acid immobilized on an insoluble immobilizing carrier, or containing the filler. The present invention has been completed by finding a method for separating, purifying and concentrating a gelatin affinity substance using a column.

That is, the present invention uses a packing material for separation, purification and concentration characterized by immobilizing hyaluronic acid on a carrier for immobilization, a column containing these packing materials and these packing materials. The present invention relates to a method for separating, purifying and concentrating a gelatin affinity substance.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION Hyaluronic acid in the present invention refers to animals such as humans, cows, monkeys, rabbits, rats, mice, pigs, dogs, sheep, guinea pigs, boars, cats, chickens, chicks, pigeons, and whales. Vitreous, aqueous fluid, umbilical cord, nuchal ligament, synovial fluid, costal fluid, skin, aorta,
Tendon, heart valve, brain, cartilage, serum, or extracted from chicken cap, abdominal cavity membrane, streptococcal capsule, etc., obtained by fermentation, derived from natural sources, artificially synthesized Or those produced by recombinant DNA technology, their salts and cross-linked hyaluronic acid.

Examples of the salt include water-soluble metal salts such as sodium salt and potassium salt. As the cross-linked hyaluronic acid, various cross-linking agents such as epichlorohydrin, resorcin diglycidyl ether, neopentyl glycol diglycidyl ether, 1,6-hexanediol diglycidyl ether and bisphenol S are used.
Diepoxy compounds such as diglycidyl ether, sorbitol polyglycidyl ether, polyglycerol polyglycidyl ether, pentaerythritol polyglycidyl ether, glycerol polyglycidyl ether, diglycerol polyglycidyl ether, triglycidyl tris (2-hydroxyethyl) isocyanurate, and other polyepoxy compounds Examples thereof include crosslinked hyaluronic acid crosslinked with an epoxy compound or the like.

The molecular weight of hyaluronic acid used in the present invention may be in the range of 1 × 10 ³ to 1 × 10 ⁸ . The hyaluronic acid used in the present invention can be chemically bonded to the immobilizing carrier in its own form, or can be chemically bonded to the immobilizing carrier via the introducing group by introducing an introducing group into a part of the immobilizing carrier, Examples of the introduction group include an amino group, a carboxyl group, a sulfuric acid group, a phosphoric acid group, a mercapto group, and the like, which can be appropriately selected and used.

The immobilization carrier used in the present invention may be one that is insoluble in an aqueous solvent or an organic solvent, for example, an organic synthetic polymer system, a silica gel system, an agarose system, a dextran system, a cellulosic system, or a polysystem. Acrylamide-based, silica bead-based, glass-based, metal-based, diatomaceous earth, celite, alumina, charcoal and the like can be mentioned and can be appropriately selected and used. Moreover, these can be used individually or as a mixture of 2 or more types.

As a method for immobilizing hyaluronic acid on a carrier for immobilization, a covalent bond method is preferable, and as immobilization conditions, a temperature is 0 to 50 ° C., particularly 1 to 30 ° C.,
The pH is preferably 1 to 14, especially pH 3 to 8, the time is 0.5 to 96 hours, particularly 1 to 24 hours, and the solution is 1M or less of carbonate, borate, phosphate or sodium hydroxide. May be mixed with an aqueous buffer solution and an aqueous solution containing, or a water-soluble organic solvent may be mixed and used.

An activation reagent can be used in the immobilization reaction between hyaluronic acid and the immobilization carrier.
N-ethyl-N '-(3-dimethylaminopropyl) carbodiimide hydrochloride, N-cyclohexyl-
Examples thereof include water-soluble carbodiimides such as N'-2- (4'-methylmorpholinium) ethylcarbodiimide-p-toluenesulfonate and N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline. .

The shape of the carrier for immobilization may be spherical, plate-like, crushed or the like, but the spherical one is particularly preferable, and the particle diameter is 0.5 to 1000 μm, preferably 5 to 500 μm. is there.

The immobilization carrier may be activated.
Examples include amino activation treatment, hydrazino activation treatment, formyl activation treatment, epoxy activation treatment, cyanogen bromide activation treatment, activation treatment with sulfonyl chloride, carbonate activation treatment or activation treatment with carbonylimidazole. However, amino activation treatment is particularly preferable.

The immobilization amount of hyaluronic acid in the hyaluronic acid-immobilized filler used in the present invention may be 10 ^{-9 to} 1 g per 1 ml of the wet volume of the carrier, and can be appropriately selected and used.

Using the packing material obtained in the present invention, column treatment or batch treatment is carried out to separate gelatin affinity substances,
It can be purified and concentrated, and examples of the material of the column include stainless steel, glass, and polymers. The pore size when packed in a column is 3 x
It is preferable to use one having a thickness of 10 ⁴ angstroms or less, preferably 50 to 4000 angstroms.

Examples of the gelatin affinity substance to be separated, purified and concentrated in the present invention include fibronectin, chondronectin, vitronectin, laminin,
Glycoproteins such as polynectin, proteoheparin, proteoheparan sulfate, proteoglycans such as proteochondroitin sulfate, acidic fibroblast growth factor,
Growth factors such as basic fibroblast growth factor, enzymes such as chondroitinase, complement factors such as Clq, platelets,
Examples thereof include those derived from natural sources such as smooth muscle cells, those artificially synthesized, and those produced by recombinant DNA technology.

The conditions for the separation, purification and concentration method of the present invention are preferably a temperature of 1 to 50 ° C. and a pH of 1 to 1.
2 is preferable, the flow rate is preferably a linear velocity of 10 ⁻³ to 10 ³ cm / min, and the eluent is pH 1 to 1 with a salt concentration of 0 to 8M.
Twelve suitable water-based solvents or water-based solvents in which 80% or less of a water-soluble organic solvent is mixed are preferable, and each can be appropriately selected and used.

[0021]

EXAMPLES Next, the present invention will be described in detail with reference to Examples. Example 1 1.0 g of amino-cellulofine (manufactured by Seikagaku Corporation) and 10 ml of distilled water adjusted to pH 5
After swelling by adding, washed with 100 ml of this distilled water and weighed 1.0 g of wet weight. A solution prepared by dissolving 20 mg of hyaluronic acid potassium salt (human umbilical cord) (manufactured by Seikagaku Corporation) in distilled water was added to the solution, and the pH was adjusted to 5 with 0.1N hydrochloric acid, followed by shaking and stirring at 4 ° C for 10 minutes. After adding 60 mg of N-cyclohexyl-N′-2- (4′-methylmorpholinium) ethylcarbodiimide-p-toluenesulfonate to this, the pH was adjusted to 5 with 0.01N hydrochloric acid.
Readjustment, and the mixture was shaken and stirred at 4 ° C. for 24 hours. After that, distilled water 100ml, 4M urea 100ml, distilled water 100m
By washing with l, a hyaluronic acid-immobilized filler having 4 mg of hyaluronic acid immobilized per 1 ml of wet volume was produced.

[Example 2] AF-Amino Toyo
Pearl 650M (manufactured by Tosoh Corporation) 1.0 g
10 ml of distilled water adjusted to pH 5 was added to the mixture to swell it, followed by washing with 100 ml of this distilled water and weighing 1.0 g of wet weight. Sodium hyaluronate (human umbilical cord)
(Seikagaku Co., Ltd.) A solution prepared by dissolving 20 mg in distilled water was added, 50 mg of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline dissolved in 40% ethanol was added, and the mixture was shaken at 25 ° C. for 16 hours. It was stirred. Then, by washing with 100 ml of distilled water, 100 ml of 4M urea, and 100 ml of distilled water, a hyaluronic acid-immobilized filler having 3.5 mg of hyaluronic acid immobilized on a wet volume of 1 ml was produced.

[Example 3] 7 ml of Sepharose
6B (Pharmacia Biotech Co., Ltd.) was washed with 500 ml of distilled water on a glass filter, and then added to a mixed solution of 5 ml of 1 M NaOH aqueous solution containing 10 mg of sodium tetrahydroborate and 5 ml of 1,4-butanediol-diglycidyl ether, After slowly stirring at 23 ° C. for 10 hours, 4 ml of epoxy-activated Sepharose 6B obtained by washing with 500 ml of distilled water
Then, 6 ml of concentrated ammonia water was added, the mixture was heated with shaking at 40 ° C. for 1.5 hours to react, and then washed with 500 ml of distilled water to obtain amino-activated Sepharose 6B.
A solution prepared by dissolving 20 mg of hyaluronic acid potassium salt (human umbilical cord) (manufactured by Seikagaku Corporation) in distilled water was added thereto, and the pH was adjusted to 5 with 0.01N hydrochloric acid, and then 10 at 4 ° C.
The mixture was shaken and stirred for a minute, and N-ethyl-N ′-(3-dimethylaminopropyl) carbodiimide hydrochloride 6 was added thereto.
After adding 0 mg, the pH was readjusted to 5 with 0.01 N hydrochloric acid, and the mixture was shaken and stirred at 4 ° C. for 24 hours. Then, distilled water 1
By washing with 00 ml, 100 ml of 4M urea and 100 ml of distilled water, a hyaluronic acid-immobilized filler having 3.5 mg of hyaluronic acid immobilized on a wet volume of 1 ml was produced.

Example 4 20 g of silica was placed in a 500 ml three-necked round bottom flask, heated under reduced pressure at 150 ° C. for 4 hours, dried, cooled and then released from the reduced pressure, and immediately put into a reflux condenser with a calcium chloride tube. Equipped with a stirrer, dehydrated toluene 3
Epoxy resin obtained by adding 00 ml, 3-glycidyloxypropyltrimethoxysilane 20 ml and dehydrated triethylamine 0.5 ml, refluxing for 16 hours with stirring, washing sequentially with toluene, acetone and ether 1 l each on a glass filter and drying under reduced pressure. 10 ml of water was added to 1.0 g of activated silica to swell it, and then washed with 100 ml of water, and 1.0 g of its wet weight was weighed. 6 ml of concentrated aqueous ammonia was added thereto, and the mixture was heated at 40 ° C. with shaking for 1.5 hours, reacted, and washed with 500 ml of distilled water to obtain amino-activated silica. To this was added a solution of 20 mg of hyaluronic acid sodium salt (pigskin) (Seikagaku Corporation) dissolved in distilled water, and N-ethoxycarbonyl-2-ethoxy-1,2-dissolved in 40% ethanol.
50 mg of dihydroquinoline was added, and the mixture was shaken and stirred at 25 ° C for 16 hours. Then, distilled water 100ml, 4M urea 10
By washing with 0 ml of distilled water and 100 ml of distilled water, a hyaluronic acid-immobilized filler having 3 mg of hyaluronic acid immobilized on a wet volume of 1 ml was produced.

Example 5 The hyaluronic acid-immobilized packing material obtained in Example 1 was applied to a column (inner diameter 4.6 mm, length 50).
mm) to prepare a column containing a hyaluronic acid-immobilized filler.

Test Example 1 Using the column obtained in Example 5, equilibrated with 20 mM Tris-hydrochloric acid buffer solution (pH 7.5) at 25 ° C., then 20 mM Tris-hydrochloric acid buffer solution (pH 7.5). A fibronectin solution, which is a gelatin affinity substance with a concentration of 100 μg / ml, was continuously fed at a flow rate of 0.5 ml / min, and the eluate from the column was subjected to front-end analysis at a wavelength of 280 nm and used as a control for the same column. 2 containing 4M urea and 2M sodium chloride
After equilibration with 0 mM Tris-HCl buffer (pH 7.5),
The concentration dissolved in 20 mM Tris-HCl buffer (pH 7.5) containing 4 M urea and 2 M sodium chloride was 100 μm.
The same procedure was performed for a g / ml fibronectin solution, and the retained amount of fibronectin per 1 mg of immobilized hyaluronic acid was determined from the difference in the breakthrough curves of both solutions. As a comparative example, a similar comparative experiment was conducted using a gelatin-cellulofine filler (made by Seikagaku Corporation) in which gelatin was immobilized. The results are shown in Table 1.

Table 1 ──────────────────────────────────── Packing agent Immobilization Ligand immobilization amount Ligand Fib ligand per mg (mg / ml gel) Lonectin retention amount (mg) ────────────────────────────────── ─── Example 1 Hyaluronic Acid 45 Comparative Example Gelatin 3-4 3 ────────────────────────────────── ───

As shown in Table 1, the hyaluronic acid-immobilized filler of the present invention was able to retain a higher amount of fibronectin, which is a gelatin affinity substance, than the existing gelatin-immobilized filler.

Test Example 2 Using the column obtained in Example 5, equilibrated with 20 mM Tris-hydrochloric acid buffer solution (pH 7.5) at 37 ° C., then 20 mM Tris-hydrochloric acid buffer solution (pH 7.5). Collagenase solution, which is a protease with a concentration of 100 μg / ml dissolved in the
The liquid was continuously sent at 1 / min for 1 hour. Then, at 25 ° C., 20% containing 4M urea and 2M sodium chloride.
20 mM Tris-hydrochloric acid buffer solution (pH 7.5) was continuously fed with 20 mM Tris-hydrochloric acid buffer solution (pH 7.5) at a flow rate of 1 ml / min for 0.5 hours, and again, 20 mM Tris-hydrochloric acid buffer solution (pH 7. After equilibration with 5), the concentration dissolved in 20 mM Tris-HCl buffer (pH 7.5) was 100 μg /
Fibronectin solution, which is a gelatin affinity substance, was continuously fed at a flow rate of 0.5 ml / min, and the eluate from the column was subjected to frontal analysis at a wavelength of 280 nm. Using the same column as a control, 4 M urea and 2 M chloride were used. After equilibration with 20 mM Tris-hydrochloric acid buffer containing sodium (pH 7.5), 20 m containing 4 M urea and 2 M sodium chloride
A fibronectin solution with a concentration of 100 μg / ml dissolved in M Tris-hydrochloric acid buffer (pH 7.5) was also used in the same manner, and the retention amount of fibronectin per 1 mg of immobilized hyaluronic acid was determined from the difference in the breakthrough curves of both solutions. I asked. As a comparative example, a similar comparative experiment was conducted using a gelatin-cellulofine filler (made by Seikagaku Corporation) in which gelatin was immobilized. The results are shown in Table 2.

Table 2 ──────────────────────────────────── Packing agent Immobilization Ligand immobilization amount Ligand Fib ligand per mg (mg / ml gel) Lonectin retention amount (mg) ────────────────────────────────── ─── Example 1 Hyaluronic acid 45 Comparative example Gelatin 3 to 40 ────────────────────────────────── ───

As shown in Table 2, even after treatment with collagenase, which is a protease, the hyaluronic acid-immobilized filler of the present invention has a higher capacity of the gelatin-affinitive substance fibronectin than the existing gelatin-immobilized filler. I was able to hold it.

[0032]

The hyaluronic acid-immobilized filler of the present invention comprises
It is a packing material that is more stable to proteases and has an excellent function of selectively separating, purifying, concentrating or quantifying a gelatin affinity substance.

Claims (3)

[Claims] 1. A packing material for separation, purification and concentration, comprising hyaluronic acid immobilized on a carrier for immobilization. 2. A column containing the packing material according to claim 1 on which hyaluronic acid is immobilized. 3. A method for separating, purifying and concentrating a gelatin affinity substance using the filler according to claim 1, wherein hyaluronic acid is immobilized.