JP2739232B2 - Method of using cellulose gel having biological affinity - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a biocompatible cellulose gel. More specifically, separation of various physiologically active substances,
The present invention relates to a cellulose gel for affinity chromatography useful for purification.

[Conventional technology and its problems]

In recent years, with the advancement of biotechnology, the importance of separation and purification techniques for trace amounts of biologically active substances and the like in vivo or produced by cell culture, genetic manipulation, and the like has been increasing. Many methods are currently used as separation and purification methods, and they can be roughly classified into the following four methods according to the principle.

1) Method using difference in solubility: organic solvent precipitation method, salting out method, isoelectric point precipitation method, etc. 2) Method using charge difference: electrophoresis method, isoelectric point fractionation method, etc. 3) Molecular analysis Methods that use differences in size and shape: gel filtration, ultracentrifugation, etc. 4) Methods that use differences in chemical or physical affinity: ion-exchange chromatography, adsorption chromatography, countercurrent distribution, etc. The separation and purification means have been variously devised and improved by the efforts and experiences of many researchers, and as a result, are widely used.

However, in these methods, the specificity of an object to be separated at the time of separation is low because the principle of separation is based on the difference in physical or chemical properties of substances. Therefore, in order to separate and purify a certain physiologically active substance, some of these different separation and purification methods must be appropriately combined.

Therefore, these separation and purification operations have many problems such as a long time and loss or denaturation of the target substance when performing many kinds of steps.

In order to solve the above-mentioned problems, affinity chromatography methods for separating and purifying by utilizing bacterial biological affinity have been actively performed.

Affinity chromatography is a specific affinity (affinity) that cannot be explained only by physical or chemical affinity at this time, such as substrates for enzymes, reaction products, specific inhibitors, coenzymes, allosteric effectors, etc. This is a method that separates and purifies the target substance using the mutually specific biological affinity that exists between substances such as antibodies to antigens, receptors for hormones, and RNAs to DNA, which is extremely simple compared to conventional methods. It is characterized in that a high-purity target substance can be obtained in a high yield by a single chromatography from a crude sample by operation.

The present invention provides a method of using a cellulose gel to which hyaluronic acid is bound as a gel that can be used for this anity chromatography. Most of the conventional gels for affinity chromatography have been produced based on a polysaccharide, agarose, by activating with promonoan and then binding a ligand.

Antibodies, antigens, enzymes, amino acids, peptides, hormones, nucleic acids and the like are used as ligands. However, since it is based on agarose, the conventional gel for affinity chromatography is soft and has low mechanical strength, and a suitable flow rate cannot be obtained when the column is packed and chromatographed. Was.

On the other hand, hyaluronic acid is a macromolecule isolated from bovine eye vitreous by Meyer et al. In 1934 and having an unbranched chain structure in which N-acetyl-D-glycosamine and D-glucuronic acid are alternately bonded. Has been confirmed. The structure is shown below.

The characteristic properties of hyaluronic acid are the viscoelasticity and water retention derived from this structure. Since the water retention is not particularly affected by the environmental humidity, in addition to basic cosmetics, intraocular injections, deformable joints It is widely used in the pharmaceutical field as a histocompatible biopolymer such as a remedy for diseases and an anti-adhesion agent after surgery.

In contrast, the present inventors have found that hyaluronic acid has specific affinity for hormones such as estradiol and prostaglandin, cell function regulators, and blood coagulation factors. In the case of performing chromatography at a high rate, the present inventors have conceived of using spherical cellulose particles having a high flow rate and bonded via a functional group, and have reached the present invention.

As apparent from the above description, an object of the present invention is to provide a method for using a cellulose gel having a biological affinity which is useful for affinity chromatography using hyaluronic acid. Another object is to provide a method for producing the cellulose gel.

[Means for solving the problem]

 The present invention has the following configurations (1) to (3).

(1) A biocompatible cellulose gel obtained by binding hyaluronic acid to spherical cellulose particles via a functional group selected from an epoxy group, an amino group, a carboxy group or a formyl group is used for affinity chromatography. A method for purifying a hormone, a cell function regulating factor, or a blood coagulation factor, comprising the steps of:

(2) The purification method according to the above (1), wherein the hyaluronic acid has been separated and purified from animal material.

(3) The purification method according to the above (1), wherein the hyaluronic acid is derived from a microorganism produced by a Streptococcus bacterium.

 The configuration and effect of the present invention will be described in detail below.

Hyaluronic acid used in the present invention is chicken cockscomb,
Those derived from animals separated, extracted and purified from fish eyes, etc., those derived from microorganisms produced by Streptococcus bacteria, or those produced by cell culture or the like can be used, and commercially available products can be used. .

Cellulose to which hyaluronic acid is bound is a true spherical particle, and its production method includes, but is not particularly limited to, the following examples.

According to the method described in JP-A-53-86749, cellulose acetate is dissolved in an organic solvent, the solution is suspended in an aqueous medium, spheroidized, and the organic solvent is evaporated to obtain cellulose ester particles. And a method of producing the cellulose particles after saponification.

JP-A-56-24429, in which porosity is adjusted by adding an aliphatic higher alcohol or the like to a solution of a cellulose acetate ester by applying the above method.

JP-A-57-159801 for granulating cellulose by dissolving it in a mixed solution of paraformaldehyde and dimethyl sulfoxide
No., method of Japanese Patent Publication No. 57-159802.

JP-A-52-11237 wherein cellulose is dissolved in aqueous ammonia of cupric hydroxide and cuprous chloride and granulated.

Japanese Patent Application Laid-Open (JP) to disperse viscose in transformer oil and granulate
Method of No. 51-5361.

JP-A-55-44312, wherein cellulose is dissolved in a calcium thiocyanate solution and granulated.

A method disclosed in JP-A-48-60754 in which a purified linter is dissolved in a copper ammonia solution and granulated.

JP-A-61-241337 discloses a method in which a viscose dispersion is produced by mixing a viscose water-soluble anionic polymer compound, and the mixture is heated and solidified.

Next, in order to bind hyaluronic acid to these spherical cellulose particles, there is a method of reacting hyaluronic acid with a hydroxyl group of cellulose or a reactive functional group separately introduced into cellulose.

The method is as follows, but is not particularly limited.

'A method in which cellulose is reacted with bisoxysilane or the like to introduce ethoxy and the ethoxy group is reacted with hyaluronic acid.

'A method in which cellulose is reacted with ω-aminoalkylamine to introduce an amino group, and this terminal amino group is condensed with a carboxyl group of hyaluronic acid.

'A method in which a formyl group is introduced into cellulose and then reacted with the amino group of deacetylated hyaluronic acid to form a Schiff base and reduce it.

'After ethoxylation of cellulose with shrimp chlorohydrin,
There is a method of aminating and reacting with succinic anhydride to introduce a carboxyl group, and condensing this terminal carboxyl group with the amino group of deacetylated hyaluronic acid.

By the way, recently, aminated-cellulofine (cellulofine is a trade name; the same applies hereinafter), formyl-cellulofine, and carboxycellulofine can be used as affinity gels with cellulose spherical particles. These can also be used in the present invention.

When the above-mentioned methods of bonding (1) to (3) are represented by a reaction formula, for example, the following is obtained.

More specific bonding conditions for the spherical cellulose particles and hyaluronic acid in the above-mentioned '~' and '~' are as follows:
It is as follows.

A suction dry product is used as the spherical cellulose particles. As a method for introducing a predetermined functional group into the suction-dried product, the section-dried product is suspended in an appropriate reaction medium, and a reaction catalyst and a functional group-introducing material are further added thereto under predetermined reaction conditions (temperature, time, etc.). ) To react.

The reaction medium, reaction catalyst and reaction conditions must be appropriately selected and combined depending on the type of the raw material for introducing a functional group. For example, in the case of the introduction of the above-mentioned ethoxy group,
A 0.1 to 10 N (preferably 0.5 to 2 N) aqueous NaOH solution is used as a medium, and NaBH ₄ is used as a catalyst. The spherical cellulose particles and the ethoxy group-containing halide are heated at 10 to 50 ° C. (preferably 15 to 50 ° C.).
(~ 35 ° C) for 1-20 hours (preferably 2-10 hours).

Further, for example, in the case of the introduction of the amino group of the above ', a KIO ₄ of 0.1 to 4M (preferably 0.2 to 2M) is used as a catalyst and medium.
Using an aqueous solution, the spherical cellulose particles are previously treated at 10 to 50 ° C (preferably 15 to 35 ° C) for 10 minutes to 5 hours (preferably 30 minutes to 2 hours). An amine for amination (for example, hexamethylenediamine) is added to the treated product after washing with water,
The reaction is carried out at 50 ° C (preferably 15 to 35 ° C) for 2 to 20 hours (preferably 3 to 12 hours).

Next, in the reaction between the functional group-containing spherical cellulose gel obtained as described above and hyaluronic acid, both are reacted in a suitable spherical medium containing a catalyst under predetermined conditions (temperature and time).

For example, in order to bind hyaluronic acid to the ethoxy-activated cellulose gel obtained by the above-mentioned method, the former suction-dried product is used as a catalyst / reaction medium in an amount of 0.05 to 2M.
(Preferably 0.1-0.5M) suspended in an aqueous solution of NaCO ₃ , and added with sodium hyaluronate as a source of hyaluronic acid to give -5.
The reaction is carried out at -20 ° C (preferably 0-10 ° C) for 2-30 hours (preferably 5-20 hours).

Further, for example, in order to bind hyaluronic acid to the aminated-cellulose gel obtained by the method described in the above method, the former section-dried product is suspended in a 0.02 to 1M (preferably 0.05 to 2M) aqueous sodium carbonate solution as a reaction catalyst. Turbid, a predetermined amount of hyaluronic acid and 1-ethyl-3- (3
-Dimethylaminopropyl) carbodiimide to give 10
The reaction is carried out at 5050 ° C. (preferably 15-25 ° C.) for 1-10 hours (preferably 2-5 hours).

The amount of hyaluronic acid when bound to the spherical cellulose gel by each of the above methods is not limited, but is 1 to 50 mg / ml (preferably 5 to 30 mg / ml).

〔The invention's effect〕

Such a cellulose gel to which hyaluronic acid is bound according to the present invention can be used as a unique separating and purifying agent.

As described above, specific proteins, viruses, hormones, and the like are purified and obtained by centrifugation, ion exchange, gel filtration, or the like, and these methods are combined or used repeatedly. By the way, when these methods are combined or repeated, a complicated operation is required for each step, a long time is required, and the loss of the target product is reduced as the steps are performed. There is.

On the other hand, the cellulose gel to which the hyaluronic acid is bound according to the present invention is very useful as a new gel for affinity chromatography because it specifically adsorbs specific hormones, cell function regulating factors and blood coagulation factors. In addition to greatly simplifying the purification process, the carrier gel is made of cellulose, which provides mechanical strength, high flow rates, and can be used on an industrial scale, so it is expected to be used as a new separation system. it can.

Further, in the hyaluronic acid-cellulose gel of the present invention, since hyaluronic acid is a macromolecule having a chain structure without branching, a site having an affinity as compared with a normal ligand is easily brought into contact with a target substance, and thus affinity chromatography is performed. It is possible to process a large amount of the target substance at a time.

〔Example〕

Hereinafter, examples of the method of binding hyaluronic acid to spherical cellulose particles and the use of the obtained carrier will be described, but the present invention is not limited to only these examples.

Example 1 100 g of suction-dried cellulose gel granulated by the method of Example 1 in JP-A-55-44312 was mixed with 1N-NaOH solution 80
ml, and the suspension further of NaBH ₄ 5 g and 12 ml 1-chloro-2,
3-Epoxypropane was added and reacted at 25 ° C. for 5 hours.

After completion of the reaction, the mixture was filtered and washed well with water. 100 g of the suction-dried product of the epoxy activated-cellulose gel thus obtained is suspended in 130 ml of 0.2 M Na ₂ CO ₃ solution, and 2.1 g of sodium auronate (Q-P Co., Ltd. HA-Q type) is added. The reaction was performed at 4 ° C. for 15 hours. After the reaction 1.
Washed with 0M NaCl.

In order to remove unreacted epoxy groups, 100 ml of 5M-hydroxyamine hydrochloride was added under neutral conditions, and the mixture was stirred and filtered. Hyaluronic acids immobilized Bitter (Anal.Biochem 4 330 (1
962)) was determined by the method of measuring uronic acid, which was 6 mg per ml of gel.

Example 2 100 g of a suction-dried cellulose gel granulated by the method of Example 1 in JP-A-56-24429 was applied to a 0.4 M KIO ₄ solution.
130 ml was added and the mixture was stirred for 1 hour at room temperature. After washing well with water,
150 ml of 1.0 M hexamethylenediamine was added and stirred for 6 hours. After completion of the reaction, it was washed with water. 100 ml of the aminated-cellulose gel thus obtained contains 150 ml of 0.1 M sodium carbonate containing 3.5 g of hyaluronic acid (hyaluronic acid Kyowa type manufactured by Kyowa Hakko Kogyo Co., Ltd.) and 1-ethyl-3- (3-dimethylamino acid). 1.0 g of propyl) carbodiimid was added and the mixture was stirred at room temperature for 3 hours. After completion of the reaction, the mixture was filtered and washed with water. The amount of immobilized hyaluronic acid was 11 mg / ml of gel.

Example 3 5 g of sodium hyaluronate (BHA-H type, manufactured by Chisso Corporation) was added to 30 ml of hydrazine hydrate, and the hydrazinolysis method (M. Fujinaga, Y. Matsushima, Bull. Chem. Soc. Jpn., 39,
185 (see 1966)).

0.2M Na ₂ HPO ₄ -NaO containing 200 g of section dried product of formyl laurofine (manufactured by Chisso Corp.) obtained by formalizing cellulose commercially available as an affinity carrier and 3 g of the above-mentioned deacetylated sodium hyaluronate
400 ml of H buffer (PH 11.0) was added, and the mixture was stirred at 30 ° C for 1 hour. This is followed by sodium cyanoborohydride (SCBH)
1 g was added and stirred overnight.

After filtration, washing with distilled water was repeated. To block unreacted formyl groups, the gel obtained by the above stirring was immersed in 500 ml of 0.2 M Tris-HCl buffer and SCBH 1 g
Was added and stirred for 2 hours. After filtration, washing with distilled water was repeated. The amount of immobilized hyaluronic acid was 5 mg / ml of gel.

Example 4 0.2 M Na ₂ containing 50 g of section-dried carboxy-cellofine (manufactured by Chisso Corporation) commercially available as an affinity carrier and 2.5 g of deacetylated sodium hyaluronate obtained by the method of Example 3 HPO ₄ -NaOH
100 ml of buffer (PH 11.0) and 1-ethyl-3- (3
1 g of-(dimethylaminopropyl) carbodiimide hydrochloride was added, and the mixture was stirred at room temperature for 8 hours. After the completion of the reaction, the mixture was filtered and sufficiently washed with water. The amount of the immobilized hyaluronic acid was 8 mg per 1 ml of the gel obtained by the above reaction.

Example 5 (Purification of Prostaglandin) The hyaluronic acid-cellulose gel prepared in Example 1 was packed in a column having a diameter of 1.2 cm × 9 cm, and a 50 mM phosphate buffer (PH 7.5) (hereinafter referred to as “starting buffer”). ).

100 ml of secretion fluid extracted from sheep seminal vesicle
Was added at a flow rate of 20 ml / hr, and 50 ml of a starting buffer was further passed to wash unadsorbed substances.

Next, a 50 mM phosphate buffer (PH6.
0) The prostaglandin adsorbed on the column was eluted by flowing 50 ml. As a result, 8 mg of prostaglandin was recovered.

This was confirmed to be a pure product by gas chromatography mass spectrometry and a decrease in the activity of adenate reductase. The recovery per unit volume of the gel was 0.8 mg / ml.

Example 6 (Purification of blood coagulation factor XII) The hyaluronic acid-cellulose gel prepared in Example 2 was packed in a column having a diameter of 2 cm x 30 cm, and the column was equilibrated with 1 liter of physiological saline. 100 ml of human plasma was flowed through this. Next, 300 ml of physiological saline was flowed to wash unadsorbed components, and the components adsorbed on the column were eluted by gradient elution in which the NaCl concentration was changed linearly from 0.16 M to 2.0 M.

This solution was fractionated with a fraction collector, and the concentration of total protein and blood coagulation factor XII was measured for each fraction. Blood coagulation factor XII was measured by enzyme immunoassay.

The results are shown in FIG. Fractions from the results in Figure 1
Blood coagulation factor XII was concentrated and eluted from No. 25 to No. 33, indicating that the product of the present invention is an excellent affinity carrier.

Example 7 (Purification of estradiol) 2 liters of the hyaluronic acid-cellulose gel prepared by the method described in Example 4 was packed in a column (diameter 8 cm × 40 cm), and 0.01 M Tris-HCl buffer (PH7.8) +0.1. 1M NaC
Equilibrated with l. To this, 10 l of pregnant horse urine was poured. 0.0
After washing 2 L of 1 M Tris-HCl buffer (PH7.8) to wash unadsorbed components, 2 L of 0.1 M Tris-HCl buffer + 2 M urea was flowed to elute estradiol adsorbed on the column.

By the above operation, 970 mg of estradiol was recovered. This was confirmed to be pure by gas chromatography mass spectrometry and biological tests. The recovery rate per unit volume of the gel was 0.5 mg / ml.

Comparative Example 1 (Measurement of Flowability of Gel) In Example 4, CH-Sepharose 4B (a carboxy group was introduced into agarose beads manufactured by Pharmacia via a functional group) was used instead of carboxy-cellulofine. The other steps were carried out under exactly the same conditions to obtain a hyaluronic acid-agarose gel. Each of this gel and the cellulose hyaluronate gel prepared in Example 4 was φ1.5 cm
Pack into a × 30cm column and add 50mM phosphate buffer (PH
7.0) was pumped, and the relationship between pressure and flow rate was measured.

The result is shown in FIG. The hyaluronic acid-agarose gel has a consolidation phenomenon in which the flow rate does not appear even when the pressure is increased, whereas the hyaluronic acid-cellulose gel of the present invention has a linear relationship between the pressure and the flow rate.

From the results, it is clear that the gel of the present invention has higher toughness than conventional agarose-based gels and can flow without any problem, which is very advantageous when used in large quantities industrially.

[Brief description of the drawings]

1 and 2 are explanatory diagrams of an embodiment of the present invention. In these figures, FIG. 1 is a diagram showing an elution curve of blood coagulation factor XII by affinity chromatography performed in Example 6, and FIG. 2 is a column A alone, a column packed with the gel of the present invention. B, Pressure and flow rate when 50 mM phosphate buffer (PH7.0) was passed through column C packed with hyaluronic acid-agarose gel.

Claims (3)

(57) [Claims] An affinity chromatograph of a biocompatible cellulose gel obtained by bonding hyaluronic acid to spherical cellulose particles via a functional group selected from an epoxy group, an amino group, a carboxy group or a formyl group. A method for purifying a hormone, a cell function regulator or a blood coagulation factor, which is used. 2. The purification method according to claim 1, wherein the hyaluronic acid is separated and purified from an animal substance. 3. The method according to claim 1, wherein the hyaluronic acid is derived from a microorganism produced by a Streptococcus bacterium.