JPH01275600A - Method for purifying antithrombin-iii - Google Patents

Abstract

PURPOSE:To effectively remove pyrogens and readily obtain the subject compound excellent in safety and useful for treating disseminated angiopathy, by treating an aqueous solution containing antithrombin-III with a hydrophobic chromatographic carrier. CONSTITUTION:An aqueous solution containing antithrombin-III is brought into contact with an insoluble carrier (e.g., phenyl type Sepharose(R)) containing hydrophobic groups as a ligand under contact conditions, e.g., pH 6-9 and 1-5 M salt concentration, and the unadsorbed fraction is then recovered to carry out purification. Furthermore, the aqueous solution containing the antithrombin-III is preferably previously heat-treated at 50-70 deg.C for 5-30hr before contact with the above-mentioned carrier.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a method for purifying antithrombin-III using hydrophobic chromatography.

[Prior art] Antithrombin-■ is a type of glycoprotein belonging to α2 globulin present in plasma, and its molecular weight is 65,000.
0 to 68,000, has protease inhibitory activity, and strongly inhibits thrombin coagulation activity.

In addition, it has an inhibitory effect not only on thrombin but also on other coagulation factors, such as activated factor X and activated factor II.

In addition, it has been reported that it has a 3-inhibitory effect on plasmin and trypsin.

It is known that these inhibitory effects generally progress more rapidly in the coexistence of heparin.

Antithrombin-■, which has such pharmacological effects,
Correction of hypercoagulopathy, specifically generalized vascular abnormality (D
It is used for the purpose of IC).

[Problem to be Solved by the Invention 8] By the way, in the purification process of antithrombin-III, there is a concern that pyrodiene or contaminant proteins may be mixed in, and various methods for removing them are being studied.

Furthermore, the presence of heat-denatured proteins produced by liquid heat treatment at 60° C. for 10 hours to inactivate hepatitis viruses and the like that may be present in plasma protein components is becoming a new problem. Recently, as a method for removing this heat-denatured protein, a method of reprocessing using immobilized heparin has been proposed (Japanese Patent Laid-Open No. 63-23896). However, it has been found that this method is not very effective for removing pyrogenes, etc.

In view of these circumstances, the present inventors conducted various studies and found that by treating an aqueous solution containing antithrombin-■ with a hydrophobic chromatographic carrier, the desired purpose was achieved and an antithrombin with superior safety was obtained. It was discovered that a thrombin-■ preparation could be prepared, and the present invention was completed.

[Means for Solving the Problems] The antithrombin-■ used in the present invention is not particularly limited as long as it is of human origin and purified to the extent that it can be used as a medicine. For example, It can be purified from human whole blood, plasma, serum, serum compressed from coagulated blood, and the like.

Starting materials for preparing antithrombin-■ include, for example, plasma, fractions IV-1, IV-4 or IV-1+IV-4 obtained by Cohn's cold ethanol method,
The residual fraction etc. after recovering blood coagulation factor (I) from citric acid-containing plasma are used.

As a method for purifying antithrombin-III, for example,
Japanese Unexamined Patent Publication No. 48-35017, Japanese Patent Publication No. 5-7693
The method disclosed in the above publication is exemplified.

Furthermore, although the purity of antithrombin-III is not particularly limited, a highly purified product is more effective within the membrane.

Antithrombin-■The protein concentration of the aqueous solution containing:
An example is about 0.1 to 10 (W/V)%.

In addition, the aqueous solution containing antithrombin-■ is subjected to heat treatment (50 to 70°C,
It is preferable to carry out the test for about 5 to 30 hours.

The insoluble carrier having a hydrophobic group as a ligand used in the present invention is as follows.

As a hydrophobic group, an alkyl group (about 1 to 10 carbon atoms,
(preferably about 3 to 5), a phenyl group which may have a substituent, and the like.

Examples of insoluble carriers include cellulose, agarose, dextran, polyacrylamide, amino acid copolymers, polyvinyl-based, polystyrene-based, and the like.

Insoluble carriers with hydrophobic groups as ligands include alkyl-type Sepharose (e.g., octyl-type Sepharose, etc.), phenyl-type Sepharose, alkyl-type polyvinyl (
For example, butyl type polyvinyl etc.) are commercially available,
Products other than these can also be easily produced by the same method as commercially available products or a method analogous thereto.

The purification step in the present invention generally involves antithrombin-
(2) This is carried out by bringing the containing aqueous solution into contact with an insoluble carrier having a hydrophobic group as a ligand, and the method may be either a column method or a batch method.

Examples of contact conditions include a pH of about 6 to 9 and a salt concentration of about 1 to 5M. Solvents that meet these conditions include 3
20mM sodium citrate buffer (pH 7.5) containing M sodium chloride, 50mM containing 2M sodium chloride
Examples include phosphate buffer (pH 7.5).

To specifically explain the purification method of the present invention, when a batch method is used, an aqueous solution containing antithrombin-1 adjusted to the above conditions is brought into contact with the hydrophobic chromatographic carrier equilibrated under the same conditions. The conditions are that the carrier 1ν!
Using the aqueous solution 1-100 if, 2-20℃
An example of this method is to mix the mixture for about 30 minutes to 2 hours. Thereafter, the supernatant is collected by centrifugation.

When using the column method, an aqueous solution containing antithrombin-■ adjusted to the above conditions is developed on the hydrophobic chromatographic carrier equilibrated under the same conditions and packed in a column,
Collect the non-adsorbed fraction.

Antithrombin-1 thus obtained is further purified, if necessary, and then formulated by a known method.

[Effects of the Invention] According to the purification method of the present invention, it is possible to effectively remove pyrogienes, contaminant proteins, heat-denatured proteins generated by heat treatment, etc. contained in the aqueous solution containing antithrombin-■, thereby improving safety. An excellent antithrombin-■ preparation can be provided.

In addition, the process is simple and the method is suitable for mass production, making it extremely useful as an industrial production method.

[Examples] Examples will be given to explain the present invention in more detail, but the present invention is not limited to these in any way.

Example 1 10 kg of paste of fraction ■-1 obtained by Cohn's cold alcohol fractionation method was suspended in 100 g of physiological saline, barium sulfate was added to give a concentration of 5 (ν/V)%, and the mixture was stirred at room temperature for 30 kg.
The mixture was stirred for a minute, and the trace amount of prothrombin present was removed by adsorption with barium sulfate.

This supernatant was adjusted to pH 6.5 and
Niall #4.000 was added to a concentration of 13 (W/V)%, the resulting precipitate was removed by centrifugation, and then the mixture was added. J:7-ru #4. OOO was added at a concentration of 30 (W/V)%, and the resulting precipitate was collected by centrifugation. Dissolve this precipitate in approximately 2 fl of cold physiological saline.
The solution was injected into a heparin-Sepharose column that had been previously adjusted with physiological saline, and antithrombin-■ was adsorbed onto the column. After washing this column with a 0.4M sodium chloride solution to remove impure proteins, a 2.0M sodium chloride solution was passed through the column and the eluted portion was collected.

Sodium citrate was added to the aqueous solution of antithrombin-III at a concentration of 0.6M to adjust the pH to 7.8, followed by heat treatment at 60°C for 10 hours. Sodium chloride (final concentration 20mM) was added to adjust the pH to 7.5. On the other hand, a butyl-type polyvinyl carrier (butyl-Toyopearl 650, Toyo Soda
After contacting the aqueous solution containing antithrombin-■ (manufactured by Alumni Co., Ltd.), the solution was developed with the above buffer and the unadsorbed fraction was collected.

Subsequently, antithrombin-III was concentrated by dialysis against 0.9% sodium chloride solution overnight.
W/V)% aqueous solution was obtained and, if necessary, filtered or centrifuged to obtain a clear liquid.

To this 1 (W/V)% aqueous solution of antithrombin-III, 2 (W/V)% mannitol and 0.2 (W/V)% sodium citrate were added, and 0.2 (W/V)% sodium chloride was added.
After diluting with a small amount of cold distilled water to a concentration of 5% and adjusting the pH to 7.6 with IN sodium hydroxide, sterilization filtering was performed using a sterilized Millipore filter, and 500 units were dispensed and freeze-dried. It was made into a dry formulation.

Example 2 Instead of fraction IV-1 paste, fraction ■-1+IV-
All treatments were carried out in accordance with Example 1, except that No. 4 was used, and the same antithrombin-■ preparation as in Example 1 was obtained.

Example 3 All treatments were carried out in accordance with Example 1, except that instead of Fraction IV-1 paste, the residual fraction after treating citric acid-containing plasma at low temperature to recover blood coagulation factor (I) was used. An antithrombin-■ preparation similar to that in Example 1 was obtained.

Experimental Example 1 (1) Removal of Pyrodiene According to Example 1, hydrophobic chromatography was performed, and two tests were conducted to examine pyrodiene in the antithrombin-■ containing fractions before and after the treatment (first sample and (referred to as the second sample). The test method was based on the pyrogen test method listed in the Japanese Pharmacopoeia. The results are shown in Table 1.

Table 1 As shown in Table 1 above, it was found that the hydrophobic chromatography treatment lowered the total temperature and sufficiently removed pyrodiene.

(2) Removal of contaminant proteins (ω specific activity. Perform hydrophobic chromatography according to Example 1, measure the total protein amount and antithrombin-■ activity in the antithrombin-■ containing fraction before and after, and compare The activity was calculated.The total protein amount was measured by absorbance at 280 nIll.Antithrombin-■ activity (AT-III activity) was determined by reacting thrombin with the sample at 28°C for 5 minutes, and adding fibrin to this. The amount of prolongation of clotting time is measured, and the titer is calculated from a pre-prepared calibration curve.However, 1 unit is normal human plasma I If
Corresponds to the amount of antithrombin contained in -■.

The results obtained are shown in Table 2.

As shown in Table 2, by hydrophobic chromatography,
An increase in AT-III activity and specific activity was observed, and in particular, the specific activity increased from 4.9 (units/mg protein) to 6°4 (units/mg protein).
mg protein).

1) Removal of contaminant plasma proteins Hydrophobic chromatography was carried out according to Example 1, and the presence or absence of contaminant plasma proteins in the antithrombin-①-containing fractions before and after the treatment was examined by the Ophthalony method. The result is the third
Shown in the table.

Table 3 (C) Gel filtration Hydrophobic chromatography was performed according to Example 1, and the antithrombin-■ containing fractions before and after the treatment were subjected to gel filtration.
The elution patterns were compared. The results are shown in FIG. Note that the conditions for gel filtration are as follows.

Carrier: TSK Gel G3000SW-XL (manufactured by Tosoh Corporation) Eluent: 0.1M phosphate buffer containing 0.2M sodium chloride (pH 6,8) Detectability: Absorbance at 280rv As shown in Figure 1 , both parts before treatment show bimodality. Among these, the high molecular fraction is considered to be based on heat-denatured proteins, judging from the elution pattern before heat treatment. It was found that this polymer fraction disappeared by hydrophobic chromatography, resulting in a negative peak.

Experimental Example 2 A freeze-dried preparation of antithrombin-III was dissolved in distilled water for injection, and an amount equivalent to 40,000 units/kg was administered to five mice via the tail vein, and observation was continued for 7 days.
No abnormality was observed. Furthermore, when the same solution was administered to rabbits at a dose of 5,000 units/kg and observed for 24 hours, no abnormalities in body temperature were observed.

[Brief explanation of the drawing]

FIG. 1 shows the gel filtration elution pattern of the antithrombin-■ containing fraction before and after hydrophobic chromatography treatment. Patent applicant Green Juji Co., Ltd. Figure 1 Processing Pre-processing Post-retention time (minutes) Retention time (minutes) Procedure Amendment (spontaneous) Commissioner of the Patent Office Yoshi 1) Moon Yi 1 Indication of the case 1988 Patent Application No. 105783 2 Name of the invention Method for purifying antithrombin-III 3 Person making the amendment (change of address on February 13, 1989) Name Mitri Cross Co., Ltd. Representative Tadakazu Suyama 5 Date of amendment order (voluntary) 6. There is no increase in the number of claims due to the amendment. 8. Contents of the amendment (1) "Zuzu" on page 9, line 15 of the specification is corrected to "zuzu". (2 Correct r40.000J on page 13, line 15 of the same book to r4.000J.

Claims (4)

[Claims] (1) Antithrombin-III, which is characterized by bringing an aqueous solution containing antithrombin-III into contact with an insoluble carrier having a hydrophobic group as a ligand, and collecting the unadsorbed fraction.
Purification method. (2) The method for purifying antithrombin-III according to claim (1), wherein the contact conditions are pH 6 to 9 and salt concentration 1 to 5M. (3) The method for purifying antithrombin-III according to claim (1), wherein the hydrophobic group is an alkyl group. (4) The method for purifying antithrombin-III according to claim (1), wherein the antithrombin-III is heat-treated.