JP2678249B2 - Antithrombin-III formulation - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial application] The present invention relates to antithrombin-II derived from human plasma.
It relates to an antithrombin-III formulation containing I, virus inactivated, and substantially free of contaminants.

The [Prior Art] antithrombin -III a kind of glycoprotein belonging to the alpha ₂ globulin present in the plasma, its molecular weight 65,000
It has a protease inhibitory activity and strongly inhibits the clotting activity of thrombin.

In addition, it has an inhibitory effect not only on thrombin but also on other coagulation factors, for example, activated factor X, activated factor IX, and the like. In addition, it has been reported that it has an inhibitory effect on plasmin and trypsin.

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

Antithrombin-III having such a pharmacological action
Is used for correction of hypercoagulability, specifically for the purpose of generalized vascular abnormalities (DIC).

[Problems to be Solved by the Invention] By the way, in the step of purifying antithrombin-III, there is a concern that pyrogen or contaminant proteins may be present, and various methods for removing the same have been studied. In addition, the presence of heat-denatured protein produced by liquid heat treatment at 60 ° C. for 10 hours, which is performed to inactivate hepatitis virus and the like that may be mixed 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. Sho 61-242).
63-23896). However, it has been found that this method is not very effective in removing pyrogens and the like.

Therefore, in view of these circumstances, the present inventors have conducted various studies, and as a result, antithrombin-II derived from human plasma has been obtained.
It was found that an antithrombin-III preparation containing I, virus inactivated, and substantially free of contaminants can be prepared, and the present invention has been completed.

[Means for Solving the Problems] The antithrombin-III preparation of the present invention has the following properties.

Contains antithrombin-III derived from human plasma.

The virus is inactivated.

Substantially free of contaminants.

The contaminants, pyrogen, heat denatured proteins, plasma proteins other than antithrombin -III (e.g., ceruloplasmin, transferrin, albumin, haptoglobin, alpha ₂ - macroglobulin, alpha ₁ - antitrypsin, IgA, etc. IgC) or the like Can be mentioned.

The specific activity of antithrombin-III is about 6 to 7 units / mg protein.

The antithrombin-III preparation of the present invention specifically uses antithrombin-III purified by a chromatographic treatment using an insoluble carrier having a hydrophobic group as a ligand for an antithrombin-III-containing aqueous solution as follows. Is prepared.

Antithrombin-III used as a raw material is of human origin and is not particularly limited as long as it is purified to the extent that it can be used as a medicine.
It can be purified from human whole blood, plasma, serum, serum expressed from coagulated blood, or the like. As a starting material for preparing the antithrombin-III, for example, plasma, Fraction IV obtained by the cold ethanol method of corn,
IV-1, IV-4 or IV-1 + IV-4, a residual fraction after collecting blood coagulation factor VIII from citrate-containing plasma, and the like are used. Examples of the method for purifying antithrombin-III include, for example, JP-A-48-35017 and JP-B-59-7693.
The method disclosed in the publication is exemplified.

The protein concentration of the antithrombin-III-containing aqueous solution is, for example, about 0.1 to 10 (W / V)%.

The antithrombin-III-containing aqueous solution is preferably subjected to a heat treatment (50 to 70 ° C., about 5 to 30 hours) before being subjected to the hydrophobic chromatography treatment.

The insoluble carrier having a hydrophobic group as a ligand used for the above purification is as follows.

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

Examples of the insoluble carrier include cellulose, agarose, dextran, polyacrylamide, amino acid copolymer, polyvinyl, and polystyrene.

As the insoluble carrier having a hydrophobic group as a ligand, alkyl-type sepharose (octyl-type sepharose, etc. when cold), phenyl-type sepharose, alkyl-type polyvinyl (eg, butyl-type polyvinyl) and the like are commercially available. The product can be easily manufactured by the same method as a commercially available product or a method analogous thereto.

The purification step using the insoluble carrier is generally carried out by contacting an antithrombin-III-containing aqueous solution with an insoluble carrier having a hydrophobic group as a ligand, and the method is a column method or a batch method. May be.

Examples of the contact conditions include a pH of about 6 to 9 and a salt concentration of about 1 to 5M. As a solvent satisfying such conditions, a 20 mM sodium citrate buffer containing 3 M sodium chloride (pH 7.
5), 50mM phosphate buffer containing 2M sodium chloride (pH7.5)
Etc. are exemplified.

The above-mentioned purification method will be specifically described. When the batch method is used, antithrombin-III adjusted to the above conditions is used.
The contained aqueous solution is brought into contact with the hydrophobic chromatography carrier equilibrated under the same conditions. The condition is 1 ml of the carrier
To 1 to 100 ml of the aqueous solution at 2 to 20 ° C. for 30 minutes
A method of mixing for about 2 hours can be used. Thereafter, the supernatant is recovered by centrifugation.

When the column method is used, the antithrombin-III-containing aqueous solution adjusted to the above conditions is developed on the hydrophobic chromatography carrier equilibrated under the same conditions and packed in a column, and a non-adsorbed fraction is collected.

Preferably, the non-adsorbed fraction obtained above is further purified by being subjected to a step of further treating with an anion exchanger to recover the adsorbed fraction.

Examples of the anion exchanger include DEAE systems (eg DEAE-agarose, DEAE-dextran, DEAE-cellulose etc.), QAE systems (eg QAE-agarose, QAE-dextran etc.) and the like.

The contact conditions are, for example, pH 6 to 8 and salt concentration of 0.01 to 0.1 M, and a solvent satisfying such conditions is 0.05
An example is 0.01 M citrate buffer (pH 7) containing M sodium chloride.

The elution conditions are, for example, about pH 6 to 8 and salt concentration about 0.1 to 0.3 M, and a solvent satisfying such conditions is 0.1.
An example is 0.01M citrate buffer (pH 7) containing 17M sodium chloride.

The method may be a column method or a batch method.

When the batch method is used, the antithrombin-III-containing aqueous solution prepared under the above contact conditions is brought into contact with the anion exchanger equilibrated under the same conditions. As the condition, 1 to 100 ml of the aqueous solution is used for 1 ml of the exchanger, and 2
After mixing for 30 minutes to 2 hours at -20 ℃, centrifuge,
Collect the exchanger. Further, the above-mentioned elution solvent is added to the exchanger. The mixing conditions are the same as for the contact, but the supernatant is recovered by centrifugation.

On the other hand, when the column method is used, the antithrombin-III-containing aqueous solution prepared under the above contact conditions is equilibrated under the same conditions and passed through the anion exchanger packed in the column to remove the non-adsorbed fraction. Discard. After washing the column as needed, the elution solvent is flowed to collect the elution fraction.

The antithrombin-III thus obtained is further formulated, if necessary, and then formulated. Examples of the preparation include an aqueous solution, a suspension, a lyophilized preparation, and the like. These preparations are prepared by appropriately mixing pharmaceutically acceptable additives (diluent, tonicity agent, surfactant, etc.). It can be carried out by the above conventional means. The preparation is used as an injection such as intravenous injection and subcutaneous injection, and the lyophilized preparation is used by dissolving it in distilled water for injection before use.

[Effects of the Invention] According to the formulation of the present invention, the antithrombin-III-containing aqueous solution contains purified antithrombin-III in which pyrogen, contaminant proteins, heat-denatured proteins produced by heat treatment, etc. are effectively removed. Therefore, an antithrombin-III preparation having excellent safety can be provided.

[Examples] The present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

Example 1 10 kg of a paste of the fraction IV-1 obtained by the cold alcohol fractionation method using corn was suspended in 100 physiological saline, and barium sulfate was added thereto at a concentration of 5 (W / V)%, and the mixture was stirred at room temperature for 30 minutes. After stirring for a minute, a small amount of prothrombin was adsorbed on barium sulfate to be removed. The pH of the supernatant was adjusted to 6.5, polyethylene glycol # 4,000 was added to 13 (W / V)%, the resulting precipitate was removed by centrifugation, and polyethylene glycol # 4,000 was further added to 30 (W / V). V)%, and the resulting precipitate was collected by centrifugation. This precipitate was dissolved in about 20 cold physiological saline and injected into a column of heparin sepharose previously adjusted with physiological saline to adsorb antithrombin-III on the column. 0.4M
After washing with sodium chloride solution to remove impure proteins,
A 2.0 M sodium chloride solution was passed through the column to collect the eluted portion.

Sodium citrate was added to the aqueous solution of antithrombin-III to a concentration of 0.6 M and adjusted to pH 7.8.
After heating for 10 hours, sodium chloride (final concentration 3M) and sodium citrate (final concentration 20 mM) were added to adjust the pH to 7.5. On the other hand, 3M sodium chloride containing 2
After contacting an aqueous solution containing antithrombin-III with a butyl-type polyvinyl carrier (butyl-Toyopearl 650, manufactured by Toyo Soda Co., Ltd.) equilibrated with 0 mM sodium citrate buffer (pH 7.5), It was developed and the unadsorbed fraction was collected. Subsequently, the solution was concentrated while performing dialysis overnight against a 0.9% sodium chloride solution to obtain a 1% (W / V) aqueous solution of antithrombin-III. If necessary, the solution was filtered or centrifuged to obtain a clear solution. And

A 1 (W / V)% aqueous solution of antithrombin-III was added to 2 (W / V)% mannitol and 0.2 (W / V) sodium citrate.
V)%, diluted with a small amount of cold distilled water so that sodium chloride becomes 0.5%, adjusted to pH 7.6 with 1N sodium hydroxide, and then sterilized and filtered with a sterilized Millipore filter.
Dispensed 500 units each, and lyophilized to give a dried preparation.

Example 2 Instead of Fraction IV-1 paste, Fraction IV-1 + IV-4
The treatment was carried out in the same manner as in Example 1 except for using, to obtain the same antithrombin-III preparation as in Example 1.

Example 3 Instead of the fraction IV-1 paste, the treatment was carried out in the same manner as in Example 1 except that the citrate-containing plasma was treated at a low temperature to collect the blood coagulation factor VIII, and the residual fraction was used. ,
The same antithrombin-III preparation as in Example 1 was obtained.

Example 4 A hydrophobic chromatographic treatment was carried out in the same manner as in Example 1, followed by a DEAE-dextran treatment.

That is, the hydrophobic chromatographically treated antithrombin-III-containing aqueous solution obtained in Example 1 was treated with sodium chloride 0.05M,
Sodium citrate was adjusted to 0.01M, pH 7, and then 0.01M citrate buffer containing 0.05M sodium chloride (pH
It was passed through DEAE-dextran (DEAE-Sephadex A-50, manufactured by Pharmacia) equilibrated in 7). After further washing the column with the same solvent, elution was performed with a 0.01M citrate buffer solution (pH 7) containing 0.17M sodium chloride, and the eluted fractions were collected. Hereinafter, formulation treatment was performed according to Example 1 to prepare an antithrombin-III formulation.

Example 5 Antithrombin-III dry powder obtained in Example 4 500 units Sodium chloride 50 mg Sodium citrate 52 mg The formulation of the above formulation was dissolved in 20 ml of distilled water for injection at the time of use to be injected intravenously or intravenously. To do. One unit corresponds to the amount of antithrombin-III contained in 1 ml of normal human plasma.

Experimental Example 1 (1) Removal of pyrogen According to Example 1, a hydrophobic chromatographic treatment was carried out, and a test for examining pyrogen in the antithrombin-III-containing fraction before and after that was performed twice (the first sample and the first sample, respectively). Referred to as a second sample). In addition, the test method was based on the pyrogenic substance test method listed in the Japanese Pharmacopoeia. Table 1 shows the results.

As shown in Table 1 above, it was found that the total temperature was lowered and the pyrogen was substantially removed by the hydrophobic chromatographic treatment.

(2) Removal of contaminating proteins (a) Specific activity A hydrophobic chromatographic treatment was carried out according to Example 1, and the total protein amount and antithrombin-III activity in the antithrombin-III-containing fraction before and after the treatment were measured. The specific activity was calculated. The total protein amount was measured by the Kjeldahl method. The antithrombin-III activity (AT-III activity) was determined by reacting thrombin with a sample at 28 ° C. for 5 minutes and measuring the extent of prolongation of coagulation time when fibrin was added thereto.
The titer was calculated from a calibration curve prepared in advance. However, one unit corresponds to the amount of antithrombin-III contained in 1 ml of normal human plasma. Table 2 shows the obtained results.

As shown in Table 2, an increase in AT-III activity and specific activity was observed by the hydrophobic chromatography treatment, and the specific activity was particularly increased from 4.9 (units / mg protein) to 6.4 (units / mg protein). .

(B) Removal of contaminating plasma proteins Hydrophobic chromatography treatment was performed according to Example 1, and the presence or absence of contaminating plasma proteins in the antithrombin-III-containing fraction before and after the treatment was examined by the Ouchterlony method. Table 3 shows the results.

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

Carrier: TSK gel G3000SW-XL (manufactured by Tosoh Corporation) Eluent: 0.1 M phosphate buffer containing 0.2 M sodium chloride (pH
6.8) Detection method: absorbance at 280 nm As shown in FIG. 1, the fraction before treatment shows bimodality. Among these, the polymer fraction is considered to be based on the heat-denatured protein in view of the elution pattern before the heat treatment. It was found that this high molecular fraction disappeared by the hydrophobic chromatography treatment and became monomodal.

(D) Removal of Contaminant Plasma Proteins An antithrombin-III-containing fraction obtained by performing hydrophobic chromatography treatment and anion exchanger treatment according to Example 4, and antithrombin-III before the above hydrophobic chromatography treatment. The contained aqueous solution was examined for the presence or absence of contaminated plasma proteins by the Ouchterlony method. It was carried out at a protein concentration of 100 mg / ml. Table 4 shows the results.

Experimental Example 2 The antithrombin-III freeze-dried preparation obtained in Example 1 was dissolved in distilled water for injection, and 4,000 units / kg equivalent amount was administered to 5 mice through the tail vein, and observation was continued for 7 days. However, no abnormality was found. The same solution was administered to rabbits at 5,000 units / kg and observed for 24 hours, but no abnormal body temperature was observed.

[Brief description of the drawings]

FIG. 1 shows gel filtration elution patterns of antithrombin-III-containing fractions before and after hydrophobic chromatography.

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Claims (4)

(57) [Claims] 1. Antithrombin-II derived from human plasma
The aqueous solution containing I is subjected to heat treatment under conditions sufficient to inactivate viruses that may be contaminated, and then treated with an insoluble carrier having a hydrophobic group as a ligand to adsorb the contaminants to the carrier. An antithrombin-III preparation in which a virus is inactivated and which is substantially free of at least a pyrogen as a contaminant and a heat-denatured protein produced by the inactivation treatment, which comprises recovering an unadsorbed fraction. 2. The antithrombin-III according to claim 1, which is substantially free of pyrogens, heat-denatured proteins and plasma proteins other than antithrombin-III as contaminants.
Formulation. 3. The claim (1) or claim (2), which is substantially free of pyrogen, heat-denatured protein, albumin, haptoglobin, α ₂ -macroglobulin, α ₁ -antitrypsin and IgA as contaminants. Antithrombin-III formulation. 4. The antithrombin-III preparation according to claim 1, wherein the hydrophobic group of the insoluble carrier having a hydrophobic group as a ligand is an alkyl group.