JPH1149799A - Antithrombin-iii perpetration and purification of antithrombin-iii - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an antithrombin(AT)-III preparation containing highly purified AT-III, and to provide a method for purifying the antithrombin III. SOLUTION: This antithrombin-III (AT-III) preparation is characterized by substantially not containing a polymer. The method for purifying AT-III comprises inserting a process for treating AT-III with a metal-chelating resin into a process for purifying AT-III. The method enables to effectively remove impure proteins contained in an AT-III raw material solution, deaturated AT-III by-produced by thermal treatments and polymers, and to give highly safe AT-III. The treatment process is simple, suitable for its production in a large amount, and extremely useful as an industrial method.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to antithrombin-I
And a method for purifying antithrombin-III.
More specifically, the present invention relates to an antithrombin-III preparation used for treating various diseases caused by a decrease in antithrombin-III, and a method for purifying the antithrombin-III.

[0002]

2. Description of the Related Art Antithrombin-III (hereinafter referred to as AT-III)
Is a type of glycoprotein belonging to α2 globulin present in plasma and has a molecular weight of 65,000 to 68,000.
It has a protease inhibitory activity and strongly inhibits the clotting activity of thrombin. In addition, it has an inhibitory effect on not only thrombin but also other coagulation factors such as activated factor X and activated IX factor. 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. AT-III having such action
Is used for correction of hypercoagulability, specifically for the treatment of various diseases caused by generalized vascular abnormalities (DIC) and decreased AT-III.

[0003]

By the way, it is known that AT-III has an affinity for heparin, and many purification methods using an immobilized heparin carrier have been reported by utilizing its characteristics. (See, for example,
No. 23896). In addition, since AT-III is a protein present in plasma, it is essential to incorporate a step of inactivating viruses that may be contaminated when used as a therapeutic drug. However, this virus inactivation process also imposes a load on proteins, and inactive AT
-III, a method in which denatured proteins such as the formation of polymers are generated, and the immobilized heparin treatment is performed again (JP-A-63-2389).
No. 6) or a hydrophobic chromatographic treatment (Japanese Unexamined Patent Publication No. 1-275).
No. 600) discloses a method for removing impurities and the like. However, in the treatment with immobilized heparin, the activity recovery rate is low even when treated alone, and in the treatment with hydrophobic chromatography, proteins such as prealbumin, transferrin, and IgG may remain as contaminating proteins. Incorporating the purification treatment again reduces the activity recovery rate. Therefore, the present inventors have conducted various studies in view of these circumstances, and as a result, while maintaining the activity of AT-III,
The inventors have found a method for removing impurity proteins such as inactive AT-III in a single treatment step, and have completed the present invention. That is, the present invention provides a novel method for purifying AT-III and an AT-III preparation containing highly purified AT-III.

[0004]

Means for Solving the Problems The gist of the present invention made to solve the above-mentioned problems is to provide an antithrombin-III preparation substantially free of a polymer; AT-III purification method, characterized by incorporating a step of treating with a metal chelate resin; Purification method as described above using copper ions as metal ions of the metal chelate resin; Heat-treated antithrombin-III solution with a metal chelate resin An AT-III preparation containing AT-III purified by any one of the above-mentioned.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention has the above constitution, and is characterized in that the AT-III preparation of the present invention contains substantially no polymer. Here, substantially not containing a polymer,
The polymer content with respect to AT-III in the preparation is 0.4% or less,
It is preferably 0.1% or less, more preferably 0%. The AT-III preparation of the present invention can be prepared by any method as long as it can achieve substantially no polymer in the preparation.
Can be achieved by incorporating a step of treating with a metal chelate resin in the purification step.

The method for purifying AT-III of the present invention is characterized by the above-mentioned method, and comprises a step of treating with a metal chelate resin in the step of purifying AT-III. More specifically, in the step of purifying AT-III from an aqueous solution containing AT-III, the aqueous solution containing AT-III is brought into contact with a metal chelate resin, and a non-adsorbed fraction is collected to obtain AT-I.
II. AT-I used in the present invention
As an aqueous solution containing II (hereinafter referred to as an AT-III-containing aqueous solution), a mixture of plasma proteins, for example, a fraction obtained by the cold ethanol method of corn (fraction, Fr.)
Illustrative examples include fractions obtained after recovering blood coagulation factor VIII from citrate-containing plasma, such as II + III supernatant and IV. However, the starting material is not particularly limited to plasma, and may be obtained, for example, by techniques such as genetic engineering. AT-III can be incorporated into a method for purifying AT-III from a solution containing AT-III.

The protein concentration of the aqueous solution containing AT-III is as follows:
About 0.1 to 5 (W / V)% is exemplified. AT-III
It is preferable to use a partially purified aqueous solution, for example, an AT-III-containing aqueous solution that has been preliminarily purified using the above-described heparin-immobilized carrier. Furthermore, A
The T-III-containing aqueous solution is subjected to a heat treatment (about 50 to 70 ° C., about 5 to 30 hours) before being subjected to the metal chelate resin treatment of the present invention.
It is preferable to perform a virus inactivation treatment in advance. When the AT-III-containing aqueous solution contains a chelating agent such as citric acid, the AT-III-containing aqueous solution from which the chelating agent has been removed by dialysis treatment or the like is used in order to prevent elution of metal ions from the metal chelating resin. It is preferred to use.

The metal chelate resin used in the present invention comprises:
The chelate resin is formed by chelate-bonding a metal ion. As the chelate resin, a commercially available chelate resin (for example, iminodiacetic acid type) can be used. More specifically, Chelating-Sepharose F.R. F.
(Pharmacia), AF-Chelate Toyopearl 65
0M (manufactured by Tosoh Corporation) and the like. The metal ion to be bound to the above chelate resin is not particularly limited as long as it is a metal ion effective for separating AT-III, and examples thereof include copper ion, nickel ion, iron ion, and cobalt ion, and in particular, purification efficiency and the like. In view of the above, it is preferable to use copper ions.

The method of the present invention is carried out by contacting an aqueous solution containing AT-III with a metal chelate resin. As the method, any of a column method and a batch method can be used, but it is preferable to use a column method from the viewpoint of purification degree, purification efficiency and the like. The contact conditions are as follows: pH 6 or more and less than 8, preferably pH 6.5 to 7.7, salt concentration 0 to 1
M, preferably about 0.05 to 0.5M.
Preferred solvents include 0.1-M phosphate buffer containing 0.05-0.5M sodium chloride (pH 7.0-7.5).
And the like, but may not contain sodium chloride. Further, in order to enhance the separability between the metal chelate resin and AT-III, an antagonist such as glycine or imidazole may be added to the above-mentioned buffer.
It is preferable to add an amount of 0 mM or less.

When the purification method of the present invention is carried out by a column method,
The AT-III-containing aqueous solution adjusted to the above conditions is packed in a metal chelate resin column equilibrated under the same conditions,
It is carried out by developing with the buffer at room temperature and collecting the non-adsorbed fraction. The amount of the metal chelate resin used can be appropriately adjusted depending on the impurity content, the AT-III content, etc. in the AT-III-containing aqueous solution.
-III-containing aqueous solution 4mL, metal chelate resin 1mL
Degree is used. The elution rate from the column can be appropriately adjusted according to the metal chelate resin filling amount, column diameter, and the like. When the batch method is used, the AT-III-containing aqueous solution adjusted to the above conditions is brought into contact with a metal chelate resin equilibrated under the same conditions at a temperature of 2 ° C to room temperature, followed by filtration, centrifugation, etc. This is performed by collecting the supernatant by a means. The contact time can be appropriately adjusted according to the impurity content, AT-III content and the like in the AT-III-containing aqueous solution, but generally, the contact may be performed for about 10 minutes to 1 hour. In some cases, metal ions may leak from the metal chelate resin. However, the metal ions are treated with a chelate resin or an ion exchanger to which no metal ions are bound, and further treated with dialysis after addition of a chelating agent such as EDTA. It can be easily removed by a suitable method.

AT-III purified by the purification method of the present invention
Has substantially no polymer, impure protein, denatured AT-III, etc., and can obtain highly purified AT-III in one step. In addition, A purified by the method of the present invention
T-III may be further purified, if necessary, by a conventional purification means, for example, treatment with an anion exchanger, treatment with an insoluble carrier having a hydrophobic group as a ligand, or the like.

AT-III purified by the method of the present invention
Is formulated according to a conventional formulation method. Examples of the preparation include an aqueous solution, a suspension, and a lyophilized preparation. These preparations are prepared by appropriately mixing pharmaceutically acceptable additives (diluent, isotonic agent, surfactant, etc.) This can be done by conventional means described above. The preparation is used as an injection for intravenous injection, subcutaneous injection and the like, and the lyophilized preparation is dissolved in distilled water for injection or the like before use.

[0013]

Since the AT-III preparation of the present invention does not substantially contain a polymer, it can provide a preparation having extremely high safety. According to the purification method of the present invention, the AT
Impurity proteins contained in the -III-containing aqueous solution, denatured AT-III and polymers produced by heat treatment can be effectively removed, and highly safe AT-III can be obtained. Moreover, since the treatment process is simple and suitable for mass production, it is extremely useful as an industrial production method.

[0014]

EXAMPLES The present invention will be described in more detail with reference to examples, but the present invention is not limited thereto. The materials and methods used in the following experiments are as follows. Materials and Methods (1) Fr. II + III supernatant and Fr. A purified solution of the purified heparin-immobilized column derived from IV was heated at 60 ° C. for 10 hours with 5% Na citrate / 2M NaCl / 30% sorbitol, pH 7.8 for virus inactivation treatment. (2) Chromatographic carrier for purification Chelating-Sepharose F.R. F. (Manufactured by Pharmacia), AF-chelate-
Toyo Pearl 650M (manufactured by Tosoh Corporation) was used.

(3) Measurement of AT-III activity The measurement was performed by a synthetic substrate method using S-2238. S-2
The 238 solution, the thrombin solution, the standard AT-III solution, the reaction terminating solution, and the like were from Test Team AT-III-2 Kit (Daiichi Kagaku). 100 μl of the thrombin solution was added to 50 μl of the sample adjusted to 1 to 10 mU / mL buffer, and the mixture was incubated at 37 ° C. for 5 minutes. Next, substrate liquid 1
After mixing 00 μl and incubating for exactly 5 minutes, the reaction was stopped by adding 1000 μl of a reaction stop solution.
The absorbance of the solution at 405 nm was measured, and the AT-III activity of the sample was determined from a standard line prepared with the standard AT-III.

(4) Gel filtration HPLC analysis G3000SW _xL (φ7.8 with System Gold (manufactured by Beckman))
mm × 30 cm, manufactured by Tosoh Corporation)
Sodium phosphate, 0.3 M NaCl, pH 7.0 was used as a developing solution. The flow rate was 0.7 mL / min, and all peaks were detected at 280 nm absorption. (5) Ouchterlony analysis The sample was appropriately concentrated so that the absorbance at 280 nm became about 30, and the analysis was performed according to a conventional method. The antibodies used are described below. Anti-human ceruloplasmin (Hoechst N-antiserum ceruloplasmin), anti-human transferrin (Hoechst N
Anti-serum transferrin), anti-human albumin (Hoechst N-antiserum albumin), anti-human prealbumin (Hoechst N-antiserum prealbumin), anti-human haptoglobin (DAKO A030), anti-human α2-macroglobulin (DAKO A033) , Anti-human α1-antitrypsin (DAKO A012), anti-human IgA
(DAKOA02621), anti-human IgG (DAKO
A04231), anti-human IgM (Hoechst anti-IgM (μ
Chain) rabbit serum), anti-human prothrombin (DAKO)
A325), anti-human fibrinogen (DAKO A0)
80).

Example 1 Chelating Sepharose F. AT-
III purified chelating-Sepharose F. F. In a 1.5 mL column (φ0.9 × 2.3 cm), CuSO ₄ .5H ₂ O dissolved at 10 mg / mL in water was equibed (1.5 m / mL).
L) Added to bind metal and washed with 2 beds (3 mL) of water. Next, 0.1 M sodium phosphate, 0.1.
The column was equilibrated with a 3-bed flow of 5M NaCl, pH 8.0. Using PD-10 on these columns,
Heat-treated AT-III concentrate (A
280 = 4.20) was added in an amount of 3 mL, and 1.5 mL / Fr was added.
The eluted fraction was collected with. After that, pH 7.5, 7.0,
6.5 of 0.1 M sodium phosphate, 0.5 M NaC
1 was added for each of three beds (4.5 mL).
The eluted fraction was collected at 5 mL / Fr. The chelate column after use was a 2-bed volume of 50 mM EDTA.2.
After removing the bound metal with Na, the water was regenerated by flowing 3 bed volumes of water.

FIG. 1 shows the results. As shown in FIG. 1, in the copper chelate column, most proteins were adsorbed at pH 8.0 and eluted at pH 7.5 or lower. Table 1 shows the results of GPC-HPLC analysis of the eluate from the copper chelate column. As shown in Table 1, the purity of the non-denatured (intact) AT-III was 88.0 before the addition of the copper chelate column.
%, But the Fr eluted from the column became 100% pure at all pH values. That is, denatured AT-III and impure proteins can be removed very efficiently by passing the present chromatogram below pH 8, preferably below pH 7.5, and these impurities are retained on the column even at pH 6.5. I knew it was there.

[0019]

[Table 1]

Embodiment 2Examination of the effect of salt concentration  To study the effect of fluctuations in salt concentration of the chromatographic buffer,
0.1 M sodium phosphate (pH 7.5) or 0.1
0.1M NaC in M sodium phosphate (pH 7.5)
Use buffer solution containing 1 or 0.5 M NaCl
And heat-treated AT-III concentrate
Mato behavior was studied. That is, the heat-treated AT-III concentrate
Solvent exchange of the condensed liquid with each buffer using PD-10,
Adjust to about 5 and equilibrate each 5 mL with each buffer
1mL copper chelate column (φ0.8 × 2cm)
Was added. Pool the first 3 mL of pass solution and
Each 1 mL was collected. After that, the column is buffered with each buffer.
After washing, the eluate at the time of washing was separated by 1 mL. Elution
The turns are shown in FIG. In addition, intact A of each eluted fraction
Table 2 shows the results of measuring the purity of T-III by GPC-HPLC.
Show.

[0021]

[Table 2]

As shown in FIG. 2, there was no significant difference in the elution patterns between when NaCl was not contained and when 0.1 M and 0.5 M NaCl were contained. Further, as shown in Table 2, the purity of intact AT-III in the eluate was 100.0% in each case. Therefore, it was clarified that the variation in the salt concentration of the buffer had little effect on the chromatographic behavior. The chromatographic behavior due to pH fluctuation was examined using a 0.1 M sodium phosphate buffer (containing 0.1 M NaCl) whose pH was adjusted to 7.3, 7.5 or 7.7. It was found that the variation had little effect on the chromatographic behavior. Also,
0.1 M sodium phosphate buffer (0.1 M NaCl
(PH 7.5), the chromatographic behavior at 4 ° C. and room temperature was examined, and it was found that temperature fluctuations in this range had little effect on the chromatographic behavior.

Example 3 Confirmation of Purification Degree in Scale-Up Experiment and Evaluation of Purification Yield Heat-treated AT-III concentrate was treated with PD-10 at 0.1 M
The buffer was exchanged with sodium phosphate, 0.1 M NaCl, pH 7.5 (A280 = 4.05), and the solution was applied to a 5 mL copper-chelated Toyopearl 650M column (φ1.5 × 2.8 cm) equilibrated with the same buffer. 25 mL was added. One half of the column bed, 2.5 mL, was discarded. Thereafter, the eluate obtained at the same time as the sample addition and the washing solution for two column beds with the equilibration buffer were pooled together (32.5 m).
L) and purified AT-III. Table 3 shows A280 and AT-III
The activity recovery rate is shown in FIG. 3 by GPC-HPL before and after chromatography.
The C pattern was shown. As shown in Table 3, the recovery rate was A280.
Was 80.3% and AT-III activity was 89.6%. As shown in FIG. 3, the column eluate in GPC-HPLC was a single peak of intact AT-III,
Polymer, modified AT-III, and other impure proteins were not included.

Furthermore, the results of the otacrony analysis of the above sample show that all the expected impure proteins (ie ceruloplasmin, transferrin, albumin, prealbumin,
Haploglobulin, α2-macroglobulin, α1-antitrypsin, IgA, IgG, IgM, prothrombin, fibrinogen) were not detected (see FIG. 4). As described above, it was found that all the impurity proteins, polymers, and denatured AT-III can be removed by the copper chelate Toyopearl 650M chromatograph at pH 7.5.

[0025]

[Table 3]

Example 4 Removal of Leaked Copper from a Copper Chelate Resin Column In copper chelate column chromatography, it is generally known that a small amount of copper bound to the column leaks with protein adsorption. As a method for removing the leaked copper, a method of connecting a small chelate resin column with no metal bond to the metal chelate resin column is recommended. Therefore, the effect of removing the leaked copper by the present method was examined as follows. 0.1M
Fr. Dissolved in sodium phosphate, 0.1 M NaCl, pH 7.5. Heat treatment AT-III derived from II + III supernatant
20 mL of the concentrated solution (A280 = 4.96) was added to a copper chelate Toyopearl column (φ1.5 ×
(2.8 cm, 5 mL) and the non-adsorbed fraction was pooled (27.5 mL, A280 = 3.18). Two of them
A metal-free chelate Toyopearl 650M column (φ0.8 × 2.0 cm, 1 m
L) and the non-adsorbed fractions were pooled (25 mL, A
280 = 2.63). Table 4 shows the results of measuring the copper content in each step.

[0027]

[Table 4]

As shown in Table 4, the copper chelate column pass fraction showed a leakage of 10300 ppb of copper. However, passing through a non-metal-bound chelate Toyopearl 650M column gave a control level of 15 ppb (13 ppb).
pb), indicating that even if copper leaks, it can be sufficiently removed. The GPC-HPLC pattern of the starting material and the metal-free chelate column eluate is shown in FIG. As shown in FIG. It was found that the raw material derived from the II + III supernatant could be purified by the copper chelate column without any clear peak other than intact AT-III.
The purity determined from the peak area was 99.9%.

Example 5 Purification on a column connecting a copper chelate and a metal-free Toyopearl
Taking into account the efficiency and automation of the yield chromatographic process, a study was conducted on a column connected with a copper chelate and a metal-free chelate column. Fr. II + III supernatant or Fr. After the heat treatment derived from IV, the AT-III concentrate was added to 0.1 M sodium phosphate, 0.1 M NaCl, pH 7.5, and then heated to 160
It was dialyzed 0-fold, concentrated and diluted with Centriprep-30, and further dialyzed 100-fold (total 160,000-fold) with the same buffer (Fr. IV: A280 = 5.23, Fr. II + I).
II supernatant: A280 = 5.50). Centriprep-
The reason for increasing the dialysis multiple at 30 is that, in 1600 dialysis, the leakage of copper increases due to the remaining citric acid. 20 mL of these solutions were equilibrated with the same buffer.
It was added to a series connection column of mL (φ1.5 × 2.8 cm) copper chelate Toyopearl and 1 mL (φ0.8 × 2.0 cm) metal-free chelate Toyopearl. Half of the total column volume of the solution eluted at the same time as sample addition was discarded, and the fraction eluted afterwards and the fraction washed with 10 mL corresponding to the 2-bed volume of the copper chelate column were pooled. As shown in Table 5, the purification yield was as follows. II +
III supernatant and Fr. A good recovery rate of 90% or more was obtained for both the raw materials derived from IV. The specific activity is also 1
It was around 0U / A280, which was equivalent to the current formulation.

[0030]

[Table 5]

Example 6 Preparation of AT-III preparation The purified AT-III obtained in Example 5 was desalted and concentrated,
After filtration through M membrane, AT-III 50 units / mL, mannitol 2% (w / v), sodium citrate 0.52%
(W / v), sodium chloride 0.5% (w / v), pH
Each component was added so as to be 7.2 to 7.5, and 1
After adjusting the pH with N sodium hydroxide, the solution was sterilized and filtered through a sterilized Millipore filter, dispensed in 500 units, and freeze-dried to obtain a freeze-dried preparation.

Example 7 Comparison of Preparation of the Present Invention with Commercial Preparation A comparative experiment was conducted between the AT-III preparation of the present invention (hereinafter referred to as the preparation of the present invention) and a commercially available AT-III preparation. Commercial AT-III
As a formulation, use a formulation sold by three companies,
These are referred to as a commercial preparation N, a commercial preparation P and a commercial preparation B, respectively. The test items compared were AT-III activity, gel filtration: high performance liquid chromatography, cellulose acetate membrane electrophoresis, immunoelectrophoresis, octalony method, two-dimensional immunoelectrophoresis, NON SDS polyacrylamide gel electrophoresis, SDS polyacrylamide gel. Electrophoresis. Hereinafter, the description will be made in the order of the experimental material, the experimental method, and the experimental result.

Experimental Material Samples (1) Sample Solution A commercially available formulation was dissolved in 10 mL / vial using the attached dissolving solution to obtain a sample solution. The final bulk liquid obtained in Example 5 was used for the preparation of the present invention. (2) Human pool plasma Human pool plasma derived from blood donation was used.

Measurement of AT-III activity The substrate solution (S-2238), the thrombin solution, the AT-III standard solution and the reaction stop solution were prepared using Test Team ATIII.
Two kits were used.

Gel filtration: High performance liquid chromatography (1) column TSK GEL G3000SWXL (Y0243) was used. (2) Development buffer: 0.05M PB, 0.3M NaC
1, pH 7.0 17.9 g of disodium hydrogen phosphate.12 water and 7.8 g of sodium dihydrogen phosphate.2 water were added to purified water 1,00
Dissolved in 0 mL. Then, using a pH meter, p
Both were mixed and adjusted so that H7.0 ± 0.1. (3) Molecular weight marker A molecular weight marker for high performance liquid chromatography manufactured by Oriental Yeast was used. The proteins contained in the molecular weight markers and the molecular weight of each protein are as follows. Glutamate dehydrogenase W. 290,000 lactate dehydrogenase W. 142,000 enolase W. 67,000 adenylate kinase W. 32,000 cytochrome CM. W. 12,400 (4) System Performed by System Gold (manufactured by Beckman).

Cellulose acetate membrane electrophoresis (1) Cellulose acetate membrane Separax SP (Fuji Film Co., Ltd.) was used. (2) Electrophoresis tank Model 51156 (manufactured by Gelman) was used. (3) Buffer for electrophoresis One bag of Veronal buffer (manufactured by Joko Co., Ltd.) is 500 m
L was used after dissolving in purified water. (4) Staining solution Ponceau S (manufactured by Germanic Science) was used. (5) Decolorization solution Purified water was added to 3 mL of glacial acetic acid to make 100 mL. (6) Power supply ATTO AE3105 was used.

[0037] the immune electrophoresis (1) a 1.2% agarose gel low EEO agarose (SIGMA Co., Ltd.) 1.2g 10
A solution prepared by adding 0 mL of veronal buffer and dissolving in a hot water bath (90 ° C. or higher) was used. (2) Buffer for electrophoresis One bag of Veronal buffer (manufactured by Joko Co., Ltd.) is 500 m
L was used after dissolving in purified water. (3) Antibody Anti-human serum (rabbit) DAKO Anti-human antithrombin III (rabbit) DAKO

Ouchterlony method (binary immunodiffusion method) (1) 1.2% agarose gel 1.2 g of low EEO agarose (SIGMA)
A solution prepared by adding 0 mL of veronal buffer and dissolving in a hot water bath (90 ° C. or higher) was used. (2) Antibodies The following antibodies were used. Anti-human IgG (rabbit, DAK
O), anti-human IgA (rabbit, manufactured by DAKO), anti-human IgM (rabbit, manufactured by Bering), anti-human albumin (rabbit, manufactured by Bering), anti-human prealbumin (rabbit, manufactured by Bering), Anti-human α2-macroglobulin (rabbit, manufactured by Bering), anti-human α1-antitrypsin (manufactured by DAKO), anti-human transferrin (manufactured by Bering), anti-human haptoglobin (manufactured by Bering), anti-human ceruloplasmin (manufactured by Bering) ), Anti-human retinol binding protein (DAKO), anti-human fibrinogen (DAKO)

Two-dimensional immunoelectrophoresis (1) 1.2% agarose gel Low EEO agarose (manufactured by SIGMA)
A solution prepared by adding 0 mL of veronal buffer and dissolving in a hot water bath (90 ° C. or higher) was used. (2) Sample volume: 10 μL (A280 nm = 0.1 ± 0.0
1) (3) Buffer for electrophoresis 500g of Veronal buffer (manufactured by Joko Co., Ltd.)
L was used after dissolving in purified water. (4) Antibody: anti-human antithrombin III (rabbit) D
AKO (5) Heparin: Heparin sodium injection GCC

NON SDS polyacrylamide gel
Electrophoresis (1) Gel Multigel 10/20 (manufactured by Daiichi Kagaku) was used.
Polyacrylamide gel, slab type: width 90 mm, length: thickness 1 m adjusted between two glass plates of 84 mm
A gel obtained by polymerizing m acrylamide, in which the concentration of acrylamide is continuously gradient from 10% to 20%. Has a groove for sample injection. (2) Electrophoresis buffer (Tris / glycine buffer) Trishydroxymethylaminomethane 103 g and 14.04 g of glycine were dissolved in purified water to make 1000 mL. (3) Sample buffer (Tris / glycerin buffer) 1.51 g of trishydroxymethylaminomethane, 12.7 g of glycerin, 0.01 g of bromophenol blue
Was dissolved in 90 mL of purified water, and pH 6.8 was added with 1N hydrochloric acid TS.
, And purified water was added to make 100 mL. (4) Staining solution 2.5 g of Coomassie Blue R-250 was dissolved in a mixed solution of purified water, methanol and glacial acetic acid (5: 4: 1), and 1,000 m
L. (5) Decolorizing solution (10% glacial acetic acid, 10% methanol) 10 mL of glacial acetic acid and 10 mL of methanol were added to purified water to make 100 mL.

SDS polyacrylamide gel electrophoresis (1) Gel Multigel 10/20 (manufactured by Daiichi Kagaku) was used. (2) Running buffer (Tris / glycine / SDS buffer) Dissolve 3.03 g of trishydroxymethylaminomethane, 14.04 g of glycine and 2.0 g of SDS in purified water,
The volume was 1,000 mL. (3) Tris / glycerin / SDS buffer 1.51 g of trishydroxymethylaminomethane, 0.014 g of glycerin, 0.00 bromophenol blue
5 g and SDS (4 g) were dissolved in purified water (90 mL), adjusted to pH 6.8 with a 1N hydrochloric acid test solution, and purified water was added thereto for 100 m.
L. (4) Staining solution 2.5 g of Coomassie Blue R-250 was dissolved in a mixed solution of purified water, methanol and glacial acetic acid (5: 4: 1), and 1,000 m
L. (5) Decolorizing solution (10% glacial acetic acid, 10% methanol) Purified water was added to 10 mL of glacial acetic acid and 10 mL of methanol to make 100 mL. (6) Molecular weight marker A low molecular weight marker (manufactured by Pharmacia) was used. The proteins contained in the molecular weight markers and the molecular weights of the proteins are as follows. Phosphorylase b W. 94,000 albumin W. 67,000 ovalbumin W. 43,000 carbonic anhydrase W. 30,000 trypsin inhibitor W. 20,100 α-lactalbumin W. 14,000

Experimental Method Appearance Visual observation was carried out according to the method for testing insoluble foreign substances in injections of the Japanese Pharmacopoeia. Measurement of AT-III activity The measurement was performed by the following method. 10 mU of standard with dilution buffer
/ ML, 5 mU / mL, 2.5 mU / mL and 1.25
It was adjusted to an accurate dilution of mU / mL and used as an AT-III standard solution. In addition, the sample is diluted with a dilution buffer to 10 to 2.5 mU / mL.
To obtain a sample solution. 50 μL each of the AT-III standard solution, sample solution and dilution buffer were collected in a plastic test tube. Next, 100 μL of the thrombin solution was added to each test tube, and heated at 37 ° C. for exactly 5 minutes.
After 100 μL of the substrate solution was added to each test tube, and the mixture was further heated at 37 ° C. for exactly 5 minutes, 1 mL of a reaction stop solution was added to stop the reaction. The absorbance (A405n) at a wavelength of 405 nm of the reaction solution obtained from each standard solution, sample solution and dilution buffer
m) was measured using purified water as a control. After plotting A405 nm on the ordinate and AT-III activity on the abscissa from the absorbance of the standard solution and preparing a standard curve, the AT-III activity was calculated from the A405 nm value of the sample solution by the following formula. Formula AT-III activity (U / mL) = (A405n of dilution buffer)
m-A405 nm of sample solution-intercept of regression equation) / (slope of regression equation) × dilution factor Specific activity calculation = AT-III activity / absorbance of sample at wavelength 280 nm (A280 nm)

Gel Filtration: High-Performance Liquid Chromatography (1) Operating Procedure of Equipment The operation manual was followed. (2) Analysis conditions Samples were adjusted to A280 nm = 5 ± 1 with physiological saline and analyzed under the following conditions. 1. Column: TSK gel G3000SWXL 2. 2. Sample injection volume: 20 μL Flow rate: 0.7 mL / min Development buffer: 50 mM containing 0.3 M NaCl
4. Phosphate buffer (pH 7.0) Analysis time: 25 minutes 6. Peak detection: A280 nm (3) Molecular weight marker A molecular weight marker for high performance liquid chromatography manufactured by Oriental Yeast Co. was used by dissolving 1 vial in 100 μL of purified water.

According to a conventional method of cellulose acetate membrane electrophoresis , the specimen was subjected to A280 nm = 5 ±
1 and 1 to 2 μL of the sample was applied to the Separax SP membrane. Next, the sample was electrophoresed for 25 minutes at a low voltage of 250 V using an electrophoresis tank (model 51156), stained with Ponceau S, and then decolorized with a decolorizing solution. Power supply is ATTO AE310
Using No. 5, the operating method followed the operating procedure manual. The electrophoresis pattern was analyzed using Appraise (digital densitometer), and the operation was performed according to the operation manual.

[0045] After the immunoelectrophoresis 1.2% agarose gel and dissolved in hot water bath (90 ° C. or higher), flushed with 7.5mL on a horizontal glass plate (7.5 × 5.0 cm), prepared agar plate did. Next, FIG.
Drill a sample hole (φ3 mm) with a punch, adjust the sample to A280 nm = 5 ± 1 with physiological saline, add 5 μL, and use a buffer for electrophoresis at a constant current (2 mA / cm width) for 120 min. Electrophoresed. After the electrophoresis, 300 μL of the antibody was added to each of the grooves (width 2 mm) for adding an antibody in the middle, and the mixture was allowed to react in a wet box at room temperature for 24 to 48 hours, and a generated sedimentation line was observed.

Ouchterlony method (binary immunodiffusion method) After dissolving 1.2% agarose gel in a hot water bath (90 ° C. or higher), 7.5 mL was placed on a horizontal glass plate (7.5 × 5.0 cm). It was poured to produce an agar plate. Next, FIG.
Drill a hole (φ3 mm) with a punch as described above, and in the center hole 10 μL of the sample concentrated to A280 nm = 30 ± 5 with an ultrafiltration membrane (10 kC), and in the outer hole about 35 μl of the antibody.
After the addition of L, the reaction was carried out at room temperature for 24 to 48 hours in a wet box, and the resulting sedimentation line was observed.

Two-dimensional immunoelectrophoresis 1.2% agarose gel was dissolved in a hot water bath (90 ° C. or higher), cooled to 50 to 60 ° C., and heparin (1,000
U / mL) was mixed at a ratio of 0.15 mL / agar 7.5 mL and poured on a horizontal glass plate (7.5 × 5.0 cm) to prepare a heparin agar plate. Next, a sample hole was made with a punch as shown in FIG.
= 0.1 ± 0.01), and electrophoresed for 120 min at a constant current (10 mA / plate) in an electrophoresis buffer. Next, a heparin agar plate (about 1 /
4), leaving 0.3 mL of anti-human AT-III / 6.0 agar
After mixing at a rate of 7.5 mL on a horizontal glass plate (7.5 ×
5.0 cm) to prepare an anti-human AT-III agar plate. Furthermore, after electrophoresis was performed at a constant current (10 mA / plate) for 120 min with a buffer for electrophoresis at right angles to the direction in which electricity was applied in one dimension, the reaction was allowed to proceed in a humid box at room temperature for 24 to 48 hours. AT-III, which has no affinity for -III and heparin, was observed.

Polyacrylamide gel electrophoresis (1) NON SDS polyacrylamide gel electrophoresis After diluting a sample to 10 units / mL with purified water, an equal volume of Tris / glycerin buffer was added to prepare a sample solution. 10 μL (0.05 unit) of this sample solution was placed in a sample groove of a polyacrylamide gel / slab type, and a tris / glycine buffer solution was used as an electrode buffer solution at a constant current of 20 mA for 90 mi.
Electrophoresed for n times. The molecular weight marker was prepared by dissolving one vial of a low molecular weight marker manufactured by Pharmacia in 100 μL of purified water and treating in the same manner as the sample solution. After the electrophoresis, the gel was taken out from the glass plate pair, immersed in the staining solution for 2 hours, washed with the decolorizing solution while changing the solution until the staining band became clear, and photographed. All operations were performed at room temperature. (2) SDS polyacrylamide gel electrophoresis (reduction) After diluting a sample to 10 units / mL with purified water, an equal volume of Tris / glycerin / SDS buffer and 1/10 volume of 2-mercaptoethanol were added. The sample was heated in a boiling water bath for minutes. Using Tris-Glycine-SDS buffer as the electrode buffer, the same operation was performed as in (1) below. (3) SDS polyacrylamide gel electrophoresis (non-reduced) After diluting the sample with purified water to 10 units / mL, add an equal volume of Tris / glycerin / SDS buffer and heat in a boiling water bath for 3 minutes. It was used as a sample solution. Tris Glycine
The SDS buffer was used as the electrode buffer, and the same operation was performed as in the following (1).

Experimental Results Appearance Preparation of the present invention: Colorless to pale yellow transparent liquid. Commercial formulation N: a colorless to pale yellow transparent liquid. Commercial formulation P: a colorless to pale yellow transparent liquid. Commercially available formulation B: a colorless to pale yellow transparent liquid.

Measurement of AT-III Activity Table 6 shows the measured AT-III activity and specific activity. As shown in Table 6, the amount of AT-III activity to be dispensed varies from company to company and is 476-594 U / vial. The specific activity of the preparation of the present invention was 9.7 U / A280.
For nm, the commercial formulation is 8.3-9.8 U / A280
A considerable width of nm was recognized.

Gel Filtration: High Performance Liquid Chromatography Table 7 shows the results of high performance liquid chromatography analysis (peak area ratio). As shown in Table 7, in the high performance liquid chromatography analysis, the preparation of the present invention showed almost a single peak, and the AT-I
The II fraction was also 99.9% or more, but in all of the commercial preparations, 0.5 to 0.9% of the dimer fraction was recognized. Thus, it was revealed that the commercial preparation contains a polymer.

Cellulose acetate membrane electrophoresis Table 8 shows the results of cellulose acetate membrane electrophoresis analysis. As shown in Table 8, in the cellulose acetate membrane electrophoresis, the preparation of the present invention and the commercial preparation N showed a single peak at the α2 globulin site, which is the migration site of AT-III. For B, 7.2 to 11.4% of the fraction shifted to the anode side from the position of α2 globulin was observed.

FIG. 9 shows the results of the immunoelectrophoresis experiment. As shown in FIG. 9, both the preparation of the present invention and the commercially available AT-III preparation showed one precipitation line at the α2 globulin site with respect to anti-human serum. Similarly, anti-human AT-III showed one sedimentation line at the α2 globulin site, and its position coincided with that of human pool plasma.

FIG. 10 shows the experimental results of the octalony method (binary immunodiffusion method) . 10 a to 10 d correspond to the preparation of the present invention, the preparation N, the preparation P and the preparation B, respectively. As shown in FIG. 10, each of the preparations of the present invention, the commercial preparation N and the commercial preparation P contained various antisera (1
No sedimentation line was observed for all of the two types),
In the commercial preparation B, albumin contamination was observed.

FIG. 11 shows the results of the two-dimensional immunoelectrophoresis experiment. 11 a to 11 d correspond to the preparation of the present invention, the preparation N, the preparation P and the preparation B, respectively. As shown in FIG. 11, the AT-III fractions of the preparations of the present invention, the commercial preparations N and the commercial preparations B all showed heparin affinity, whereas the commercial preparation P exhibited AT-III without heparin affinity. Fractions were found to be 12-14%.

FIG. 12 shows the results of the polyacrylamide gel electrophoresis experiment. 12A to 12C are respectively:
NON SDS polyacrylamide gel electrophoresis, SD
The patterns of S polyacrylamide gel electrophoresis (reduction) and SDS polyacrylamide gel electrophoresis (non-reduction) are shown. Each lane is composed of a commercial preparation B, a commercial preparation P, a commercial preparation N, a preparation of the present invention and a marker in order from the right side. is there. As shown in FIG. 12A, in the NON SDS polyacrylamide gel electrophoresis, the preparation of the present invention and the commercially available preparation N
Showed a single band that was slightly wider, while Commercial Formulation P and Commercial Formulation B showed two broad bands. In addition, as shown in FIGS. 12 b and c, in the SDS polyacrylamide gel electrophoresis, each of the preparations of the present invention, the commercial preparations N and the commercial preparations B shows a single band near 58 KD in both reduced and non-reduced forms. However, in the case of the commercial preparation P, one or two minor bands were observed in both reduced and non-reduced forms.

Conclusion As described above, the physicochemical properties of each company's AT-III preparation were analyzed, and the following results were obtained. 1) The properties of each formulation were colorless to pale yellow, transparent liquids. 2) The amount of AT-III activity to be dispensed differs according to each company.
6-594 U / vial, the formulation of the present invention is 9.7 U / A
For 280 nm, 8.3-9.
8 U / A 280 nm. 3) In the high performance liquid chromatography analysis, the preparation of the present invention showed almost a single peak, and the AT-III fraction was 99.9%.
As described above, all of the commercially available preparations contained a polymer fraction of 0.1%.
5-0.9% was recognized. 4) In the cellulose acetate membrane electrophoresis, the preparation of the present invention and the commercial preparation N showed a single peak at the α2 globulin site, which is the migration site of AT-III. The fraction shifted from the position to the anode side was found to be 7 to 11%. 5) In the immunoelectrophoresis analysis, each of the AT-III preparations showed one sedimentation line at the α2 globulin site for anti-human serum and anti-human AT-III, and the position coincided with that of human pool plasma. . 6) In the Ouchterlony method, no sedimentation line was observed for each of the twelve types of plasma components used in each of the preparation of the present invention, the commercial preparation N and the commercial preparation P, but albumin was mixed in the commercial preparation B. Admitted. 7) In the two-dimensional immunoelectrophoresis, the preparation of the present invention and the commercially available preparation N
All the AT-III fractions of the various preparations of the commercial preparation B showed affinity for heparin, but the commercial preparation P contained 12 to 14% of the AT-III fraction having no heparin affinity. 8) In the NON SDS polyacrylamide gel electrophoresis analysis, the preparation of the present invention and the commercial preparation N showed a slightly broad single band, whereas the commercial preparations P and B showed two broad bands. Was. 9) In the SDS polyacrylamide gel electrophoresis, each of the preparation of the present invention, the commercial preparation N and the commercial preparation B showed a single band around 58 KD for both reduced and non-reduced preparations. , Minor band is 1-2
This was recognized.

Based on the above results, the preparation of the present invention showed AT-I
Since the II activity is as high as 9.7 U / A 280 nm and the pattern of cellulose acetate membrane electrophoresis or high performance liquid chromatography shows a sharp symmetric peak, it is a relatively uniform protein, It was not substantially contained, and the purity as α2 globulin was 99.9% or more. Further, no impurities were detected by the analysis by the Ouchterlony method or the immunoelectrophoresis method, and all the AT-III fractions showed affinity for heparin by the two-dimensional immunoelectrophoresis analysis. Therefore, it became clear that the preparation of the present invention had extremely high purity as compared with the commercially available AT-III preparation. In contrast, the commercial preparation has a polymer fraction of 0.5 to
0.9% was found, and a formulation containing an impure protein was also found.

[Brief description of the drawings]

FIG. 1. AT-I from a chelating resin bound with copper ions
FIG. 2 is a view showing an elution pattern of II.

FIG. 2: AT-I from a chelating resin bound to copper ions
FIG. 9 is a diagram showing the effect of salt concentration on the elution pattern of II.

FIG. 3. AT-I using a chelating resin bound with copper ions
It is a figure which shows the HPLC-GPC pattern before and after purification of II.

[Fig. 4] AT-I using a chelating resin bound with copper ions
FIG. 4 is a diagram showing impurity analysis by the Ouchterlony method before and after purification of II.

FIG. 5: HPLC-G before and after purification of AT-III using a chelating resin bound to copper ions + a chelating resin not binding to a metal.
It is a figure showing a PC pattern.

FIG. 6 is a schematic diagram of an apparatus used for immunoelectrophoresis.

FIG. 7 is a schematic diagram of an apparatus used for the Ouchterlony method.

FIG. 8 is a schematic diagram of an apparatus used for two-dimensional electrophoresis.

FIG. 9 shows the results of analysis of the preparation of the present invention and a commercially available preparation by immunoelectrophoresis.

FIG. 10 is a graph showing the results of analysis of the preparation of the present invention and a commercially available preparation by the Ouchterlony method.

FIG. 11 is a view showing the results of analysis of the preparation of the present invention and a commercial preparation by two-dimensional electrophoresis.

FIG. 12 is a view showing the results of analysis of the preparation of the present invention and a commercially available preparation by polyacrylamide gel electrophoresis.

Claims (5)

[Claims] 1. An antithrombin-III preparation substantially free of a polymer. 2. A method for purifying antithrombin-III, wherein the step of purifying antithrombin-III includes a step of treating with a metal chelate resin. 3. The purification method according to claim 2, wherein copper ions are used as metal ions of the metal chelate resin. 4. Heat-treated antithrombin-III
3. The method of claim 2, further comprising the step of treating the solution with a metal chelate resin.
Purification method as described. 5. An antithrombin-III containing the antithrombin-III purified according to any one of claims 2 to 4.
Formulation.