KR19980042603A - Preparation method of antithrombin-III, purification method thereof and preparation containing the same - Google Patents

Abstract

(a) heating the antithrombin-III-containing aqueous solution in the presence of a stabilizer to maintain at least 85% of the antithrombin-III activity before heating after heating and to maintain the ratio of antithrombin-III monomer after heating to at least 95% before heating. And (b) treating the antithrombin-III containing aqueous solution with a metal chelate resin and recovering the purified antithrombin-III to produce antithrombin-III from an aqueous antithrombin-III containing solution. How to do this is described.

Description

Preparation method of antithrombin-III, purification method thereof and preparation containing same

The present invention relates to a method for preparing antithrombin-III, a method for purifying the same, and a preparation containing the same, in particular, antithrombin-III, which is used to treat various diseases caused by causes such as decreased use of antithrombin-III in the body. The preparation and purification method of this invention, and the preparation containing the produced and purified antithrombin-III are related.

Antithrombin-III (hereinafter referred to as AT-III) is present in plasma as a glycoprotein belonging to α ₂ globulin. AT-III has a molecular weight of 65,000 to 68,000, which acts to inhibit protease and strongly inhibits the coagulation action of thrombin.

AT-III also inhibits not only thrombin but also other coagulation factors such as activating factor X and activating factor IX. AT-III has also been reported to inhibit plasmin and trypsin.

Such inhibitory action generally proceeds at a faster rate in the presence of heparin.

Inhibitory AT-III has been used to treat abnormal overcoagulation, in particular Disseminated Intravascular Coagulation (DIC) and other diseases resulting from reduced use of AT-III.

In addition, many AT-III purification methods using immobilized heparin have been reported based on the affinity of heparin to AT-III (e.g., JP-A-63-23896; the JP-A is a published Japanese patent application without examination). Means).

AT-III is a protein present in plasma, so in order to use it as a therapeutic agent, it is necessary to inactivate a virus that may be contained therein.

As the virus inactivation treatment method, a liquid phase heat treatment method which is treated at 60 ° C. for 10 hours is known. However, AT-III is reported to have a 80% decrease in activity when stable at pH 6-8 and heat treated at 56 ° C. for 6 hours (J. Biol. Chem., 246: 3694 (1971)). On the other hand, there have been many reports that the activity of AT-III remains stable when subjected to liquid phase heat treatment at 60 DEG C for 10 hours in the presence of 14.7 w / v% sodium citrate (Thrombolsis Research, 22: 233 (1981). and J. Biol. Chem, 256: 12140 (1981)). However, when AT-III was heated in the liquid phase in the presence of 0.35 to 1.5 M (10.29 to 44.12 w / v%) sodium citrate, AT-III was observed to be denatured by immunoelectrophoresis assay (see Vox Sang, 48: 325 (1985).

In this virus inactivation process, denatured proteins such as inactivated AT-III, polymerized protein, etc. are produced, so that fixed heparin treatment is performed again (JP-A-63-23896) or hydrophobic chromatography treatment (JP -A-1-275600) A method for removing these impurities has been reported. However, when fixed heparin treatment is carried out, a single treatment alone has low activity recovery rate, and hydrophobic chromatography can leave contaminating proteins such as prialbumin, transferrin, IgG, etc. Activity recovery is reduced.

It is an object of the present invention to maintain a stable AT-III activity when AT-III is heated in a liquid phase and to reduce AT-III denaturation (structural modifications such as dimerization, polymerisation, etc.), which is suitable for use in medical treatment. It is to provide a method for producing AT-III.

Another object of the present invention is to provide a method for removing contaminating proteins such as inactivated AT-III in a single step while maintaining the activity of AT-III.

That is, the present invention relates to a method for preparing AT-III, a method for purifying the same, and a preparation containing the same.

In particular, these or other objects of the present invention are directed to stabilizers such that (a) even after heating, the activity of AT-III maintains at least 85% before heating and the AT-III monomer ratio after heating is at least 95% before heating. Heating at least an aqueous solution containing AT-III in the presence of at least one step; and (b) treating the AT-III containing aqueous solution with a metal chelate resin and recovering purified AT-III. It is realized by the method of manufacturing AT-III from -III containing aqueous solution.

These or other objects of the present invention are obtained by a pharmaceutical composition comprising AT-III or a pharmaceutically acceptable salt thereof prepared according to the above process as an active ingredient, optionally together with a pharmaceutically acceptable carrier.

1 is a graph showing the elution pattern of antithrombin (AT) -III from a copper ion-bonded chelate resin.

2 is a graph showing the effect of salt concentration on the elution pattern of AT-III from a copper ion-bonded chelate resin.

3 is a graph showing the HPLC-GPC pattern before and after AT-III purification by a copper ion-bonded chelate resin.

4 is a graph showing the results of impurity analysis by the Ouchterlony method performed before and after AT-III purification from a copper ion-bonded chelate resin.

FIG. 5 is a graph showing the HPLC-GPC pattern before and after AT-III purification by copper ion-bonded chelate resin + metal non-bonded chelate resin.

(I) starting material

The aqueous solution containing AT-III (hereinafter referred to as AT-III aqueous solution) includes the fraction obtained by Cohn cold ethanol method (Fr.) II + III supernatant and the remaining fraction from which blood coagulation factor VII was recovered from IV and citrate-containing plasma. Same mixtures of plasma proteins; However, the starting material is not particularly limited to the plasma content, so AT-III containing solutions obtained by techniques such as genetic engineering may be used in the AT-III purification method.

Purification methods for obtaining AT-III from blood or plasma are described in US Pat. No. 3,842,061 (JP-A-48-35017), US Pat. No. 4,340,589 (JP-A-54-95715), EP-A-339919 (JP-A-1 -275600) and EP-A-551084. For example, a method of further purifying the cold ethanol fractions IV-1, IV or II + III of the supernatant obtained by removing the cold precipitate from plasma is further purified by heparin affinity chromatography.

In addition, cell culture methods (e.g. EP-A-53165 (JP-W-57-500768); the JP-W means Japanese international patent applications published without examination) or genetic engineering techniques (e.g. EP-A- AT-III manufactured by 90505 (JP-A-58-162529) may be used.

(II) heat treatment

In the heat treatment of the present invention, the AT-III aqueous solution is heat-treated for an appropriate time at an appropriate temperature sufficient to inactivate contaminating viruses that may be contained in the aqueous solution. In particular, the heat treatment is carried out at 50 to 70 ° C., preferably at about 60 ° C. for 10 minutes to 20 hours, preferably about 10 hours. The pH of the solution is 5 to 10, preferably 6.5 to 9.0, more preferably adjusted to a suitable buffer. The AT-III aqueous solution contains AT-III at a rate of 10 to 500 units / ml, preferably 1 to 200 units / ml, more preferably 25 to 100 units / ml. In the present invention, 1 unit of AT-III means the activity of AT-III corresponding to the amount of AT-III contained in 1 ml of normal human plasma. The activity of AT-III is measured with an AT-III activity measurement kit (Test Team AT-III-2-Kit manufactured by Daiichi Pure Chemicals Co., Ltd.) using a synthetic substrate.

In the heat treatment, the total activity of AT-III after the heat treatment is maintained at 85% or more, preferably 95% or more of the total activity before the heat treatment, and the ratio of the AT-III monomer in the total amount of the AT-III aqueous solution is 95% or more, Preferably at least 99%.

The proportion of AT-III monomers was determined by high performance liquid chromatography (HPLC) using a G 3,000 SW _XL column (manufactured by Tosoh Corp.) previously equilibrated with 0.3 M sodium chloride-0.05 M phosphate buffer (pH 7.0). can do.

In order to meet such residual activity and monomer ratio conditions, the heat treatment is carried out in the presence of a stabilizer as described below.

(III) stabilizers

The stabilizer for heat treatment according to the invention is preferably selected from organic acids or salts thereof, saccharides and sodium chloride.

The organic acid used in the present invention is an aliphatic or aromatic, saturated or unsaturated monobasic acid (monocarboxylic acid), dibasic acid (dicarboxylic acid) or tribasic acid (tricarboxylic acid). Preferably, the organic acid has 2 to 10, preferably 2 to 6 carbon atoms. Monobasic acids include monobasic amino acids such as glycine, alanine, valine, leucine and isoleucine. Dibasic acids include saturated aliphatic dicarboxylic acids (e.g. oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid), saturated aliphatic dicarboxylic acids (e.g. maleic acid, fumaric acid), aromatic dicarboxylic acids (e.g. phthalic acid), dibasic Amino acids (eg aspartic acid, glutamic acid), dibasic hydroxy acids (eg malic acid, tartaric acid). Tribasic acids include tribasic hydroxy acids such as citric acid. Of these, malic acid, tartaric acid, maleic acid, aspartic acid and citric acid are preferable, and malic acid and citric acid are more preferable.

In addition, the organic acid may be used in the form of a salt. Organic salts include alkali metal salts such as sodium salts and potassium salts, alkaline earth metal salts such as calcium salts and organic salts such as ammonium salts. Sodium and calcium salts are preferred. Preferred organic acid salts used in the present invention are sodium malate and sodium citrate, with sodium citrate being most preferred.

The organic acid or salt thereof is added in the range of 3 to 20 w / v%, preferably 3 to 10 w / v% and most preferably 3 to 5 w / v%. If the amount added is 3w / v% or less, the stabilizing effect is reduced.

Organic acids or salts and saccharides thereof are used essentially. Sodium chloride may not be used. The addition of saccharide or sodium chloride reduces the amount of organic acid added, thereby inhibiting denaturation (dimerization and polymerization of AT-III), which inactivates AT-III activity. The saccharides include monosaccharides, disaccharides, sugar alcohols and amino sugars. Monosaccharides include glucose, fructose, galactose, mannose, arabinose and inositol. Disaccharides include saccharose, lactose and maltose. Sugar alcohols include mannitol, sorbitol and xylitol. Amino sugars include glucoseamine and N-acetyl-D-glucosamine, which is an amino sugar derivative. Among them, saccharose, lactose, sorbitol, inositol, maltose, N-acetyl-D-glucosamine and mannitol are preferable.

The saccharide is added in the range of 10 to 60 w / v%, more preferably 20 to 50 w / v%, most preferably 20 to 30 w / v%. If the amount added is less than 10w / v% stabilization effect is reduced, if more than 60w / v% stabilization increases but there is a disadvantage that must also stabilize the contaminating virus.

Sodium chloride is added in the range of 0.5 to 3.0M, more preferably 1.0 to 2.5M, most preferably 1.5 to 2.0M. If the added amount is 0.5M or less, the stabilization effect is reduced, if more than 3.0M stabilization is increased, but there is a disadvantage that the solubility of AT-III decreases.

However, according to the present invention, the amount of stabilizer added is only in the above range as long as the AT-III total activity after heat treatment maintains 85% or more of the total activity before heat treatment and the ratio of AT-III monomers after heat treatment maintains 95% or more. It is not limited.

(IV) tablets

The AT-III purification method of the present invention includes treating AT-III with a metal chelate resin in the AT-III purification step. In particular, the process consists of contacting the aqueous solution of AT-III with a metal chelate resin in the purification of AT-III from the aqueous solution containing AT-III and recovering the unadsorbed fraction.

The aqueous AT-III solution has a protein concentration of about 0.1-5 w / v%. It is also preferable to use an AT-III aqueous solution purified to some extent, preferably an AT-III aqueous solution pre-purified using the heparin-fixed carrier described above. In addition, the virus inactivation treatment of the AT-III aqueous solution is preferably performed by heat treatment (at 50 to 70 ° C. for 5 to 30 hours) or preferably before the solution is treated with the metal chelate resin according to the present invention. .

In the case where the AT-III aqueous solution contains a chelating agent such as citric acid, it is preferable to use the AT-III aqueous solution after removing the chelating agent by a method such as dialysis treatment to prevent the metal ions from eluting from the metal chelate resin.

Metal chelate resins used in the present invention are obtained by chelate-bonding metal ions to a chelate resin, and include commercially available chelate resins, such as iminodiacetic acid forms. Specific examples of metal chelate resins are Chelate-Sepharose F.F. (manufactured by Pharmacia) and AF-chelate Toyopearl 650 M (manufactured by Tosoh Corp.).

The present invention does not specifically limit the metal ions bound to the chelate resin as long as the metal ions are effective for AT-III separation. The metal ions include copper ions, nickel ions and cobalt ions, and copper ions are particularly preferable in view of purification efficiency.

The process according to the invention is carried out by contacting an aqueous solution of AT-III with a metal chelate resin. In such a contact method, it is effective to use a column method or a batch method, and it is preferable to use a column method in view of purity and purification efficiency.

In contact conditions, the reaction occurs when the pH is less than 6 to 8, preferably 6.5 to 7, and the salt concentration is 0 to 1 M, preferably 0.05 to 0.5 M. Preferred solvents include 0.1 M phosphate buffer (pH 7.0 to 7.5) containing 0.05 to 0.5 M sodium chloride, although sodium chloride is not necessary. Antagonists such as glycine or imidazole can be added to the buffer to improve the separation efficiency between the metal chelate resin and AT-III. The antagonist is preferably added in an amount of 20 mM or less.

When the purification method according to the present invention is a column method, the AT-III aqueous solution meeting the above conditions is applied to a metal chelate resin column in equilibrium under the same conditions (using the same buffer as the developing solution) maintained at 2 ° C to room temperature. After addition, the non-adsorbed fraction is recovered. The amount of the metal chelate resin used is controlled according to the impurity content and the content of AT-III contained in the AT-III aqueous solution, and generally about 1 ml of the metal chelate resin is used per 4 ml of the AT-III aqueous solution. The elution rate from the column is controlled by the amount of metal chelate resin and the size of the column.

When the purification method according to the present invention is a batch method, the supernatant is recovered by centrifugation after contacting the AT-III aqueous solution meeting the above conditions with a metal chelate resin in equilibrium condition under the same conditions at 2 ° C to room temperature. The contact time is adjusted according to the impurity content and the content of AT-III and the like contained in the aqueous solution containing AT-III, and generally, about 10 minutes to 1 hour is sufficient.

In some cases, metal ions are released from the metal chelate resin, either by treating the metal ions with a chelate resin or ion exchanger that is not bound to the metal ions, or by adding a chelating agent such as EDTA and then using dialysis. Metal ions can be removed easily.

The AT-III purified by the purification method according to the present invention is free of protein impurities, denatured AT-III, AT-III polymers, etc. and can be obtained by immobilizing AT-III in a single step.

Moreover, AT-III purified by the purification method according to the present invention can be further purified by conventional purification methods such as anion exchanger treatment or treatment with an insoluble carrier having a hydrophobic group as a ligand.

(V) post-treatment

AT-III treated and / or purified by virus inactivation by the method according to the invention is made into pharmaceutical preparations according to the formulation methods generally used. Such pharmaceutical formulations include aqueous solutions, suspensions and lyophilized formulations prepared in a conventional manner, optionally in admixture with pharmaceutically acceptable carriers or additives (eg, diluents, tonics, surface active agents, etc.). Administration of the AT-III preparations is carried out according to conventional prescriptions such as AT-III injections or intravenous drip injections by slow intravenous injection or drip injection of liquid formulations.

AT-III prepared and / or purified according to the present invention is useful for treating thrombus formation by propagating vascular coagulation syndrome (DIC) accompanied by coagulating AT-III deficiency and a decrease in AT-III in the body.

In general, AT-III is administered at a rate of 1,000 to 3,000 units (or 20 to 60 units per kg of body weight) per day, such dosage may vary depending on conditions such as age, weight, symptoms and the like. In addition, when the agent according to the invention is used for obstetric or surgical DIC emergency treatment, it is preferable to administer 40 to 60 units per kg of body weight per day. The osmotic pressure of the liquid formulation is preferably equal to or close to the physiological conditions in the human body or animal body.

The invention is explained in more detail with reference to the following examples which do not limit the invention. The materials and methods used in the following experiments are as follows.

Material and method:

(1) AT-III material

Starting materials used were Cohn's Fr. II + III supernatant or Fr. After concentrating the solution derived from IV, the solution was prepared by virus inactivation treatment which was heated at a temperature of pH 7.8 and 60 ° C. for 10 hours in the presence of 5% sodium citrate, 2M sodium chloride and 30% sorbitol.

(2) Chromatography Carriers Used for Purification

Chelate-Sepharose F.F. (manufactured by Pharmacia) or AF-chelate Toyopearl 650M (manufactured by Tosoh Corp.) was used as the chelate chromatography carrier.

(3) AT-III activity measurement and residual ratio calculation

Activity measurement of AT-III was performed by the synthetic substrate method using S-2238. S-2238 and the reaction termination solution were used in the test team AT-III-2-Kit (Daiichi Pure Chemicals Co., Ltd.). A bottle of S-2238 (20 mg) was dissolved in 21 ml of water, and the thrombin solution (1.2 U / ml) and standard AT-III solution (1 U / ml) prepared by The Green Cross Corporation were prepared and stored at -40 ° C. When used, it was dissolved again and used. 100 μl of thrombin solution 50 μl of each sample to be tested adjusted to 1-10 mU / ml with a solution containing 40 mM Tris, 60 mM EDTA, 0.14 M NaCl, 0.2% HSA (human serum albumin), heparin 2.4 U / ml, pH 8.4 And then incubated at 37 ° C. for 5 minutes. Then, the mixture was mixed with 100 µl of substrate solution and incubated for exactly 5 minutes, and then the reaction was terminated by adding 1000 µl of reaction termination solution. The absorbance of the solution was measured at 405 nm and the AT-III activity of the sample was calculated from a standard straight line prepared using a standard AT-III solution.

The residual ratio of AT-III activity was calculated as the percentage of total AT-III activity of each AT-III sample after heat treatment to the total AT-III activity [U / ml × liquid volume (ml)] before heat treatment. Listed in

(4) AT-III intrinsic activity

The amount of protein present in each sample was calculated by measuring the absorbance at A _{280 nm} (hereinafter referred to as A ₂₈₀ ). The intrinsic activity (U / A ₂₈₀ ) of each sample was calculated by dividing the AT-III activity obtained above by the amount of protein, and the results are shown in Table 1.

(5) Gel Filtration HPLC Analysis

This assay was performed with System Gold (manufactured by Bechkman) with a G3000 SW _XL (Φ 7.8 mm × 30 cm, manufactured by Tosoh) column using a solution containing 50 mM sodium phosphate and 0.3 M NaCl (pH 7.0) as the development solution. . The flow rate was 0.7 ml / min and in all cases was detected at an absorbance of 280 nm.

The proportion of AT-III monomers obtained by HPLC analysis is shown in Table 1. In addition, the results of Table 1 show the degree of denaturation (structural modification such as dimerization or polymerization) of AT-III.

(6) Ouchterlony Analysis

This analysis was carried out in a general manner in which each sample was concentrated to have an absorbance of about 30 at 280 nm. The antibodies used were as follows (number corresponding to the antibodies in FIG. 4): anti-human ceruloplasmin (1) (Hoechst N-antiserum ceruloplasmin); Antihuman transferrin (2) (Hoechst N-antiserum transferrin); Antihuman albumin (3) (Hoechst N-antiserum albumin); Antihuman prialbumin (4) (Hoechst N-antiserum prialbumin); Antihuman heptoglobin (5) (DAKO A030); Antihuman α ₂ macroglobulin (6) (DAKO A033); Antihuman α ₂ antitrypsin (7) (DAKO A012); Antihuman IgA (8) (DAKO A02621); Anti-human IgG (9) (DAKO A04231); Antihuman IgM (10) (Hoechst anti-IgM (μ chain) rabbit serum); Antihuman prothrombin (11) (DAKO A325); And anti-human fibrinogen 12 (DAKO A080).

Example 1

10 kg of paste of fraction IV-1 obtained by Cohn cold ethanol fractionation was suspended in 100 l of physiological saline and barium sulfate was added to the suspension to a concentration of 5 w / v%. The mixture was stirred at room temperature for 30 minutes to remove prothrombin by adsorption with barium sulfate. The supernatant thus obtained was adjusted to pH 6.5 and mixed with polyethylene glycol # 4,000 (average molecular weight test: 2,600 to 3,800, manufactured by Nippon Soda Co., Ltd.) 13 w / v%, and the obtained precipitate was removed by centrifugation; The supernatant was again mixed with 30 w / v% polyethylene glycol # 4,000 and the precipitate formed was recovered by centrifugation.

The precipitate thus recovered was dissolved in about 20 L of cold physiological saline and added to a Heparin-Sepharose (Pharmacia) column prepared in advance in equilibrium with physiological saline to adsorb AT-III to the column. The prepared column was washed with 0.4 M sodium chloride solution to remove protein impurities and passed through the column with 2.0 M sodium chloride solution to recover the eluted AT-III portion.

The aqueous AT-III solution thus obtained was mixed with the stabilizers shown in Table 1 adjusted to pH 7.8 and then heat treated at 60 ° C. for 10 hours. The treated AT-III aqueous solution was contacted with a butyl polyvinyl carrier (butyl Toyopearl 650, manufactured by Tosoh Corp.) in advance to equilibrate with 20 mM sodium citrate buffer (pH 7.5) containing 3M sodium chloride. And the same buffer as above was used as a developing solution for recovering the fraction which was not adsorbed. Then, dialysis was performed overnight against 0.5% sodium citrate buffer (pH 7.5) to obtain purified AT-III.

After the heat treatment, the activity of each purified AT-III sample was measured to calculate the residual ratio of the activity of the AT-III before heat treatment. The proportion of monomer in each AT-III sample was also obtained.

Residual Ratio (%): Percentage of Total AT-III Activity After Heat Treatment to Total AT-III Activity Before Heat Treatment

HPLC analysis (%): ratio of AT-III monomers.

A synergistic effect was observed when sodium citrate and sodium chloride were added to prevent dimerization and polymerization, and a stabilizing effect was observed at low concentrations of sodium citrate. In the table, Na-Cit represents sodium citrate and Sorb. Represents sorbitol.

Example 2

Purification of AT-III using Chelate-Sepharose F.F .:

To a 1.5 ml column of Chelate-Sepharose FF (Φ 0.9 × 2.3 cm) was added to the same bed volume (1.5 ml) of CuSO ₄ · 5H ₂ O dissolved in water at a concentration of 10 mg / ml and bound to a metal. The column was washed with two bed volumes (3 ml) of water. The column was then equilibrated through a three bed volume solution (pH 8.0) containing 0.1 M sodium phosphate and 0.5 M NaCl. Using a PD-10 column (manufactured by Pharmacia), 3 ml of heat-treated concentrate of AT-III (A ₂₈₀ = 4.20) exchanged with the same buffer was added to the column and the eluted fractions were recovered at a rate of 1.5 ml / Fr. . From this, a solution containing 0.1 M sodium phosphate and 0.5 M NaCl, adjusted to pH 7.5, 7.0 and 6.0, was added to the column by 3 bed volumes (4.5 ml), respectively, and the eluted fraction was added in 1.5 ml / Fr Recovered at a rate. In addition, the bound chelate column was regenerated by removing the binding metal in a two bed volume of 50 mM EDTA.2Na and passing through a three bed volume of water.

The results are shown in FIG. As can be seen in FIG. 1, most of the protein was adsorbed at pH 8.0 and eluted in a copper chelate column below pH 7.5.

Table 2 shows the results of GPC-HPLC analysis of the eluate eluted from the copper chelate column. As shown in Table 2, the purity of the denatured (raw) AT-III was 88.0% before addition to the copper chelate column, but the purity in the fraction eluted from the column was 100% at all pH values. That is, when the solution is passed through the column at a pH of less than 8, preferably at pH 7.5 or less, denatured AT-III and protein impurities are efficiently removed, and these impurities are retained by the column even at pH 6.5.

Fraction number 1-5 6 7 8 9 10 11 12 13 14 pH-modified AT-III purity (%) 8.0- 7.5100 7.0100 6.5100

Purity of the original AT-III of the sample added to the column: 88.0%

Example 3

Effect of Salt Concentration:

To test the effect of salt concentration in the chromatography buffer, the copper cultivation chromatographic tendency of the heat-treated and concentrated AT-III solution was 0.1M in 0.1M sodium phosphate buffer (pH 7.5) or 0.1M sodium phosphate (pH 7.5). Examination was performed using buffer obtained by addition of NaCl or 0.5M NaCl. That is, the heat-treated and concentrated AT-III solution was solvent exchanged into each buffer by PD-10 column, and after adjusting A ₂₈₀ to about 5, 5 ml of each solution was equilibrated with the same buffer, 1 ml of copper. It was added to a chelate column (Φ 0.8 × 2 cm). The initial 3 ml of the solution passed through were collected and the rest was recovered in 1 ml portions. The column was then washed with each buffer and the eluent at wash was recovered in 1 ml portions. The elution pattern is shown in FIG. Table 3 shows the results of GPC-HPLC measurements of native AT-III purity in each elution fraction.

Fraction number Total volume of eluent (ml) Original AT-Ⅲ Purity (%) NaCl concentration (M) 0 0.1 0.5 1 2 3 4 5 34567 100.0100.0100.0100.0100.0 100.0100.0100.0100.0100.0 100.0100.0100.0100.0100.0

Purity of intact AT-III of the sample added to the column: 81.15%

As shown in FIG. 2, there was no significant difference in the elution pattern between the case containing no NaCl and the case containing 0.1M or 0.5M NaCl. As shown in Table 3, the purity of the intact AT-III in the eluent was 100.0% in all cases. Thus, it was found that the change in salt concentration of the buffer did not affect the chromatographic propensity.

In addition, the change in pH on chromatographic propensity was examined using 0.1 M sodium phosphate buffer (containing 0.1 M NaCl) adjusted to pH 7.3, 7.5 or 7.7. As a result, it was shown that the pH change within this range did not affect the chromatographic tendency. The chromatographic propensity at 4 ° C. and room temperature was examined using 0.1 M sodium phosphate buffer (containing 0.1 M NaCl, pH 7.5), and it was found that temperature changes within this range did not significantly affect the chromatographic propensity.

Example 4

Confirmation of Purity and Evaluation of Purification Yield in Scale Up Tests:

The heat-treated and concentrated AT-III solution was buffer exchanged with 0.1 M sodium phosphate containing 0.1 M NaCl, pH 7.5 by PD-10 column (A ₂₈₀ = 4.05), and 25 ml of solution was then equilibrated with the same buffer. 5 ml of chelate Toyopearl 650M column (Φ 1.5 × 2.8 cm) was added. 2.5 ml portions corresponding to half the column bed volume were discarded and the sample was added and the eluent obtained was combined with water from a two column bed washed with equilibration buffer and collected as 32.5 ml roll purified AT-III.

The A ₂₈₀ value and AT-III activity recovery rate are shown in Table 4, and the GPC-HPLC pattern before and after chromatography is shown in FIG.

As shown in Table 4, A ₂₈₀ showed excellent recovery of 80.3% and AT-III activity of 89.6%. As shown in FIG. 3, the column eluent of GPC-HPLC showed a single peak of intact AT-III and contained no polymer, denatured AT-III and other protein impurities.

In addition, Ouchterlony analysis of the sample showed that protein impurities that may be contained (ie, ceruloplasmin, transferrin, albumin, prialbumin, hepatoglobin, α ₂ macroglobulin, α ₂ antitrypsin, IgA, IgG , IgM, prothrombin and fibrinogen) were not detected (see FIG. 4).

Thus, it was found that all protein impurities, polymer and denaturated AT-III can be removed by copper chelate Toyopearl 650M chromatography at pH 7.5.

step Volume (ml) A ₂₈₀ AT-III activity A ₂₈₀ A ₂₈₀ x volume yield(%) U / ml Total activity U / A ₂₈₀ yield(%) Addition column 25 4.05 101.3 80.3 29.9 748 7.38 89.6 Passing column 32.5 2.50 81.3 20.6 670 8.26

Example 5

Removal of Copper Leaked from Copper Chelate Resin Columns

In copper chelate column chromatography, the copper bound to the column flows out in small amounts as the protein is adsorbed. A method of removing the spilled copper involves connecting a column with a metal chelate resin column, such as a metal unbound chelate resin (eg, a chelate may bind to a metal but no metal). The spilled copper removal effect of this method was examined as follows. Fr. 20 ml of a heat-treated concentrated AT-III solution (A ₂₈₀ = 4.96) obtained in II + III supernatant was dissolved in 0.1 M sodium phosphate buffer (pH 7.5) containing 0.1 M NaCl and equilibrated with the same buffer. After addition to Toyopearl column (Φ 1.5 × 2.8 cm, 5 ml), an unadsorbed fraction was obtained (27.5 ml, A ₂₈₀ = 3.18). 23.5 ml of this was added to a metal-free chelate Toyopearl 650M column (Φ 0.8 × 2.0 cm, 1 ml) equilibrated in the same manner to obtain an unadsorbed fraction (25 ml, A ₂₈₀ = 2.63). The results of measuring copper content at each step are shown in Table 5.

step Copper content (ppb) Copper Chelate Column Addition Copper Chelate Column Pass Metal Unbound Chelate Column Pass Negative Control (Chromato Buffer) 32010,3001513

As shown in Table 5, the outflow of copper was reduced to 15 ppb, close to the control level (13 ppb), so that 10,300 ppb of copper was leaked out of the copper chelate column pass, but the leaked copper was passed through a metal unbonded chelate Toyopearl 650M column into solution. It can be seen that it was removed.

The GPC-HPLC pattern of the eluent of the starting material and the metal free chelate column is shown in FIG. 5. As shown in Fig. 5, since no peak was detected except for the original AT-III peak, Fr. It can be seen that the material derived from the II + III supernatant can be purified by a copper chelate column. The purity of the material, calculated from the peak area, was 99.9%.

Example 6

Purification Yield by Linked Columns of Copper Chelate and Unbonded Toyopearl Metals

Considering the improvement of purification efficiency and the automation of the chromatographic step, the connection column of copper chelate and metal free chelate was examined. Fr. II + III supernatant or Fr. The solution of AT-III obtained from IV and heat-treated and concentrated was concentrated and diluted with Centriprep 30 (manufactured by Amicon) (Fr. IV: A ₂₈₀ = 5.23, Fr. II + III supernatant: A ₂₈₀ = 5.50) to 0.1M. Dialysis was performed 1,600 times against 0.1 M sodium phosphate buffer (pH 7.5) containing NaCl and again dialysis 100 times (total 160,000 dialysis) against the same buffer. The amount of dialysis was increased with Centriprep 30 because the amount of copper outflow was increased by 1,600 dialysis due to residual citric acid. 20 ml of each of these solutions was added to a tandem column of copper chelate Toyopearl (Φ 1.5 × 2.8 cm, 5 ml) and metal unbound chelate Toyopearl (Φ 0.8 × 2.0 cm, 1 ml) equilibrated with the same buffer. In the solution eluted when the sample was added, half the volume of the total column capacity was discarded and the eluted fraction was then combined with 10 ml of the washed fraction corresponding to the two bed volume of the copper chelate column.

From the purification yields shown in Table 6 Fr. II + III supernatant and Fr. It can be seen that both materials derived from IV showed good activity recovery of 90% or more. In addition, the intrinsic activity is approximately 10 U / A ₂₈₀ , which is a value corresponding to the intrinsic activity of the pharmaceutical agent used.

Source of matter step Liquid amount (ml) A ₂₈₀ A ₂₈₀ All A ₂₈₀ yield(%) Ⅱ + Ⅲ supernatant Column addition 20.0 5.50 110 100 Pass through column 27.0 3.22 86.9 79.0 Ⅳ Column addition 20.0 5.23 105 100 Pass through column 26.0 2.92 75.9 72.3

Source of matter step AT-III activity U / ml Overall activity yield(%) Intrinsic Activity (U / A ₂₈₀ ) Ⅱ + Ⅲ supernatant Column addition 49.0 981 100 8.9 Pass through column 33.9 914 93.2 10.5 Ⅳ Column addition 40.9 818 100 7.8 Pass through column 29.1 757 92.5 10.0

Example 7

Preparation of AT-III Pharmaceutical Formulations:

Purified AT-III obtained in Example 5 was desalted, concentrated and filtered through a BMM (Bemberg Microporous Membrane) membrane (manufactured by Asahi Chemical Industry, Co., Ltd.). 50 units / ml of the AT-III sample prepared in this way were mixed with mannitol 2w / v%, 0.52w / v% sodium citrate and 0.5w / v% sodium chloride, and the mixture was adjusted to pH 7.2-7.5 with 1N sodium hydroxide. Sterilized by filtration, filtered through a sterile Millipore filter, divided into 500 unit portions, and lyophilized to obtain a lyophilized pharmaceutical formulation.

Although the present invention has been described in detail with reference to specific embodiments, it will be apparent that various changes and modifications can be made therein without departing from the spirit and scope of the invention.

This application is based on Japanese Patent Application No. 96-309281 for which it applied on November 20, 1996, and 97-163426 for which it applied on June 4, 1997, and the general content is referred to in this invention.

When AT-III was heated in the liquid phase for virus inactivation according to the present invention, the activity of AT-III remained stable and the denaturation of AT-III (structural modifications such as dimerization, polymerization, etc.) was reduced, resulting in medical In view of the above, it is possible to manufacture AT-III which is very safe and useful.

In addition, according to the purification method of the present invention can effectively remove the protein impurities contained in the AT-III aqueous solution and the modified AT-III and the polymer formed by heat treatment, it is possible to obtain a very safe AT-III. In addition, since the purification step is simple and suitable for mass production, it is also useful as an industrial production method.

Since the pharmaceutical preparation according to the present invention includes AT-III purified by the above-described method, it is possible to prepare an AT-III preparation having excellent safety.

Claims (20)

(a) heating the antithrombin-III-containing aqueous solution in the presence of a stabilizer to maintain at least 85% of the antithrombin-III activity after heating and to maintain the ratio of antithrombin-III monomer after heating, at least 95% before heating. Steps and (b) treating antithrombin-III containing aqueous solution with a metal chelate resin and recovering purified antithrombin-III. The method of claim 1 wherein the stabilizer is an organic acid or salt thereof and saccharide optionally containing sodium chloride. The organic acid or salt thereof according to claim 2, wherein the organic acid or salt thereof is glycine, alanine, valine, leucine, isoleucine, oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, maleic acid, fumaric acid, phthalic acid, aspartic acid, glutamic acid, malic acid , Tartaric acid, citric acid and salts thereof. The method of claim 2, wherein the organic acid or salt thereof is sodium citrate. 3. The saccharide of claim 2 wherein the saccharide is glucose, fructose, galactose, mannose, arabinose, inositol, saccharose, lactose, maltose, mannitol, sorbitol, xylitol, glucoseamine and N-acetyl-D-glucosamine How is chosen from. The method of claim 2, wherein the saccharide is sorbitol. The method of claim 2, wherein the organic acid or salt thereof is contained in the solution in an amount of 3 to 20 w / v%. The method of claim 2 wherein the saccharide is contained in the solution in an amount of 10 to 60 w / v%. The method of claim 2 wherein sodium chloride is contained in the solution in an amount of 0.5 to 3.0 mol / l. The method of claim 1 wherein the metal ions of the metal chelate resin are selected from copper ions, nickel ions and cobalt ions. The method of claim 1, wherein the metal ions of the metal chelate resin are copper ions. The method of claim 1 wherein the antithrombin-III containing aqueous solution is heat treated before step (b). 13. The method of claim 12, wherein the heat treatment is performed according to step (a). 13. The method of claim 12, wherein the antithrombin-III containing aqueous solution is subjected to dialysis between the heat treatment and step (b). The method of claim 1 wherein the heating is carried out at a temperature of 50 to 70 ° C. for 10 minutes to 20 hours. The method of claim 1, wherein at least 95% of the antithrombin-III activity after heating is maintained after heating. The method of claim 1, wherein the ratio of antithrombin-III after heating is maintained at 99% or more. The method of claim 1 wherein the antithrombin-III containing aqueous solution is treated with the metal chelate resin by contact treatment with the metal chelate resin. The method of claim 1, wherein the protein concentration of the antithrombin-III containing aqueous solution is 0.1 to 5 w / v%. A pharmaceutical composition comprising an antithrombin-III or a pharmaceutically acceptable salt thereof prepared according to the method of claim 1 as an active ingredient, optionally together with a pharmaceutically acceptable carrier.