JPH07268003A - Purification of antithrombin iii - Google Patents

Abstract

PURPOSE:To produce a heparin gel having a high ability to combine with antithrombin III coupling a highly concentrated heparin solution with a formylated carrier at a specified temperature. CONSTITUTION:A highly concentrated heparin solution (preferably a solution having a heparin concentration of at least 100mg/ml) is coupled with a formylated carrier at 20 deg.C or higher, preferably 37 deg.C or higher, to produce a heparin gel having a high ability to combine with antithrombin III (referred to as AT-III hereafter), suitably at least 30 units/ml gel. This gel has a high ability to combine with AT-III, is useful for efficient purification of AT-III, and can give a better effect with a less gel quantity than in conventional processes. Therefore, AT-III can be purified at a low cost with a small-scale installation.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to antithrombin-III.
The present invention relates to a heparin gel useful for purifying (hereinafter referred to as AT-III), a method for producing the heparin gel, and a simple and efficient method for purifying AT-III by using the heparin gel.

[0002]

Prior Art / Problems to be Solved by the Invention AT-II
I is a kind of glycoprotein belonging to α ₂ globulin existing in plasma, and its molecular weight is 60,000 to 68,000.
It has protease inhibitory activity and strongly inhibits thrombin coagulation activity. Further, it has not only an inhibitory effect on thrombin but also an inhibitory effect on other coagulation factors such as activated factor X and activated factor IV. In addition, it has been reported that it has an inhibitory effect on plasmin and trypsin. Families with congenital AT-III deficiency have a high incidence of thrombosis. Attempts have been made to administer AT-III as a treatment for patients with thrombotic disorders.

Traditionally, AT from human plasma and plasma pastes by various chromatographic combinations
Several attempts to purify -III have been reported. 197
Since MIller-Anderson et al. Used affinity chromatography using purified heparin as a solid phase binding ligand in 4 years, purification of AT-III has become relatively easy (Thromb. Res., 5:43
9, 1974).

At present, many immobilized heparins are commercially available, but none of them has a high AT-III binding ability.

An object of the present invention is to provide a heparin gel having a high binding ability for AT-III. Another object of the present invention is to provide a highly reproducible method for producing a heparin gel having high AT-III binding ability. Still another object of the present invention is to provide a simple and efficient method for purifying AT-III, which is characterized by using a heparin gel having a high binding ability to AT-III.

[0006]

As a result of intensive studies to achieve the above object, the present inventors succeeded in producing a heparin gel having a high binding ability to AT-III, and obtained the heparin gel with good reproducibility. The present invention has been completed by confirming that it is effective and highly useful in highly purifying AT-III.

That is, the present invention has high AT-III binding ability, preferably 30 units / ml (hereinafter,
U / ml) gel or more, more preferably having a binding capacity of 4
It relates to a heparin gel which is 0 U / ml gel or more. The present invention also relates to a method for producing a heparin gel having high AT-III binding ability, which comprises coupling a high-concentration heparin solution with a carrier. Concentration is preferably 100m
It relates to a method for producing a heparin gel, which comprises coupling a heparin solution of g / ml or more with a carrier having a formyl group at a specific temperature. Furthermore, the present invention relates to a method for purifying AT-III, which comprises using the above-mentioned heparin gel or the heparin gel produced by the above-mentioned method.

(1) Heparin gel The carrier for immobilizing heparin is not particularly limited as long as it is a commonly used insoluble carrier, preferably a carrier for affinity, hydrophilic vinyl alcohol polymer, crosslinked dextran (trade name Sephadex), agarose. (Product name Sepharose), cellulose (Product name Cellulofine) and the like are exemplified. Further, the reaction active group for immobilizing the heparin which is a ligand on the affinity carrier is preferably a formyl group. As such a ligand-immobilizing carrier, commercially available products such as AF-formyl Toyopearl 650M (manufactured by Tosoh Corporation) and formyl-cellulofine (manufactured by Chisso Corporation) are preferably used.

The heparin to be immobilized on the carrier is not particularly limited, and ordinary commercially available heparin can be conveniently used. Heparin having a large amount of amino groups is preferable. Further, heparin in a salt form, particularly in a sodium salt form is preferably used. Further, those having a high solubility in water or a buffer solution and containing almost no impurities are preferable.

The heparin gel of the present invention is characterized by high AT-III binding ability. Usually, heparin gel has an ability to bind about 10 to 20 U of AT-III to 1 ml of gel. On the other hand, the heparin gel of the present invention has an ability to bind 30 U or more, preferably 40 U or more of AT-III to 1 ml of gel, and is useful as an AT-III affinity gel in the purification treatment of AT-III. Is.

AT-III 1U is a unit showing AT-III activity in 1 ml of plasma (usually AT-III).
(About 140 μg / 1 U), and can be measured by thrombin inhibitory activity using a synthetic substrate S-2238, that is, heparin cofactor activity.

(2) Method for producing heparin gel Heparin gel is usually produced by the following steps or in accordance with the steps. The ligand immobilization carrier (gel) is washed with distilled water and a coupling buffer. Add the heparin solution to the washed gel, mix,
Stir before. NaCNBH ₃ (sodium cyanoborohydride) is added to the mixture, and the mixture is reacted by stirring for several hours or longer. After the reaction, the unadsorbed heparin solution is removed from the gel by filtration or the like. Tris-HCl and Na to block excess active groups
Add CNBH ₃ and react at room temperature for 1 hour. Wash the gel with water and buffer.

The method for producing a heparin gel of the present invention is characterized in that a carrier having a formyl group as an active group is used as a carrier for immobilizing a ligand in the step. By using the carrier having the formyl group as an active group, it is possible to produce a heparin gel with less loss of ligand and relatively high stability to alkali.

Further, as the heparin solution used in the above steps and the coupling reaction of the steps, a solution having a high heparin concentration of 50 to 300 mg / ml, preferably 100 mg / ml or more of heparin is used. Conventionally, due to the low solubility of heparin in water or buffer, 50 mg / ml heparin was used for the coupling reaction.
Since the heparin solution before and after was used, only heparin gel with low AT-III binding ability was obtained. The present invention is
The fact that heparin can be practically dissolved in water or a buffer solution at a concentration higher than ever recognized, and according to the present invention, a high concentration heparin solution is used for the reaction. As a result, the effect that a heparin gel having high AT-III binding ability can be produced can be obtained.

Furthermore, the method of the present invention is characterized in that the temperature used in the step reaction (coupling reaction) is 20 ° C. or higher, preferably 37 ° C. or higher, more preferably 60 ° C. Conventionally, the coupling reaction is carried out at 4 ° C. or room temperature for reasons such as avoiding thermal denaturation of the ligand. The present invention is contrary to the conventional recognition, and according to the present invention, a heparin gel having a high AT-III binding ability can be produced by carrying out the coupling reaction under the above temperature condition. .

The method for producing a heparin gel of the present invention includes all methods including the steps having the above characteristics.

(3) Purification of AT-III The method for purifying AT-III of the present invention is AT using the heparin gel of the present invention or the heparin gel produced by the method of the present invention.
-Treating a plasma fraction containing III. The heparin gel treatment is a conventional method for purifying AT-III,
Alternatively, any of the AT-III purification methods to be used in the future can be performed at a desired process step. Preferably, supernatant I and supernatant II + III in the Cohn fractionation method of plasma
It is desirable to carry out at the stage of from the viewpoint of efficiency and reproducibility. The method for purifying AT-III of the present invention includes all methods for purifying AT-III including the step of treating with heparin gel of the present invention.

The AT-III targeted by the method of the present invention is AT-III derived from plasma of mammals, preferably human, AT-III prepared by a genetic engineering method, and the starting material of the present invention is AT-III. Examples thereof include human plasma fractions, for example, supernatant II + III by the Cohn fractionation method, supernatant I, and a residual fraction after collecting blood coagulation factor VIII from citrate-containing plasma. Among them, supernatant II by the Cohn fractionation method
+ III is preferred.

Treatment with heparin gel The treatment of AT-III with the heparin gel according to the present invention is
It is carried out by contacting the heparin gel of the present invention with AT-III at any stage of the AT-III purification step.

The treatment with the gel basically comprises the following steps. In addition, the said process is applied to any method, such as a batch method and a column method. (A) A plasma fraction containing AT-III (eg, supernatant I,
Supernatant II + III) is obtained. (B) The above AT-III-containing solution is brought into contact with and mixed with heparin gel at -7 ° C to 4 ° C to bond AT-III and heparin gel. (C) Separate the heparin gel from the solution and wash the gel if necessary. (D) AT-III is eluted from the heparin gel to obtain purified AT-III.

In the method for purifying AT-III of the present invention, the AT-III-containing plasma fraction is contacted with the heparin gel matrix at about -7 ° C to 4 ° C. When such treatment is carried out in a batch system, the ligand-bound AT-III and the plasma supernatant can be separated from the mixture with the gel matrix by a conventional method such as a centrifugation method or a filtration method.

AT-III is washed with 0.1 to 1 M NaCl or the like, and particularly preferably after the washing operation is repeated by gradually increasing the salt concentration, and then the AT-III is added to a high salt concentration buffer such as 2 M NaCl. It is eluted from the heparin gel matrix by conventional methods such as contacting.

In the heparin treatment, when the AT-III is brought into contact with the heparin gel of the present invention, the mixing ratio of AT-III and the heparin gel of the present invention is AT-III 1 mg.
On the other hand, gel is exemplified by 0.3 to 3 g, preferably 0.5 to 1 g.

The purity of AT-III obtained through the heparin gel treatment of the present invention is 95% or more.

The obtained purified AT-III solution is further subjected to hydrophobic chromatography treatment, anion exchanger treatment and the like, if necessary, to remove impurities such as other plasma proteins, pyrogens and heat denatured proteins. In addition, highly pure AT-III can be purified and recovered.

[0026]

INDUSTRIAL APPLICABILITY The heparin gel of the present invention has a high AT-III binding ability and is useful for efficient purification treatment of AT-III. That is, according to the heparin gel of the present invention, an effect equal to or higher than that of the conventional method can be obtained with a small amount of gel, and therefore AT-III can be purified with a small-scale facility and at low cost. Further, according to the method for producing a heparin gel of the present invention, a gel having a high AT-III binding ability can be produced with good reproducibility. Furthermore, according to the method for purifying AT-III of the present invention, AT-III can be purified easily, efficiently and with a reproducible degree of purification.

[0027]

EXAMPLES In order to explain the present invention in more detail, examples will be given, but the present invention is not limited thereto.

Example 1 (1) Method for producing heparin gel Influence of concentration of heparin used for coupling Heparin sodium (manufactured by Welding) at a concentration of 5,1
Coupling buffer (0.2M phosphate buffer, 0.1M NaCl, p) so that the concentration becomes 0, 20, 30, 40%.
It was dissolved in H7) to prepare a white turbid heparin solution.
This heparin solution was centrifuged to remove white turbidity, and the heparin concentration in the obtained heparin solution was determined by the Azure A method described in Reference Example 1. The heparin solution is added to the coupling buffer as described above at a heparin concentration of 5,
Re-preparation was made to 10%, 15% and 20%, and 4 ml of each solution was added to 2 g of a carrier AF-formyl-TOYOPEARL 650M (manufactured by Tosoh Corporation), and NaCNBH ₃ was added to NaCNBH _3.
/ Heparin (mg / mg) was added at a ratio of 0.13 and reacted at 20 ° C. for 24 hours with stirring. The AT-III binding ability of each manufactured heparin gel was measured according to the method of Reference Example 2. The results are shown in Fig. 1. From this, it was confirmed that the higher the heparin concentration, the higher the AT-III binding ability (about 40 to 46 U / ml gel). Further, from these results, the optimum concentration of heparin at the time of coupling was 150 mg / ml or more, and the heparin content (the amount of heparin relative to the gel) was 300 mg / ml gel.

Heparin content and heparin concentration In accordance with the method described above, heparin in heparin solution
Keep the content constant at 300 mg / ml gel and concentrate on heparin.
The coupling reaction was carried out at different temperatures [NaCNBH ₃/ Hepa
Phosphorus (mg / mg) = 0.25, room temperature, 60 hours]. The results are shown in Figure 2.
You From the results shown in FIG. 2, the heparin content of the gel was 300.
If the heparin concentration is low, even if the gel / mg gel is kept constant,
It was confirmed that the pulling efficiency was poor.

Effect of Heparin Content on Coupling Efficiency With the heparin concentration kept constant at 150 mg / ml, the coupling reaction [NaCNBH ₃ / heparin (mg / mg) = 0.13, varying the amount of the heparin solution added to the gel]. At room temperature for 16 hours], the influence of the coupling content at the time of coupling was examined. The results are shown in Fig. 3.

Effect of Heparin Concentration and Coupling Temperature on Coupling Efficiency According to the method of Example 1, 25, 50, 100, 150
And a 200 mg / ml concentration of heparin solution were prepared in duplicate. AF-formy carrier 4ml of each solution
l-TOYOPEARL 650M (manufactured by Tosoh Corporation) in addition to 2g, NaC
NBH ₃ was added at a ratio of NaCNBH ₃ / gel (mg / ml gel) of 20 and one sample was reacted at room temperature (RT) for 24 hours, and another sample was reacted at 37 ° C. for 16 hours with stirring. For each heparin gel produced,
The AT-III binding ability was measured according to the method of Reference Example 2, and 2
The case of reaction at 0 ° C and the case of reaction at 37 ° C were compared. The results are shown in Fig. 4. As a result, a temperature-raising effect was not observed at low heparin concentration, but at the optimum heparin concentration, a gel having high AT-III binding ability was prepared even when the stirring time was short.

Next, the other AT-III binding capacities were compared by changing the coupling reaction temperature to 20 ° C., 37 ° C. and 60 ° C. while keeping other conditions constant. Ie 150
Using a heparin solution with a concentration of mg / ml at the above temperature,
Coupling was performed for 24 hours, and the resulting heparin gel was examined for AT-III binding ability. The results are shown in Table 1.

[0033]

[Table 1]

From this result, the coupling temperature was 60 ° C.
Was determined to be the most preferable.

Reference Example 1 Quantification of heparin (Azur
eA method) A heparin sample was added with 0.05 M Tris-HCl (pH 8.
It was diluted with 0) to obtain a measurement sample. Prepared by dissolving Azure A powder (manufactured by Sigma) in 0.2 ml of this sample 2
After adding 1 ml of 0 μg / ml Azure A solution and stirring the mixture sufficiently, the absorbance at a wavelength of 500 nm was measured. The heparin concentration of the measurement sample was calculated and calculated from the standard curve.

Reference Example 2 Test of AT-III binding ability Heparin-TOYOPEARL 650M gel AT-II was applied to a column of 1 ml.
Apply the I solution (containing about 60 to 120 U) to 0.1
2M NaCl, 0.01M citrate buffer (pH
Washed with 7), 0.4 M NaCl, 0.01 M citrate buffer (pH 7.2) and 0.7 M NaCl,
After further washing with 0.01 M citrate buffer (pH 7.2), AT-III was eluted with 2 M NaCl and 0.01 M citrate buffer (pH 7.2). AT-III
The binding capacity is 0.7M NaCl solution and 2M
It was calculated as the amount of AT-III recovered in the NaCl solution.

Example 2 Plasma Cohn Fraction Supernatant II + III
Method of Purifying AT-III from -21 ° C, 21% alcohol, 0.12M NaC
2.5 ml of the supernatant II + III fraction containing AT-III was applied to 100 ml of heparin gel which had been equilibrated with 0.01M citrate buffer (pH 6.8).
12M NaCl solution, 0.4M NaCl solution, 0.
After sequentially washing with 7M NaCl solution, 2M NaCl
AT-III was eluted with 1 solution. The AT-III solution was concentrated by ultrafiltration. Virus inactivation is 19%
60 in the presence of sodium citrate, 2M NaCl solution
It was carried out by applying a heat treatment at ℃ for 10 hours. After heating, heat denatured proteins and impurities were removed by hydrophobic chromatography and ion exchange chromatography. The obtained AT-III solution (500 U / vial) was lyophilized under aseptic conditions.

[Brief description of drawings]

FIG. 1 is a diagram showing the effect of the concentration of heparin used in a coupling reaction on the AT-III binding ability of a produced heparin gel.

FIG. 2 is a diagram showing the effect of the concentration of heparin used in the coupling reaction on the AT-III binding ability of the produced heparin gel when the heparin content is fixed at 300 mg / ml gel.

FIG. 3 is a diagram showing the effect of the heparin content used in the coupling reaction on the AT-III binding ability of the produced heparin gel when the heparin concentration is fixed at 150 mg / ml.

FIG. 4 is a graph showing the influence of the temperature of the coupling reaction on the AT-III binding ability of the produced heparin gel.

FIG. 5 is a diagram showing one embodiment of a method for producing a heparin-TOYOPEARL 650M gel.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Yoshinobu Honda Innovator Yoshinobu Honda, 3-5-44, Miyakojima-dori, Miyakojima-ku, Osaka City Midori Cross Miyakojima Plant Co., Ltd. 3-5-44 Tsudori Co., Ltd. Midori Cross Miyakojima Factory

Claims (9)

[Claims] 1. A heparin gel having high antithrombin-III binding ability. 2. The binding ability of antithrombin-III is 30.
The heparin gel according to claim 1, which has a unit / ml gel or more. 3. The binding ability of antithrombin-III is 40.
The heparin gel according to claim 1, which has a unit / ml gel or more. 4. The method for producing a heparin gel according to claim 1, wherein the high-concentration heparin solution and the carrier are coupled. 5. The method for producing a heparin gel according to claim 4, wherein the carrier is a carrier having a formyl group. 6. The concentration of the heparin solution is 100 mg / ml.
It is above, The manufacturing method of the heparin gel of Claim 4 or 5 characterized by the above-mentioned. 7. The method for producing a heparin gel according to claim 4, wherein the coupling temperature is 20 ° C. or higher. 8. The method for producing a heparin gel according to claim 4, wherein the coupling temperature is 37 ° C. or higher. 9. A method for purifying antithrombin-III, which comprises using the heparin gel according to any one of claims 1 to 3 or the heparin gel obtained by the method according to any one of claims 4 to 8.