CS196349B2 - Process for preparing heparine - Google Patents

Abstract

The present invention provides a method for the preparation of heparin of high specific activity, which comprises contacting heparin with an adsorbent which is matrix-bound heparin-binding plasma proteins.

Description

The invention relates to a process for the preparation of very pure heparin of high specific activity.

Heparin has been used for therapeutic purposes for several decades. It is one of the best known anticoagulants and is widely used, inter alia, to prevent thrombosis.

Heparin is a sulphated polysaccharide that can be prepared from animal intestinal mucosa or lung by relatively complex methods. Heparin formulations used in current clinical practice contain a substance with considerable scattering in molecular sizes, from about 5000 to about 35,000. The specific activity is usually about 130 J / mg. The currently available commercial preparations are highly heterogeneous. Side effects occur very rarely with heparin therapy, but if such cases occur, they present serious problems. Some of these side effects are probably due to impurities in the heparin formulation.

Thus, there are good reasons for making heparin preparations cleaner than those commonly used. In connection with studies on antithrombin, a heparin cofactor, the possibility of protein binding to the matrix has been investigated. Surprisingly, it has been found that antithrombin bound to the matrix can specifically bind fragments of the therapeutically used heparin molecules. which acts as a carrier of the clinically valuable preventive effect of heparin against thrombosis. Exceptionally, adsorption and desorption conditions can be prepared appropriately. - pure heparin. The specific activity achieved was 200 to 270 J / mg compared to approximately 130. j / mg of starting material. - The molecular weight scatter for specifically purified heparin was more limited than in the original material. In addition to the heparin cofactor antithrombin, other heparin binding proteins, such as an inter-β-trypsin inhibitor having a molecular weight of about --150,000, can be advantageously used. during the isolation of coagulation factor IX (B-factor). Matrix-bound antithrombin achieved the best heparin quality with a specific activity of up to 270 J / mg.

Heparin with a very high degree of purity and specific activity can be produced by the process of the present invention, which is simple and applicable to the industrial production of heparin with a higher specific activity than that of existing formulations. Determination of specific heparin activity was performed according to Denson and Bonnar - (1975, Brit. J. Haematol. 30, p. 139).

The invention is illustrated by the following examples.

Example 1

Purification of heparin on carrier-bound antithrombin. Column procedure.

The adsorption gel was prepared as follows:

Activation: 3 g of cyanogen bromide were dissolved in 30 ml of distilled water. 50 ml of sedimented agarose (Sepharose.RTM. 4B, Pharmacia, Fine Chemicals, Uppsala, Sweden) was added to the cyanogen bromide solution, and the mixture was cooled in an ice bath with stirring. The gel was then washed with cold distilled water and 0.2 M sodium bicarbonate solution.

Binding: 200 mg antithrombin, - purified according to Miier-Anderson sp. (1974, Thromb. Res. S, pp. 439-452), was dissolved in 50 ml of a 0.2 M sodium bicarbonate solution, pH 9.0 and added to the gel. The gel-protein suspension was stirred gently. at room temperature overnight, then it was thoroughly washed with high ionic strength buffers, alternately at high and low pH values.

Heparin Purification: The adsorption gel containing antithrombin bound to the matrix was packed in a column and equilibrated with a pH 7.4 buffer containing 0.05 M Tris and - 0.15 M sodium chloride. 300 mg of heparin (Vitrum, specific activity - 130 J / mg) was - dissolved in 20 ml of pH 7.4 buffer containing 0.05 M Tris and 0.15 M sodium chloride and poured through the column. The gel was then washed with the buffer, and the adsorbed heparin was desorbed with a pH 7.4 buffer containing 0.05 M Tris and 1.0 M sodium chloride.

Salt was removed from the eluted heparin by gel filtration in distilled water on a column packed with Sephadex (R) G 25 (Pharmacia) and then the heparin was lyophilized. Purified heparin had a specific activity of 270 J / mg. Molecular weight scattering studies show that the molecular weight of purified heparin has a much more limited scatter than the original heparin.

Carbohydrate analysis was performed by carbazole-sulfuric acid.

Example - 2

Purification of heparin on matrix-bound antithrombin. Interrupted procedure.

The antithrombin matrix was prepared as in Example 1. Heparin purification: 500 mg heparin (Vitrum, specific activity 130 j / mg) was - dissolved in 300 ml of pH 7.4 buffer containing 0.05 M Tris and 0.15 M chloride 300 ml of sedimented antithrombin bound to the sedimented matrix were equilibrated with the buffer and sucked dry The adsorbent was added to the heparin solution, which was then stirred at room temperature for 1 hour, then the gel was suction dried on a glass filter and washed 10 times 200 ml of pH 7.4 buffer containing 0.05 M Tris and 0.15 M sodium chloride The adsorbed heparin was then desorbed from the gel by washing 3 times with 200 ml of pH 7.4 buffer containing 0.05 M Tris and 1.0 M sodium chloride The eluted heparin was concentrated by lyophilization and the remaining salt was removed by gel filtration on Sehpadex G 25 in distilled water. heparin was then lyophilized. The specific activity of the purified heparin of 250 U / mg.

Example 3

Purification of heparin on a gel adsorbent containing an - - inter -? - trypsin - - - inhibitor - matrix bound.

Preparation of adsorption gel.

During purification of coagulation factor IX (B-factor) [L.—O. Andersson et al., Thromb. Res. (1975), pp. 451-459], a protein that binds heparin, an inter-α-trypsin inhibitor, was obtained. Its molecular weight is approximately 150,000. 250 mg of this protein was dissolved in 200 ml of pH 9.0 buffer containing 0.2 M sodium bicarbonate and added to 50 ml of sedimented Sepharose®-4B activated with cyanogen bromide as described in of Example 1. The gel-protein suspension was stirred gently at room temperature overnight. Then the gel was washed as in Example 1.

Heparin Purification: The adsorption gel containing matrix bound protein was packed into a column. 300 mg of heparin (Vitrum) was dissolved in 20 ml of pH 7.4 buffer containing 0.05 M Tris and 0.15 M sodium chloride and the solution was poured onto a column. The gel would be washed with the above buffer and the adsorbed heparin was then desorbed with a pH 7.4 buffer containing 0.05 M Tris and 1.0 M sodium chloride. The salt was removed from the heparin by gel filtration on Sephadex G 25 in distilled water and the heparin was then lyophilized. The specific activity of purified heparin was 230 J / mg.

Claims (4)

A method for the preparation of heparin of high specific activity, characterized in that heparin is adsorbed on a gel adsorbent containing bound plasma proteins capable of binding heparin. 2. The method of claim 1, wherein the protein is antithrombin. OF THE INVENTION 3. The method of claim 1, wherein the plasma protein is an inter-α-trypsin inhibitor. Method according to any one of the preceding claims, characterized in that an agarose gel is used as the gel adsorbent.