DE2709500C2 - Method for purifying heparin - Google Patents

Description

The invention relates to a method for purifying heparin, in which the heparin is very pure Shape and with a high specific activity

Heparin has been used in medical treatments for several decades and is one of the best known Anticoagulants and is widely used, e.g. B. to prevent thrombosis. Heparin is a sulfated polysaccharide. the relatively complicated process from animal intestinal mucosa or lungs can be made or isolated. The heparin preparations used clinically today contain Substances with a wide range of variation in molecular weight from about 5,000 to about 35,000. The specific activity is usually about 130 units / mg. Thus, the currently commercially available Preparations quite heterogeneous. The frequency of side effects with heparin therapy is although relatively minor, when such cases occur they often lead to serious problems. Some These side effects are believed to be caused by impurities in the heparin supplements available.

The task is therefore to provide purer, more specifically active heparins than are currently available. In connection with studies of antithrombin, a cofactor of heparin, the possibilities of binding the protein to a matrix were investigated. Surprisingly, it has been shown that antithrombin bound to a matrix was able to specifically bind the molecular fraction of therapeutically used heparin which acts as a carrier of the clinically valuable thrombosis-preventing effect of heparin. Under appropriately selected adsorption and desorption conditions, it is possible to obtain extremely pure heparin. The specific activities that could be obtained were 200 to 270 units / mg versus 130 units / mg in the starting material. The molecular weight distribution in the specifically purified heparin was narrower than in the original material. In addition to the heparin cofactor antithrombin, inter- «- trypsin inhibitor with a molecular weight of approximately 150,000, which has been obtained during the isolation of coagulation factor IX (B factor), can advantageously be used. The best heparin could be obtained with matrix-bound antithrombin with specific activities up to 270 units / mg.

The subject of the invention is thus the method characterized in the claim. The inventive The process is simple and well applicable for the industrial production of heparin with a larger one specific activity than can be achieved by known methods. Determining the specific The activity of heparin took place according to the method given by Denson and Bonnar (Brit. J. Haematol,

Volume 30, 1975, page 139).

The invention is illustrated in more detail by the following examples:

For example

Purification of heparin with
matrix-bound antithrombin (column method)

ίο The adsorption gel was made as follows:

activation

3 g of BrCN were dissolved in 30 ml of distilled water. 50 ml of the BrCN solution were deposited Agarose (Sepharose® 4B, Pharmacia Fine Chemicals. Uppsala, Sweden). The mixture was under Stirring in an ice bath, cooled by adding 4 M NaOH, the pH was increased to 11.2 and 10 minutes kept constant. The gel was then cold washed distilled water and 0.2 M NaHCOj solution.

binding

200 mg antithrombin, purified according to Miller-Andersson et al. (Thromb. Res. Volume 5.1974, pages 439 to 452), in 50 ml of 0.2 M NaHOC ₃ pH 9.0, were added to the gel, the gel-protein suspension was stirred gently overnight at room temperature and then carefully washed with buffers of high ionic strength with alternating high and low pH values.

Purification of heparin

The adsorption gel containing matrix-bound antithrombin was placed in a column and equilibrated with 0.05 M Tris / 0.15 M NaCl buffer, pH 7.4. 300 mg of haperin (specific activity 130 units / mg) were dissolved in 20 ml of 0.05 M Tris / 0.15 M NaCl buffer, pH 7.4, and pumped through the column. The gel was then washed with the said buffer and then the adsorbed heparin is desorbed with a 0.05 M Tris / 1.0 M NaCl buffer, pH 7.4. The salt was separated from the haperin by gel filtration in distilled water in a column containing Sephadex® G 25 (Pharmacia) and the heparin was then freeze-dried. The purified heparin had a specific activity of 270 units / mg. Investigations of the molecular weight distribution showed that the molecular weight was in a much narrower range than that of the starting heparin.
Carbohydrate analysis was carried out according to the carbazole H2SO4 method.

Example 2

Purification of heparin with

matrix-bound antithrombin

(Approach method)

The matrix-bound antithrombin was produced according to Example 1.

Purification of heparin
500 mg heparin (specific activity 130 units /

mg) were in 300 ml of 0.05 M Tris / 0.15 M NaCl buffer, pH 7.4, dissolved 300 ml of deposited matrix-bound antithrombin were treated with the above Buffer equilibrated and sucked dry. The absorbent was added to the heparin solution, which was then stirred for 1 hour at room temperature. The gel was sucked dry on a glass filter and with 10 χ 200 ml of a 0.05 M Tris / 0.15 M NaCl buffer solution, pH 7.4, washed. The adsorbed heparin was then removed from the gel using 3 × 200 ml of a 0.05 M Tris / 1.0M NaCl buffer solution, pH 7.4, desorbed The eluted heparin was concentrated by freeze-drying. The remaining salt was passed through Gel filtration over Sephadex® G 25 m distilled water removed and the heparin then freeze-dried. The specific activity of the purified heparin was 250 units / mg.

Example 3

20th

Purification of heparin ir.it
matrix-bound inter-ar-trypsin inhibitor

25 Preparation of the absorption gel

During the purification of coagulation factor IX (β-factor) (L O. Andersson et al., Thromb. Res, Volume 7, 1975, pages 451-459) the heparin-binding protein inter-trypsin inhibitor was obtained. The molecular weight is approximately 150,000,250 mg of this protein was dissolved in 20 ml of a 0.2 M Nütf CO ₃ buffer, pH 9.0, and then added to 50 ml of settled Sepharise® 4B prepared with BrCN according to that described in Example 1 Procedure had been activated. The gel-protein suspension was stirred gently overnight at room temperature. The gel was then washed according to Example 1.

40

Purification of heparin

The adsorption gel containing the matrix-bound protein was placed in a column. 300 mg of heparin were dissolved in 20 ml of a 0.05 M Tris / 0.15 M NaCl buffer, pH 7.4, and pumped into the column. That Gel was washed with the buffer given above and the absorbed haperin was then washed with a 0.05 M Tris / 1.0M NaCl buffer solution, pH 7.4, desorbed. The salt was removed from the heparin by gel filtration over Sephadex® G 25 in distilled water and then freeze-dried the heparin. The specific activity of the purified heparin was 230 Units / mg.

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Claims (2)

Patent claims: 1. A method for purifying heparin, characterized in that the heparin with the help of antithrombin or inter-ar-trypsin inhibitor, which are bound to a matrix, absorbed and then desorbed 2. The method according to claim 1, characterized in that agarose is used as the matrix