DE2709500A1 - METHOD FOR CLEANING UP HEPARIN - Google Patents

Description

DR. ING. F. WDKSTJIOKK

I) H. K ν. ΡΚΟΠΜ TO N DK. ING. D. BEnHKNS DIPL. ING. R. GOETZ

PATEKTAH WlLTE

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PHITIOTP1TIIIIT M C NC HKN

1A-49

Applicant: Aktiebolaget KABI

London 133

S-112 87 Stockholm / Sweden

Title: Methods of Purification of

Heparin

70983 ^ / 0979

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description

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

Heparin has been used for medical treatments for several decades. It is one of the best known anticoagulants and is widely used, for example to prevent thrombosis. Heparin is a sulfated polysaccharide that can be produced or isolated from animal intestinal mucosa or lungs by relatively complicated processes. The heparin preparations used clinically today contain substances with a wide range of variation in molecular weight from approximately 5000 to approximately 35,000. The specific activity is usually approximately 130 units / mg. Thus, the currently commercially available preparations are quite heterogeneous. The incidence of side effects associated with heparin therapy is relatively low, but when such cases occur they often lead to serious problems. Some of these side effects are believed to be caused by contaminants that are present in the heparin preparation.

There are therefore important reasons to produce purer, specifically active heparin preparations than they are for Time to be applied. In connection with studies of antithrombin, a cofactor of heparin, the Investigated ways to bind the protein to a matrix. Surprisingly, it has been found that a matrix bound antithrombin specifically the molecular

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Could bind fraction of therapeutically applied heparin, which as a carrier of the clinically valuable thrombosis-preventing Effect of heparin. Under appropriately selected adsorption and desorption conditions, it is possible to produce a to obtain extremely pure heparin. The specific activities that could be obtained are from 200 to 270 units / mg versus 130 units / mg in the starting material. The molecular weight distribution in the specific purified heparin was narrower than in the original material. In addition to the heparin cofactor antithrombin, others can heparin-binding proteins are advantageously used, such as an inter-iV trypsin inhibitor with a molecular weight of approximately 150,000 obtained during the isolation of coagulation factor IX (B-factor). With one Matrix bound antithrombin the best heparin could be obtained with specific activities up to 270 units / mg.

Heparin with a very high purity and specific activity can be obtained with the aid of the invention Process which is simple and well applicable for industrial purposes for the production of heparin with a greater specific activity than can be achieved with known methods. Determining the specific Heparin activity was carried out according to the procedure given by Denson and Bonnar (1975, Brit. J. Haematol, Volume 30, page 139).

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

Example 1 Purification of heparin with matrix-bound antithrombin. Pillar method

The gel adsorbent was prepared as follows:

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activation *

3 g of BrCN were dissolved in 30 ml of distilled water. 50 ml of settled agarose were added to the BrCN solution (Sepharose ^ B, Pharmacia Fine Chemicals, Uppsala, Sweden). The mixture was cooled in an ice bath with stirring. The pH was increased to 11.2 by adding 4m NaOH and kept constant for 10 minutes. The gel was then using cold distilled water and 0.2m NaHCO, solution.

binding

200 mg antithrombin, purified according to Miller-Andersson et al (1974, Thromb Res Volume 5, pages 439 to 452), in 50 ml of 0.2 M NaHCO 3, pH 9.0, was added to the gel. The gel-protein suspension was stirred gently overnight at room temperature and then carefully with high ionic strength buffers washed with alternating high and low pH values.

Purification of heparin

Containing the antithrombin bound to the matrix Gel adsorbent was placed in a column and equilibrated with 0.05M Tris, 0.15M NaCl, pH 7.4, buffer. 300 mg Heparin (Vitrum, specific activity 130 units / mg) were dissolved in 20 ml of 0.05m Tris and 0.15m NaCl buffer pH 7.4 and pumped through the column. Subsequently, the gel was washed with the above-mentioned buffer, and then the adsorbed one Heparin is desorbed with a 0.05m Tris, 1.0m NaCl buffer pH 7.4. The salt was distilled by gel filtration in Water in a column containing Sephadex ° ü25 (Pharmacia) separated from the heparin and the heparin then freeze-dried. The purified heparin had a specific one Activity of 270 units / mg. Investigations of the molecular weight distribution showed that the molecular weight in was in a much narrower range than with the original heparin.

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Carbohydrate analysis was carried out by the carbazole H _{2 SO ^ method.}

Example 2 Purification of heparin with matrix-bound

Antithrombin. Approach process

The antithrombin matrix was prepared according to Example 1 manufactured.

Purification of heparin

500 mg of heparin (Vitrum specific activity 130 units / mg) were dissolved in 300 ml of 0.05m Tris, 0.15m NaCl buffer, pH 7-4. 300 ml of settled matrix-bound antithrombin were equilibrated with the above-mentioned buffer and sucked dry. The absorbent was added to the heparin solution, which was then stirred at room temperature for 1 hour. The gel was sucked dry on a glass filter and washed with 10 ml of a χ 200 0.05M Tris, 0.15M NaCl buffer solution ^1, washed 7 ». 4 The adsorbed heparin was then desorbed from the gel with the aid of 3 x 200 ml of a 0.05m Tris, 1.0m NaCl buffer solution, pH 7.4. The eluted heparin was concentrated by freeze drying. The remaining salt was removed by gel filtration through Sephadex G 25 in distilled water and the heparin was then freeze-dried. The specific activity of the purified heparin was 250 units / mg.

Example 3 Purification of Heparin with a Geladsoroens,

containing matrix-bound inter-ot-trypsin inhibitor

Manufacture of the gel adsorbent

During purification of coagulation factor IX (B-factor) (L.-0. Aneersson et al., Thromb Res, Volume 7 (1975 »pages 451-459) the heparin-binding protein inter-or-trypsin inhibitor was obtained. The molecular weight is approximately 150,000. 250 mg of this protein was dissolved in 200 ml of 0.2 M NaHCO 3, puffed ice, pH 9.0 and then added 50 ml of settled Sepharoser ^ 4B are given, which after with BrCN

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Λ-

the procedure described in Example 1 had been activated. The gel-protein suspension was left overnight at room temperature slightly stirred. The gel was then washed according to Example 1.

Purification of heparin

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

709836/0979

Claims (3)

Claims 1. J method for purifying heparin, characterized in that the heparin is adsorbed with the aid of heparin-binding plasma proteins which are bound to a matrix and then desorbed. 2. The method according to claim 1, characterized in that there is used as the adsorbent matrix-bound antithrombin or matrix-bound inter-tf-trypsin inhibitor used. 3. The method according to claim 1 or 2, characterized in that the matrix is agarose used. 6231 709836/097 9