DK155606B - METHOD OF CLEANING HEPARIN - Google Patents

Description

DK 155606B

The present invention relates to a process for the preparation of heparin with high specific activity; the process according to the invention is characterized by the characterizing part of claim 1.

5 Heparin has been used in medicine for several decades. It is one of the best known anticoagulants and is widely used i.a. to prevent blood clots. Heparin is a sulfated polysaccharide that can be prepared from animal gut or lung by a relatively complicated process. The heparin preparations used clinically nowadays contain material of very varied molecular size, from ca. 5000 up to approx. 35,000. The specific activity is usually approx. 130 units per mg. The commercially available commercially available preparations are thus quite heterogeneous. The frequency of side effects with heparin therapy is quite low, but the cases that occur cause problems that are difficult to solve. Some of these side effects are probably attributable to contaminants in the heparin preparation.

Therefore, there are good reasons to make cleaner, more specifically effective heparin preparations than those currently used. In connection with anti-thrombin co-factor to heparin studies, the possibility of the matrix binding protein was investigated. Surprisingly, it was found that matrix-bound (sucrose-bound) antithrombin could specifically bind the molecular fraction of the drug used as a drug, the carrier of the clinically valuable thrombosis prophylactic effect of heparin.

By suitable choice of adsorption conditions and conditions, an exceptionally pure heparin can be prepared. The specific activities gained were 200-270 units / mg, which should be compared with approx. 130 units / mg in the starting material. The molecular weight distribution in the purified heparin was much narrower than that of the starting material. In addition to the cofactor anti-thrombin of heparin, a heparin-binding protein can also be used

DK 155606 B

2, such as molecular weight inter-α-trypsin inhibitor, ca.

150,000, obtained during the isolation of coagulation factor IX (B factor). The best quality heparin was obtained with matrix-bound antithrombin with specific activities 5 up to 270 units / mg.

Thus, heparin of extremely high purity and specific activity can be prepared in the manner set forth herein. The process is simple and industrially applicable to produce heparin with higher specific activity than 10 existing preparations. Determination of the specific activity of heparin has been performed according to Denson and Bonnar (1975, Brit J Haematol, 30, page 139).

Some examples serve to elucidate the method of the invention.

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Example 1

Purification of heparin with matrix-bound antithrombin. Column Procedure_ 2Q The gel sorbent was prepared as follows:

Activation 3 g of BrCN was dissolved in 30 ml of distilled water. 50 ml of agarose ("Sepharose" 4B) was added to the BrCN solution. The mixture was cooled on an ice bath with stirring.

2 By the addition of 4M NaOH, the pH was raised to 11.2 and kept constant there for 10 minutes. The gel was then washed with cold distilled water and 0.2 M NaHCO 3.

Coupling 2Q 200 mg of antithrombin purified according to Miller-

Andersson et al. (1974, Thromb Res 5, pages 439-452) and dissolved in 50 ml of 0.2M NaHCO 3, pH 9.0, was added to the gel. The protein solution and gel were stirred at room temperature for one night and then washed thoroughly with high ionic strength buffer 2, alternating high and low pH.

DK 155606B

3

Heparinrensning

The gel sorbent containing matrix bound antithrombin was packed on a column and equilibrated with 0.05M Tris, 0.15M NaCl, pH 7.4. 300 mg of heparin (Vitruro, specific activity 130E / mg) was dissolved in 20 ml of 0.05M Tris, 0.15M NaCl, pH 7.4 buffer and pumped through the column.

The gel was washed with the above buffer and the adsorbed heparin was then desorbed with 0.05M Tris, 1.0M NaCl, pH 7.4 buffer. The eluted heparin is freed from salt

AED

by gel filtration on a column packed with "Sephadex" ^ G-25 in distilled water and freeze-dried. The purified heparin had a specific activity of 270 U / mg.

Examination of the molecular weight distribution of this heparin shows that it has significantly narrower molecular weight distribution than the starting heparin used.

Carbohydrate analysis by gel filtration was done by the carbazole® SO 2 method.

Example 2

Purification of heparin on matrix-bound antithrombin. Batch Process_

The antithrombin matrix was prepared as described in Example 1.

Heparin Purification 500 mg of heparin (Vitrum, specific activity 130 U / mg) was dissolved in 300 ml of 0.05M Tris, 0.15M NaCl, pH 7.4 buffer. 300 ml of sedimented antithrombin containing the gel absorbent was equilibrated in the above buffer and sucked dry. The gel was added to the heparin solution followed by stirring at room temperature for 1 hour. The gel was sucked dry on glass filter and washed with 10 x 200 ml 0.05M Tris, 0.15 NaCl, pH 7.4 buffer. The adsorbed heparin is then desorbed from the gel with 3 x 200 ml 0.05M Tris, 1.0M NaCl, pH 7.4. The eluted hepa-

DK 155606 B

4 rinse was concentrated by freeze drying. Residual salt was removed by gel filtration on nSephadex, in G 25 in distilled water followed by freeze drying. The specific activity of the purified heparin was 250 U / mg.

5

Example 3

Purification of heparin on gelase sorb containing matrix-bound inter-α-trypsin inhibitor pages 451-459), the heparin-binding protein in-ter-α-trypsin inhibitor was obtained. Its molecular weight is approx. 150,000.

250 mg of this protein was dissolved in 200 ml of 0.2 M NaHCO 3, pH 9.0 buffer and then added to 50 ml of sedimented "Sepharose" ® 4B activated with BrCN as described in Example 1. The protein solution and gel were stirred at room temperature overnight. over. The gel was then washed ana-2Q log with the gel of Example 1.

Heparinrensning

The gel sorbent containing the matrix bound protein was packed onto a column. 300 mg of heparin (Vitrum) dissolved in 20 ml of 0.05M Tris, 0.15M NaCl, pH 7.4 buffer was pumped into the column. The gel was washed with the above buffer and the adsorbed heparin was then desorbed with 0.05M Tris, 1 OM NaCl, pH 7.4 buffer. The heparin was liberated from salt by gel filtration on Sephadex, containing G 25 30 (crosslinked dextran) in distilled water and then lyophilized. The specific activity of the purified heparin was 230 U / mg.

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Claims (2)

A process for the preparation of heparin with high specific activity, characterized by the use of a water-insoluble '. gel matrix to which 5 is bound antithrombin or inter-α-trypsin inhibitor having a molecular weight of approx. 150,000 to adsorb a heparin fraction with specific antithrombin cofactor activity, after which the gel-bound heparin factor is desorbed. Process according to claim 1, characterized in that an agarose gel is used as a gel matrix. 15 20 25 30 35