FI62103C - REQUIREMENTS FOR THE REQUIREMENT OF HEPARIN MED HOEG SPECIFIC ACTIVITIES - Google Patents

Description

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Sweden-Sweden (SE) 76030lt02 (71) Aktiebolaget Kabi, S-LOU 25 Stockholm, Sweden-Sweden (SE) (72) Lars-Olov Andersson, Knivsta, Erik Yngve Holmer, Bromma,

Swedish-Swedish (SE) (7 * 0 0y Roister Ab (5U) Method for the preparation of heparin with high specific activity - Förfarande för framställning av heparin med hög specif aktivitet

The invention relates to a process for the preparation of heparin having a high specific activity. The process according to the invention is characterized in that a water-insoluble gel matrix containing agarose, to which an antithrombin or an interprinin inhibitor with a molecular weight of about 150,000 is bound, is used for a heparin fraction with a specific activity of 200-270 U / mg. to selectively adsorb the thione from a starting material having a specific activity of 100-160 U / mg, after which the gel-bound heparin fraction is desorbed.

Heparin has been used in the medical field for several decades. It is one of the best known coagulation inhibitors and is widely used, e.g. to prevent blood clots. Heparin is a sulfated polysaccharide that can be prepared from the intestinal mucosa or lungs of animals by a relatively complex method. The heparin preparations currently in clinical use contain substances with very variable molecular sizes, from about 5,000 to about 35,000. The specific activity is usually about 130 units / mg.

2 62103

The commercial preparations now available are thus quite heterogeneous. There are relatively few side effects associated with heparin treatment, but the cases that do occur often cause problems that are difficult to resolve. Some of these side effects are likely to be due to impurities in heparin preparations.

It is therefore justified to produce heparin preparations that are purer and more specific than those currently used. Studies with the antithrombin component of the heparin component examined the potential for binding to the protein matrix. It was surprisingly found that antithrombin bound to the matrix (agarose) was specifically able to bind a molecular fraction in heparin used as a drug with a clinically valuable thrombosprophicactic effect of heparin. By appropriately selecting the adsorption and desorption conditions, excellent pure heparin can be prepared. The specific activities obtained were 200-270 units / mg compared to about 130 units / mg in the starting material. The molecular weight distribution in purified heparin was much narrower than in the starting material. In addition to the heparin component antithrombin, other heparin-binding proteins, such as the inter-trypsin inhibitor having a molecular weight of about 150,000 and obtained in isolation of coagulation factor IX (factor B), may also be advantageously used. Qualitatively, the best heparin was obtained with matrix-bound antithrombin, with specific activities increasing to 270 units / mg.

Thus, the process according to the invention makes it possible to prepare heparin which is extremely pure and has a high specific activity. The method is simple and industrially applicable and the heparin obtained has a higher specific activity than the existing preparations. Determination of heparin specific activity has been performed according to Denson and Bonnar and Bonnar (1975, Brit. J. Haematol, 30, p. 139). The following examples illustrate the invention.

Example 1

Purification of heparin with matrix-bound antithrombin

Kolonnimenetelmä

The gel adsorbent was prepared as follows:

Activation 3 g of BrCN was dissolved in 30 ml of distilled water. 50 ml of sedimented agarose (Sepharose 4B, Pharmacia Fine Chemicals) was added to the BrCN solution. The mixture was cooled in an ice bath with stirring. The pH was raised to 11.2 by the addition of 4M NaOH and kept constant for 10 minutes at 3 62103. The gel was then washed with cold distilled water and 0.2 M NaHCO 3.

Combination 200 mg of antithrombin purified according to Miller-Andersson et al. (1974, Thromb Res. 5, pages 439-452) and dissolved in 50 ml of 0.2 M NaHCO 3 pH 9.0 was added to the gel. The protein solution and gel were mixed at room temperature overnight and then washed thoroughly with high ionic strength and variable buffers; correspondingly high and low pH. !

Heparin purification

A gel adsorbent containing matrix-bound antithrombin was packed on a column and equilibrated with 0.05 M Tris buffer (0.15 M NaCl, pH 7.4). 300 mg of heparin (Vitrum, specific activity 130 units / mg) was dissolved in 20 ml of 0.05 M Tris buffer, 0.15 M NaCl, pH 7.4 and pumped through the column. The gel was washed with the above buffer and the adsorbed heparin was then desorbed with 0.05 M Tris, 1.0 M NaCl, pH 7. The eluted heparin was liberated from the salt by gel filtration on a column packed with Sephadex® G-25 (Pharmacia) in distilled water. and lyophilized. The specific activity of purified heparin was 270 units / mg.

Studies of the molecular weight distribution for this heparin showed that it has a significantly narrower molecular weight distribution than the starting heparin.

Carbohydrate analysis in gel filtration has been performed by the carbazole-H 2 SO 4 method.

Example 2 »

Purification of heparin by matrix-bound antithrombin11a

Input method '

An antithrombin matrix was prepared according to Example 1.

i

Purification of heparin: 500 mg of heparin (Vitrum, specific activity 130 units / mg) was dissolved in 300 ml of buffer 0.05 M Tris, 0.15 M NaCl, pH 7.4).

300 ml of sedimented antithrombin containing gel adsorbent 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 suction filtered through a glass filter and washed 10 times with 200 ml of 0.05 M Tris buffer, 0.15 NaCl, pH 7.4. The adsorbed heparin was then desorbed from the gel three times with 200 ml of buffer (0.05 M Tris, 1.0 M NaCl, pH 7.4). The eluted heparin was concentrated by freeze-drying. The remaining salt was removed by gel filtration in water distilled with Sephadex ®c 25, followed by freeze-drying. The specific activity of purified heparin was 250 units / mg.

Example 3

Purification of heparin with a gel adsorbent containing matrix-bound inter-cK-trypsin inhibitor

Preparation of gel adsorbent:

Purification of coagulation factor IX (factor B) (L.-O. Andersson et al., 1975, Tromb Res, 7, pages 451-459) provided a heparin-binding protein, an inter-β-trypsin inhibitor. The weight of the protein is about 150,000. 250 mg of this protein was dissolved in 200 ml of buffer 0.2 M NaHCO 3 pH 9.0 and then added to 50 ml of sedimented Sepharose® 4B activated with BrCN according to Example According to Method 1. The protein solution and gel were mixed at room temperature overnight, and the gel was then washed in the same manner as the gel in Example 1.

Heparin purification:

A gel adsorbent containing matrix-bound protein was packed into a column. 300 mg of heparin (Vitrum) dissolved in 20 ml of buffer 0.05 M Tris, 0.15 M NaCl, pH 7.4 was pumped onto the column. The gel was washed with the above buffer and the adsorbed heparin was then desorbed with 0.05 M Tris, 1.0 M NaCl, pH 7.4. Heparin was freed from the salts by gel filtration in water distilled with Sephadex G 25 and then lyophilized. The specific activity of purified heparin was 230 units / mg.