IE44907B1 - Method for the purification of heparin - 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

THIS INVENTION relates to a method for the purification of heparin to give a heparin which is very pure and of high specific activity.

Heparin has been used in medical treatment 5 for several decades. It is one of the best-known anti-coagulants and in extensive use, e.g. in the prevention of thrombosis. Heparin is a sulphated polysaccharide which can be prepared from animal intenstinal mucus or lungs by comparatively complicated methods.

The heparin preparations used clinically to-day contain material with a great variety in molecular size, from about 5,000 to about 35,000. The specific activity usually is about 130 units per mg. Thus, the commercial preparations available to-day are quite heterogeneous.

The frequency of side effects in heparin therapy is rather low but where cases occur, they often present severe problems. Some of these side effects are probably caused by impurities in the heparin preparation.

There are, therefore, good arguments for producing purer, more specifically active heparin preparations than those in current use. Our invention arises from studies of antithrombin, a co-factor of heparin, during which the possibilities of binding the protein to a matrix were investigated. Surprisingly, we found that matrix - bound antithrombin could specifically - 2 .j, bind the molecule fraction of therapeutically used heparin that acts as a carrier of the clinically valuable; thrombose-prophylactic effect of heparin.

The present invention provides a method for the preparation of heparin of specific activity greater than 130 units/mg, wherein a heparin containing preparation is brought into contact v/ith a matrix-bound heparin-binding plasma protein, the protexn carrying adsorbed heparin is separated from the preparation and the heparin is then released from the protein. The units which are the measure of specific activity are determined as described in t'he Denson & Bonner method for measuring specific activity (1975, Brit J. Haematol, 30 p.l3S).

With suitably selected adsorption and desorption conditions, an extraordinarily pure heparin can be prepared. We have secured specific activities of 200-270 units/mg. compared to approximately 130 units/mg of the starting material. 'The distribution of molecular v/eights in our purified heparin was far more limited than in the primary material. We find it practical to carry out the adsorption and desorption at a pH in the range 5.0-9.5 by passing a solution of the impure heparin in a buffer of desired pH through a column of the matrix-bound protein and then eluting the column, carrying the - 3 heparin, with a buffer of desired pH. The pH during adsorption and desorption can be the same, generally in the region of 7.0, and Tris/NaCl buffers of pH 7.A are particularly suitable. Other buffer solutions compatible with heparin, can also be used. The buffer solution used for desorption may contain the same salutes and be of the same pH as that used in adsorption but will have a higher ionic strength so as to salt out the heparin. For example;,' the ionic strength in the adsorption buffer can be 0-0.6 M while that in the desorption buffer would then be above 0.6 M.

Xn addition to the heparin co-factor antithrombin, other heparin-binding proteins which can be used include inter-a-trypsin inhibitor preferably with a molecular weight of about 150,000, obtained during isolation of coagulation factor IX (B-factor).

We find that the best quality of heparin was· obtained, with specific activities up to 270 units per mg. using matrix-bound antithrombin.

The heparin binding protein can be bound to any convenient insoluble polymeric matrix,for exanple agarose, one of the various modified dextrans sold as a Sephadex (Registered Trade Mark) gel or a polyacrylamide.

The present invention also includes a pharmaceutical composition comprising a heparin obtained by the method of the present invention together with 49 0 7 a pharmaceutically acceptable carrier.

The following Examples illustrate the invention: The determination of the specific activity of heparin has been made according to Denson and Bonnar (1975, Brit J Haematol, 30, p. 139).

EXAMPLE 1 Purification of heparin on matrix-bound antithrorobin Column procedure The gel adsorbent was prepared as follows: Activation g. of BrCN was dissolved in 30 ml. of distilled water 50 ml. of settled agarose (Sepharose (Registered Trade Mark) ZiB, Pharmacia Fine Chemicals, Uppsala, Sweden) was added to the BrCN-solution. The mixture was cooled on an ice bath while stirred. By adding Z|M NaOH, the pH was raised to 11.2 and kept constant for 10 minutes. The gel was then washed with cold distilled water and 0.2 M NaHCO^.

Linking 200 mg. Antithrcmbin purified according to Miller-Andersson et al (197Z), Thromb Res 5 pp Z|39-Z|52) and dissolved in 50 ml. 0.2 M NaHCO^, pH 9.0 was added to the gel. The gel protein suspension was gently •stirred in room temperature Overnight and then carefully ’washed with buffers of.high ion strength with alternately high and low pH values. - 5 4-4®®^ I Purification of heparin The agarose containing bound antithrombin was packed in gel form in a column and equilibrated with 0.05 M Tris, 0.15 M NaCl, pH 7.4 buffer. 300 mg. heparin (Vitrum, specific activity 130 units/mg.) was dissolved in 20 ml. 0.05 M Tris, 0.15 M NaCl, pH 7.4 buffer, and pumped through the column. Thereafter, the gel was washed with the abovementioned buffer and then the adsorbed heparin was desorbed with a 0.05 M Tris, 1.0 M NaCl, pH 7.4 buffer. The salt was removed from the eluted heparin by gel filtration in distilled water in a column packed with Sephadex G 25 (Pharmacia) and after that the heparin was freezedried. The purified heparin had a specific activity of 270 units/mg. Studies of the distribution of molecular weights show that it has a considerably more limited molecular weight distribution than has the primary heparin.

Carbohydrate analysis was performed with the carbazoleH2SO4 method.

EXAMPLE 2 ?Hti£ication_Qf_hegarin_on_matrix-bound_antithrombin.

Batch procedure The matrix-bound antithrombin was prepared as described in Example 1.

Purification of heparin 500 mg. heparin (Vitrum. specific activity 130 units/mg.) was dissolved in 300 ml. 0.05 M Tris, 0.15 M NaCi, pH 7.4 buffer. 300 ml. of settled matrixbound antithrombin was equilibrated in the abovementioned buffer and suction-dried. The matrix-bound antithrombin gel was added to the heparin solution which v/as then stirred in room temperature for one hour. The gel was suction dried on a glass filter and washed v/ith 10 x 200 ml. of a 0.05 M Tris 0,15 M NaCb pH 7.4 buffer. The adsorbed heparin v/as then desorbed from the gel by 3 x 200 ml. 0,05 M Tris, 1.0 M Had, pH 7.4 buffer. The eluted heparin was concentrated by freeze-drying.

The remaining salt was removed by gel filtration on Sephadex G 25 in distilled water, and then the heparin was freeze dried. The specific activity of ths purified heparin was 250 units/mg, SIMPLE 3 E2li£i2S£i2S_2£_'2£B2£i2~22-22=-S-22£-2®Si:_222iSi2i2S ?Ktrix2bound_inter-gHtrY£sin_inhibifcor Preparation of gel adsorbent During the purification of coagulation factor IX (B-f actor) (33.-0. Andersson et al., Thromb Res 7. (1975), pp. 451-459), the heparin-binding protein interα-trypsin inhibitor was obtained. Its molecular1 weight - 7 is approximately 150,000. 250 mg. of this protein was dissolved in 200 ml. of a O^'MNaHCOg, pH 9.0 buffer and then added to 50 ml. of settled agarose (Sepharose 4B) activated with BrCN by the procedure described in Example 1. The gel-protein suspension was gently stirred in room temperature overnight. Then the gel was washed as described in Example 1.

Purification of heparin The gel comprising matrix-bound protein was packed in a column. 300 mg. heparin (Vitrum) was dissolved in 20 ml. of a 0.05 M Tris, 0.15 M NaCl, pH 7.4 buffer and pumped into the column. The gel was washed with the abovementioned buffer and the adsorbed heparin was then desorbed with a 0.05 M Tris, 1.0 M NaCl, pH 7.4 buffer. The salt was removed from the heparin by gel filtration on Sephadex G25 in distilled water gnd then the heparin was freese-dried. The specific activity of the purified heparin was 230 units/mg.

Claims (5)

1. A method for the preparation of heparin of specific activity greater than 130 units (determined as hereinbefore defined) per mg, wherein a heparin 5 containing preparation is brought into contact with a matrix-bound heparin-binding plasma protein, the protein carrying adsorbed heparin is separated from the preparation and the heparin is then released from the protein. 10 2. A method according to claim 1 wherein the protein is matrix-bound antithrombin. 3. A method according to claim 1, wherein the protein is matrix-bound inter-a-trypsin inhibitor. Zj. A method according to any one of the preceding claims, wherein the matrix to which the protein is bound is agarose. 5. A method according to any one of the preceding claims wherein a solution of heparin in a first buffer of pH 5.0-9.5 is passed through

2. Q a column of the matrix-bound protein and the heparin is then removed from the matrix-bound protein by passing through the column of matrix-bound protein a second buffer of PH 5.0-9.5 and of higher ionic strength than the first buffer.

3. 6. A method according to Claim 1 substantially as hereinbefore described v/ith reference to any one of the Examples.

4. 7. Heparin of high specific activity obtained by a method according to any one of the preceding Claims.

5. 8, A pharmaceutical composition comprising heparin according to Claim 7 together with a pharmaceutically acceptable carrier.