CA1079272A - Method for the purification of heparin - Google Patents
Method for the purification of heparinInfo
- Publication number
- CA1079272A CA1079272A CA271,731A CA271731A CA1079272A CA 1079272 A CA1079272 A CA 1079272A CA 271731 A CA271731 A CA 271731A CA 1079272 A CA1079272 A CA 1079272A
- Authority
- CA
- Canada
- Prior art keywords
- heparin
- bound
- gel matrix
- matrix
- plasma protein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
Abstract
ABSTRACT OF THE DISCLOSURE
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.
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
` `- 1079Z72 .
The present invention relates to a method ~or the purification of heparin to produce heparin that is very pure and possesses high specific activity.
Heparin has been used in medical treatment for several decades. It is one of the best-known anticoagulants and in extensive use, i.e., for the prevention of thrombosis. Heparin is a sulfated polysaccaride which can be prepared from animal ~-intestinal mucus or lungs by relatively complicated methods. The heparin preparations used clinically today contain material with a great range 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 today are quite heterogenous.
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. In connection with studies of antithrombin, a co-factor of heparin, the possibilities of binding the protein to a matrix were investigated. Surprisingly, it was found that matrix-bound antithrombin could specifically bind the molecule fraction of therapeutically used heparin that acts as a carrier of the clinically valuable, thrombose-prophylactic effect of heparin. With suitably selected adsorption and desorption conditions, an extraordinarily pure heparin can be prepared.
The specific activlties obtained were 200 - 270 units/mg. versus approximately 130 units/mg. of the starting material. The distribution of molecular weights in the specifically purified heparin was far more limited than in the primary material. In addition to the heparin co-factor antithrombin, other heparin-binding proteins can be used to advantage, such as inter-~-trypsin '''' ' - 1 - ~ :.
'' ' ' ~, ' : ' :, 107927~:
, inhibitor with a molecular weight of about 150,000, obtained during isolation of coa~ulation factor IX (B-factor). With matrix-bound antithrombin, the best quality of heparin was obtained, with specific activities up to 270 units per mg. -;~
Thus, heparin with a very high degree of purity and specific activity can be produced by the process of the present invention which is a simple one and applicable for industrial use for the preparation of heparin with greater specific activity than is the case with existing preparations. The determination of the specific activity of heparin has been made according to Denson and Bonnar (1975, Brit J Haematol, 30, p. 139).
According to-the present invention therefore there is provided 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.
The following examples illustrate the invention:
Example 1 Purification of he~arin on matrix-bound ~ . Column ~rocedur~
The yel adsorbent was prepared as follows:
Activation 3 g. of BrCN was dissolved in 30 ml. of distilled water. 50 ml. of settled agarose (Sepharose~ 4B, Pharmacia Fine Chemicals, Uppsala, Sweden) was added to the BrCN-solution. The mixture was cooled on an icebath while stirred. By adding ~ M NaOH, 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 NaHCO3.
Linkinq 200 mg. antithrombin purified according to Miller-Andersson et al (1974, Thromb Res 5 pp 439-452) and dissolved in 50 ml. 0.2 M NaHCO3, ph 9.0 was added to the gel. The gel protein suspension was gently stirred in room temperature over niqht and then carefully washed with buffers of high ion strength with alternately high and low pH values.
Purification of he~arin -The gel adsorbent containing matrix-hound antithrombin was packed in a column and equilibrated with 0.05 M Tris, 0.15 M NaCl, pH 7.4 buffer. 30b mg. heparin (Vitrum, specific activity 130 units/mg.) was dissolved in 20 ml. 0.05 M Tris, B
0.15 M NaCl, pH 7.4 buffer, and pumped through the column. There-after, the gel was washed with the abovementioned buffer and then the adsorbed heparin was desorbed with a 0.05 r~ Tris, 1~.0 M
NaCl, pH 7.4 buffer. The salt was removed from the eluted he~arin by gel filtration is distilled water in a column packed with SephadexR G 25 (Pharmacia) and after that the heparin was freeze-dried. 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 limlted molecular weight distribution than has the primary heparin. Carbon hydrate analysis was performed with the carbazol-H2SO4 method.
Example 2 Purification of heparin on matrix-bound antithrombin. Batch ~rocedure The antithrombin matrix was prepared according to Example 1.
Purification of heparin 500 mg. heparin (Vitrum, specific activity 130 unitsjmg.) was dissolved in 300 ml. 0.05 M Tris, 0.15 M NaCl, pH 7.4 buffer.
300 ml. of settled matrix-bound anti-thrombin was equilibrated in the abovementioned buffer and suction-dried. The adsorbent :-was added to the heparin solution which was then stirred in room temperature for one hour. The gel was suction-dried on a glass filter and washed with 10 x 200 ml. of a 0.05 M Tris 0.15 M NaCl, pH 7.4 buffer. The adsorbed heparin was then desorbed from the gel by 3 x 200 ml. 0.05 M Tris, 1.0 M NaCl, pH 7.4. buffer.
- 10792~2 :-The eluted heparin was concentrated by freeze-drying. The remaining salt was removed by gel filtration on Sephadex ~, 25 (a trademark) in distilled water, and then the heparin was freeze-dried. The specific activity of the purified heparin was 250 units/mg.
Example 3 Purification of heparin on ~el adsorbent containin~ matrix-__________________ ________ __.,.__________________.~________ bound inter-~-trypsin inhibitor ________________ _____________ Preparation of gel adsorbent During the purificationof coagulation factor IX(~factor) (L.-O. Andersson et al., Thromb Res 7 (1975), pp. 451-459), the heparin-binding protein inter-~-trypsin inhibitor was obtained. Its molecular weight is approximately 150,000. 250 mg.
of this protein was dissolved in 200 ml. of a 0.2 M NaHCO3, pH
9.0 buffer and then added to 50 ml. of settled SepharoseR 4B
activated with BrCN according to the procedure as described in Example 1. The gel-protein suspension was gently stirred in room temperature over night. Then the gel was washed as the gel according to Example 1.
Purification of heparin:
The gel adsorbent containiny 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 filtrationon Sephadex G25 in distilled water and then the heparin was freeze-dried. The specific activity of the purified heparin was 230 units/mg.
The present invention relates to a method ~or the purification of heparin to produce heparin that is very pure and possesses high specific activity.
Heparin has been used in medical treatment for several decades. It is one of the best-known anticoagulants and in extensive use, i.e., for the prevention of thrombosis. Heparin is a sulfated polysaccaride which can be prepared from animal ~-intestinal mucus or lungs by relatively complicated methods. The heparin preparations used clinically today contain material with a great range 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 today are quite heterogenous.
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. In connection with studies of antithrombin, a co-factor of heparin, the possibilities of binding the protein to a matrix were investigated. Surprisingly, it was found that matrix-bound antithrombin could specifically bind the molecule fraction of therapeutically used heparin that acts as a carrier of the clinically valuable, thrombose-prophylactic effect of heparin. With suitably selected adsorption and desorption conditions, an extraordinarily pure heparin can be prepared.
The specific activlties obtained were 200 - 270 units/mg. versus approximately 130 units/mg. of the starting material. The distribution of molecular weights in the specifically purified heparin was far more limited than in the primary material. In addition to the heparin co-factor antithrombin, other heparin-binding proteins can be used to advantage, such as inter-~-trypsin '''' ' - 1 - ~ :.
'' ' ' ~, ' : ' :, 107927~:
, inhibitor with a molecular weight of about 150,000, obtained during isolation of coa~ulation factor IX (B-factor). With matrix-bound antithrombin, the best quality of heparin was obtained, with specific activities up to 270 units per mg. -;~
Thus, heparin with a very high degree of purity and specific activity can be produced by the process of the present invention which is a simple one and applicable for industrial use for the preparation of heparin with greater specific activity than is the case with existing preparations. The determination of the specific activity of heparin has been made according to Denson and Bonnar (1975, Brit J Haematol, 30, p. 139).
According to-the present invention therefore there is provided 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.
The following examples illustrate the invention:
Example 1 Purification of he~arin on matrix-bound ~ . Column ~rocedur~
The yel adsorbent was prepared as follows:
Activation 3 g. of BrCN was dissolved in 30 ml. of distilled water. 50 ml. of settled agarose (Sepharose~ 4B, Pharmacia Fine Chemicals, Uppsala, Sweden) was added to the BrCN-solution. The mixture was cooled on an icebath while stirred. By adding ~ M NaOH, 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 NaHCO3.
Linkinq 200 mg. antithrombin purified according to Miller-Andersson et al (1974, Thromb Res 5 pp 439-452) and dissolved in 50 ml. 0.2 M NaHCO3, ph 9.0 was added to the gel. The gel protein suspension was gently stirred in room temperature over niqht and then carefully washed with buffers of high ion strength with alternately high and low pH values.
Purification of he~arin -The gel adsorbent containing matrix-hound antithrombin was packed in a column and equilibrated with 0.05 M Tris, 0.15 M NaCl, pH 7.4 buffer. 30b mg. heparin (Vitrum, specific activity 130 units/mg.) was dissolved in 20 ml. 0.05 M Tris, B
0.15 M NaCl, pH 7.4 buffer, and pumped through the column. There-after, the gel was washed with the abovementioned buffer and then the adsorbed heparin was desorbed with a 0.05 r~ Tris, 1~.0 M
NaCl, pH 7.4 buffer. The salt was removed from the eluted he~arin by gel filtration is distilled water in a column packed with SephadexR G 25 (Pharmacia) and after that the heparin was freeze-dried. 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 limlted molecular weight distribution than has the primary heparin. Carbon hydrate analysis was performed with the carbazol-H2SO4 method.
Example 2 Purification of heparin on matrix-bound antithrombin. Batch ~rocedure The antithrombin matrix was prepared according to Example 1.
Purification of heparin 500 mg. heparin (Vitrum, specific activity 130 unitsjmg.) was dissolved in 300 ml. 0.05 M Tris, 0.15 M NaCl, pH 7.4 buffer.
300 ml. of settled matrix-bound anti-thrombin was equilibrated in the abovementioned buffer and suction-dried. The adsorbent :-was added to the heparin solution which was then stirred in room temperature for one hour. The gel was suction-dried on a glass filter and washed with 10 x 200 ml. of a 0.05 M Tris 0.15 M NaCl, pH 7.4 buffer. The adsorbed heparin was then desorbed from the gel by 3 x 200 ml. 0.05 M Tris, 1.0 M NaCl, pH 7.4. buffer.
- 10792~2 :-The eluted heparin was concentrated by freeze-drying. The remaining salt was removed by gel filtration on Sephadex ~, 25 (a trademark) in distilled water, and then the heparin was freeze-dried. The specific activity of the purified heparin was 250 units/mg.
Example 3 Purification of heparin on ~el adsorbent containin~ matrix-__________________ ________ __.,.__________________.~________ bound inter-~-trypsin inhibitor ________________ _____________ Preparation of gel adsorbent During the purificationof coagulation factor IX(~factor) (L.-O. Andersson et al., Thromb Res 7 (1975), pp. 451-459), the heparin-binding protein inter-~-trypsin inhibitor was obtained. Its molecular weight is approximately 150,000. 250 mg.
of this protein was dissolved in 200 ml. of a 0.2 M NaHCO3, pH
9.0 buffer and then added to 50 ml. of settled SepharoseR 4B
activated with BrCN according to the procedure as described in Example 1. The gel-protein suspension was gently stirred in room temperature over night. Then the gel was washed as the gel according to Example 1.
Purification of heparin:
The gel adsorbent containiny 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 filtrationon Sephadex G25 in distilled water and then the heparin was freeze-dried. The specific activity of the purified heparin was 230 units/mg.
Claims (15)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A method for the preparation of heparin of high specific activity which comprises contacting heparin with an absorbent which is a gel matrix-bound heparin-binding plasma protein selected from antithrombin and inter-.alpha.-trypsin inhibitor.
2. A method according to Claim 1, in which the adsorbent is matrix-bound antithrombin.
3. A method according to Claim 1, in which the adsorbent is matrix-bound inter-.alpha.-trypsin inhibitor.
4. A method according to Claim 1, 2 or 3, in which the matrix is an agarose.
5. A method for purifying a crude heparin preparation to provide a heparin preparation of enhanced heparin specific activity, which method comprises the combination of steps of (a) contacting the starting heparin preparation with a water-insoluble gel matrix :
having adsorbently bound to it a heparin-binding plasma protein selected from antithrombin and inter-.alpha.-trypsin inhibitor to provide a gel matrix with adsorbently bound said plasma protein and the heparin bound thereto, and (b) separating the heparin from said gel matrix having said heparin-binding plasma protein bound to it.
having adsorbently bound to it a heparin-binding plasma protein selected from antithrombin and inter-.alpha.-trypsin inhibitor to provide a gel matrix with adsorbently bound said plasma protein and the heparin bound thereto, and (b) separating the heparin from said gel matrix having said heparin-binding plasma protein bound to it.
6. The method as claimed in claim 5, wherein antithrombin is the heparin-binding plasma protein bound to the gel matrix.
7. The method as claimed in claim 5, wherein inter-.alpha.-trypsin inhibitor is the heparin-binding plasma protein bound to the gel matrix.
8. The method as claimed in claim 5, wherein the water-insoluble gel matrix is the beaded agarose gel prepared by having an about 4% aqueous solution of agarose in bead form.
9. The method as claimed in claim 8, wherein said agarose gel beads are activated by activation treatment with a cyanogen halide.
10. The method as claimed in claim 9, wherein the cyanogen halide is cyanogen bromide.
11. The method as claimed in claim 5, wherein the initial contact between the starting heparin preparation and the gel matrix with said adsorbently bound plasma protein is conducted by feeding said heparin preparation dissolved in a compatible aqueous buffer solution through a column packed bed of said gel matrix.
12. The method as claimed in claim 5, wherein the initial contact between the starting heparin preparation and the gel matrix with said adsorbently bound plasma protein is conducted by admixing said heparin preparation dissolved in a compatible buffer solution with said gel matrix.
13. The method as claimed in claim 5, wherein said heparin is separated from said gel matrix having the heparin bound to the gel matrix with heparin-binding plasma protein bound to it, by desorbing the heparin therefrom by elution with an aqueous solution containing about one-half percent of sodium chloride dissolved in it.
14. The method as claimed in claim 13, wherein sodium chloride in the separated heparin is removed therefrom by gel filtration.
15. The method as claimed in claim 5, wherein the separated heparin is dried by lyophilization.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| SE7603040A SE431218B (en) | 1976-03-05 | 1976-03-05 | HEPARIN PURIFICATION PROCEDURE |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1079272A true CA1079272A (en) | 1980-06-10 |
Family
ID=20327240
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA271,731A Expired CA1079272A (en) | 1976-03-05 | 1977-02-14 | Method for the purification of heparin |
Country Status (21)
| Country | Link |
|---|---|
| JP (1) | JPS52108012A (en) |
| AT (1) | AT356818B (en) |
| AU (1) | AU508430B2 (en) |
| CA (1) | CA1079272A (en) |
| CS (1) | CS196349B2 (en) |
| DE (1) | DE2709500C2 (en) |
| DK (1) | DK155606C (en) |
| ES (1) | ES456196A1 (en) |
| FI (1) | FI62103C (en) |
| FR (1) | FR2342991A1 (en) |
| GB (1) | GB1539332A (en) |
| HU (1) | HU176811B (en) |
| IE (1) | IE44907B1 (en) |
| IL (1) | IL51346A (en) |
| NL (1) | NL186385C (en) |
| NO (1) | NO144388C (en) |
| NZ (1) | NZ183480A (en) |
| PL (1) | PL103049B1 (en) |
| SE (1) | SE431218B (en) |
| SU (1) | SU736860A3 (en) |
| ZA (1) | ZA771267B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| SE449753B (en) * | 1978-11-06 | 1987-05-18 | Choay Sa | MUCOPOLYSACCARIDE COMPOSITION WITH REGULATORY EFFECTS ON COAGULATION, MEDICINAL CONTAINING ITS SAME AND PROCEDURE FOR PREPARING THEREOF |
| DE3531101A1 (en) * | 1984-08-31 | 1986-03-13 | Nicolás Huberto Olivos Behrens | A NON-THROMBIC HEPARINE AND A METHOD FOR ITS PRODUCTION |
| WO2015112121A1 (en) * | 2014-01-21 | 2015-07-30 | The Board Of Trustees Of The University Of Arkansas | Heparin affinity tag for use in protein purification |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US2623001A (en) * | 1949-04-07 | 1952-12-23 | Bengt E G V Sylven | Preparing heparin |
| DE1195010B (en) * | 1962-05-12 | 1965-06-16 | Ormonoterapia Richter S P A | Method for purifying heparin by the chromatographic method |
-
1976
- 1976-03-05 SE SE7603040A patent/SE431218B/en not_active IP Right Cessation
-
1977
- 1977-01-27 IL IL51346A patent/IL51346A/en unknown
- 1977-02-14 CA CA271,731A patent/CA1079272A/en not_active Expired
- 1977-02-15 FI FI770491A patent/FI62103C/en not_active IP Right Cessation
- 1977-02-22 NL NLAANVRAGE7701882,A patent/NL186385C/en not_active IP Right Cessation
- 1977-02-23 ES ES456196A patent/ES456196A1/en not_active Expired
- 1977-02-25 JP JP2016477A patent/JPS52108012A/en active Pending
- 1977-03-02 NZ NZ183480A patent/NZ183480A/en unknown
- 1977-03-03 IE IE469/77A patent/IE44907B1/en not_active IP Right Cessation
- 1977-03-03 ZA ZA00771267A patent/ZA771267B/en unknown
- 1977-03-04 DE DE2709500A patent/DE2709500C2/en not_active Expired
- 1977-03-04 FR FR7706469A patent/FR2342991A1/en active Granted
- 1977-03-04 AU AU22953/77A patent/AU508430B2/en not_active Expired
- 1977-03-04 HU HU77KA1485A patent/HU176811B/en unknown
- 1977-03-04 SU SU772457090A patent/SU736860A3/en active
- 1977-03-04 AT AT144577A patent/AT356818B/en not_active IP Right Cessation
- 1977-03-04 PL PL1977196438A patent/PL103049B1/en unknown
- 1977-03-04 GB GB9275/77A patent/GB1539332A/en not_active Expired
- 1977-03-04 NO NO770764A patent/NO144388C/en unknown
- 1977-03-04 DK DK096077A patent/DK155606C/en not_active IP Right Cessation
- 1977-03-07 CS CS771523A patent/CS196349B2/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| JPS52108012A (en) | 1977-09-10 |
| SU736860A3 (en) | 1980-05-25 |
| AT356818B (en) | 1980-05-27 |
| NZ183480A (en) | 1979-08-31 |
| GB1539332A (en) | 1979-01-31 |
| FI770491A (en) | 1977-09-06 |
| SE431218B (en) | 1984-01-23 |
| DE2709500C2 (en) | 1986-09-18 |
| ATA144577A (en) | 1979-10-15 |
| IE44907B1 (en) | 1982-05-19 |
| FR2342991B1 (en) | 1981-10-30 |
| CS196349B2 (en) | 1980-03-31 |
| PL103049B1 (en) | 1979-05-31 |
| NO144388B (en) | 1981-05-11 |
| SE7603040L (en) | 1977-09-06 |
| FI62103B (en) | 1982-07-30 |
| DK155606C (en) | 1989-09-11 |
| FR2342991A1 (en) | 1977-09-30 |
| IL51346A0 (en) | 1977-03-31 |
| NL186385B (en) | 1990-06-18 |
| NL186385C (en) | 1990-11-16 |
| DK96077A (en) | 1977-09-06 |
| HU176811B (en) | 1981-05-28 |
| ES456196A1 (en) | 1978-07-01 |
| DK155606B (en) | 1989-04-24 |
| IL51346A (en) | 1979-11-30 |
| IE44907L (en) | 1977-09-05 |
| AU508430B2 (en) | 1980-03-20 |
| ZA771267B (en) | 1978-01-25 |
| NO770764L (en) | 1977-09-06 |
| NL7701882A (en) | 1977-09-07 |
| NO144388C (en) | 1981-08-19 |
| DE2709500A1 (en) | 1977-09-08 |
| AU2295377A (en) | 1978-09-07 |
| FI62103C (en) | 1982-11-10 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| MKEX | Expiry |