CA1340217C - Process for the isolation and preparation of pasteurized alpha2-antiplasmin product - Google Patents
Process for the isolation and preparation of pasteurized alpha2-antiplasmin productInfo
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- CA1340217C CA1340217C CA000616377A CA616377A CA1340217C CA 1340217 C CA1340217 C CA 1340217C CA 000616377 A CA000616377 A CA 000616377A CA 616377 A CA616377 A CA 616377A CA 1340217 C CA1340217 C CA 1340217C
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- antiplasmin
- mol
- solution
- sucrose
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Abstract
A process for the pasteurization of an antiplasmin solution, which comprises adjusting an antiplasmin solution to a pH of 6 to 9.5 and heating it in the presence of a monosaccharide or disaccharide, sugar alcohol or an organic dicarboxylic or tricarboxylic acid and, where appropriate, an amino acid, for at least one hour at a temperature of 40 to 90°C.
Description
... 13~2~7 Thi~ Application i~ a Divi~ional of rAn~ n Patent Application Serial No. 528,511, Filed 29 January 1987.
A process for the isolation and preparation of a pasteurized alpha2-antiplasmin product The invention relates to a process for the isolation of alpha2-antiplasmin from a body fluid, a tissue or a cell culture, and for the preparation of a pasteurized product thereof. Preparations of this type can be used for therapeutic purposes.
Alpha2-ant;plasmin is designated antiplasmin hereinafter.
Antiplasmin is an inhibitor of the fibrinolytic system and acts, in particular, as an inhibitor of plasmin. It is a glycoprotein ~ith a molecular ~eight of about 65,000 Dalton and is present in a concentration of about 70 mg/l in blood. Its properties make it a potentially interesting therapeutic agent for the treatment of both congenital and acquired disease states, in particular of hyperfibrinolysis. The lo~ plasma concentration and the properties of the molecule have hitherto prevented isola-tion on the large scale industrially.
Processes for the isolation of the inhibitor from plasma have, ho~ever, already been described (Biochem. J. 159 (1976) 545-553; Eur. J. Biochem. 78 (1977) 19-26;
J.B.C. 251, No. 19 (1976) 5956-5965). These processes require process steps ~hich cannot be used on the large scale in ~ trially, such as the use of concanavalin A-Sepharos or gel filtration.
The therapeutic use of proteins from body fluids, tissues or cell cultures is associated ~ith the risk of trans-mission of viral diseases. Heat-treatment of plasma pro-teins at 60~C for ten hours has proved suitable for virus inactivation. An inactivation of this type can be carried out only if the protein is stable or can be stabilized.
~ 2 - 1 3 4 0 2 1 7 However, it ;s known that prot-eins, and in particular inhibitors of enzymes, may be unstable to heat.
. .
Hence the object of the present invention was to develop a process for the preparation, on the large scale indus-trially, of anantiplasmin concentrate which is pasteurized where appropriate.
It has been found, surprisingly, that this object can be achieved by modification of known purification steps and removal of contaminating proteins by precipitation in one dialysis step and heat-treatment with the addition of stabilizers, without essential loss of antiplasmin activity.
The invention relates to a process for the purification and, where appropriate, isolation and, where appropriate, pasteurization of a solution of antiplasmin.
For the purification, dialysis against an aqueous solu-tion of low ionic strength is carried out, and the result-ing sediment is removed.
The dialysis is preferably carried out against a low-concentration salt solution of pH 6 to 9, preferably 6.5 to 8.5, and of a conductivity equal to or less than 5, pre-ferably 1 to 2, mSi (20~C) until a corresponding conduc-tivity is reached in the dialyzed solution.
The precipitated impurities can be removed by filtration or centrifugation.
This dialysis brings about an improvement in the purity such as can be achieved only with the aid of several other purification steps which, by reason of their elaborateness and the loss of yield, have proven unfavorable.
The antiplasmin can be precipitated from the dialyzed solution using a neutral salt, for example using 0.1 to . ._.
13 40 2 ~ 7 134~217 0.77 kg/l, preferably 0.18 to~0.56 kg/l, ammonium sulfate.
However, the dialyzed solution can also be contacted with an anion exchanger and, by stepped gradient elution, the S antiplasmin separated from concomitant proteins and iso-lated.
For this purpose, the antiplasmin solution can be con-tacted with an anion exchanger, for example DEAE-Sephade the anion exchanger which is loaded with antiplasmin can be washed with, for example, a buffer containing 0.02 mol/l phosphate and O.OS mol/l NaCl, pH 6 to 9, preferably 6 to 8, and the antiplasmin can be eluted with a buffer containing, for example, 0.02 mol/l phosphate, pH 6 to 9, and 0.1 to 1 mol/l NaCl, preferably 0.2 to 1 mol/l NaCl.
To isolate a particularly pure antiplasmin, it is possi-ble to make use of its known property of binding to plasminogen. For this purpose it is possible to contact an antiplasmin-containing solution, which ought to be low in plasminogen, preferably plasma which is low in plas-minogen, with a plasminogen which is bound to a solid phase, to wash the solid phase ~hich is loaded with anti-plasmin, to elute the antiplasmin and, where appropriate,to precipitate the antiplasmin from the solution using a neutral salt. The latter measure can be carried out before or after the dialysis which is described above.
For the removal of plasminogen from the antiplasmin-containing solution, for example of 60 to 80% of the plasminogen present in plasma or a plasma fraction, it is possible to adsorb the plasminogen on a lysine-resin, for example on lysine-Sepharose~ It is then possible to con-tact the antiplasmin-containing solution with carri~-bound plasminogen, preferably plasminogen-Sepharose~ at 4~C to 30~C, for 10 minutes to 24 hours, to remove impurities from the loaded affinity material by washing with a buffer, for example a O.OS mol/l phosphate solution 1 3 ~ O 2 1 7 ~340~17 of pH 6 to 9, and to elute the antiplasmin with a buffer, for example a phosphate buffer containing NaCl, pH 6 to 9, preferably 7 to 8, and a conductivity of 20 to 60 mSi (20~C) s The invention also relates to a process for inactivating viruses in an antiplasmin solution, which comprises ad-justing the antiplasmin solution to a pH of 6 to 9.5, preferably 7 to 8.5, and heating it in the presence of a monosaccharide or disaccharide, sugar alcohol or an or-ganic dicarboxylic or tricarboxylic acid and, where appro-priate, an amino acid, for at least one hour, preferably 8 to 20 hours, at a temperature of 40 to 90~C, preferably 50 to 70~C.
It is preferable to add 0.5 to 2 kg/l sucrose or 0.5 to 2 kg/l sorbitol or 0.1 to 4 mol/l potassium citrate and 0.2 to 1.5 kg/l sucrose and, where appropriate, a water-soluble amino acid, preferably 1 to 114 g/l glycine, and to heat at 60~C for 10 hours. It is also possible to use mixtures of the said additives.
A purified and, where appropriate, pasteurized antiplasmin solution can then, where appropriate, be concentrated, sterilized and freeze-dried. ~efore the lyophilization it is possible to add stabilizers, for example glycine, albumin, a sugar or gelatin.
A product prepared by the process described is distinguished by the yield being two to five times that of products pre-pared according to the state of the art. Furthermore, a product of this type is very stable and has high activity.
The invention is to be illustrated in detail by the examples which follow.
Example 1 10 l of Cohn fraction I supernatant were dialyzed against , ,,.. . . , _, ~ .. .... . . . .. ...... .
1 3 ~,O 2 17 1340~17 - water at 4~C for 15 hours until~the conductivity of the dialysate was 0.5 mSi (20~C). The precipitate which had formed was removed by centrifugation.
This dialysis brought about a tripling of the specific activity and a loss of 2% of the yield.
Example 2 10 l of Cohn fractig~ I supernatant were mixed with 1 kg of lysine-SepnaroseW (Pharmacia, Sweden) and stirred at room temperature for 80 minutes. The supernatant which was low in plasminogen was mixed with 0.4 kg of plasmino-gen-Sepharose~ and stirred at 4~C for 2 hours. The affinity material was then washed with 0.05 mol/l phos-phate, pH 7, and the antiplasmin was eluted with a solu-tion containing 0.05 mol/l phosphate and 0.5 mol/l NaCl, pH 7. The eluate was mixed with 0.5 kg/l ammonium sul-fate, and the resulting precipitate was taken up in 0.05 mol/l phosphate, pH 7, and dialyzed against 0.02 mol/l phosphate and 0.05 moL/l NaCL, pH 7, at 4~C for 15~ ours.
The solution was mixed with 0.035 kg DEAE-Sephadex~ The anion exchanger was separated off, washed with a solution containing 0.02 mol/l phosphate and 0.05 mol/l NaCl, pH 7, and the antiplasmin eluted with a buffer containing 0.02 mol/l phosphate and 0.2 mol/l NaCl, pH 7.
The eluate was concentrated, sterilized by filtration and freeze-dried.
Example 3 A phosphate-buffered (0.02 mol/l) antiplasmin solution obtained as in Example 1 or 2 was mixed with 1 kg/l sucrose and 37 g/l glycine, adjusted to pH 8, and heated at 60~C for 10 hours. 83~ of the initial activity remained.
- Example 4 An antiplasmin solution was adjusted to pH 7.5 with 0.02 mol/l phosphate and 0.15 mol/l NaCl, and 0.5 mol/l glycine and 1.5 kg/l sucrose were added, and the mix-ture was heated at 60~C for 10 hours. The initial ac-tivity remained after the heating. The activity like-wise remained when, in place of sucrose, use was made of the same concentration of sorbitol or, in place of glycine and 1.5 kg/l sucrose, use was made of 4 mol/l potassium citrate and 0.5 kg/l sucrose.
Example S
A buffered antiplasmin solution was mixed with 1 kg/l sucrose and 37 g/l glycine, adjusted to pH 6.5, 7, 7.5, 8, 8.5, 9 or 9.5 and heated at 60~C for 10 hours.
The following results were found after the heating:
Activity before heating: 100%
Activity after heating: pH 6.5 46.8%
pH 7.0 51.1X
pH 7.5 71.1X
pH 8.0 83.3X
pH 8.5 63.8X
pH 9.0 56.4%
pH 9.5 56.9%
Example 6 An ~2-antiplasmin solution was adjusted to pH 7.5 with a buffer containing 0.02 mol/l phosphate and 0.15 mol/l NaCL, and 0.5 mol/l glycine and various amounts of sucrose, sorbitol or potassium citrate were added, and the mixture was heated at 60~C for 10 hours.
The following results were found:
Activity after heating in %
Additive (100% before heating) 5 0.1 g/ml sucrose 0%
0.25 g/mL " 0%
0.5 g/ml " 0%
0.75 g/ml " 35%
1.0 g/ml " 63%
1.5 g/ml " 100%
0.1 g/ml sorbitol 0%
0.25 g/ml " 0%
0.5 g/ml " 20%
0.75 g/ml " 45%
1.0 g/ml " 60%
1.5 g/ml " 100%
0.1 mol/l potassium citrate + 1 mg/ml sucrose 30%
20 0.5 mol/l " " 1 mg/ml " 33X
1 mol/l " " 1 mg/ml " 34X
2 mol/l " " 0.5 mg/ml " 50%
A process for the isolation and preparation of a pasteurized alpha2-antiplasmin product The invention relates to a process for the isolation of alpha2-antiplasmin from a body fluid, a tissue or a cell culture, and for the preparation of a pasteurized product thereof. Preparations of this type can be used for therapeutic purposes.
Alpha2-ant;plasmin is designated antiplasmin hereinafter.
Antiplasmin is an inhibitor of the fibrinolytic system and acts, in particular, as an inhibitor of plasmin. It is a glycoprotein ~ith a molecular ~eight of about 65,000 Dalton and is present in a concentration of about 70 mg/l in blood. Its properties make it a potentially interesting therapeutic agent for the treatment of both congenital and acquired disease states, in particular of hyperfibrinolysis. The lo~ plasma concentration and the properties of the molecule have hitherto prevented isola-tion on the large scale industrially.
Processes for the isolation of the inhibitor from plasma have, ho~ever, already been described (Biochem. J. 159 (1976) 545-553; Eur. J. Biochem. 78 (1977) 19-26;
J.B.C. 251, No. 19 (1976) 5956-5965). These processes require process steps ~hich cannot be used on the large scale in ~ trially, such as the use of concanavalin A-Sepharos or gel filtration.
The therapeutic use of proteins from body fluids, tissues or cell cultures is associated ~ith the risk of trans-mission of viral diseases. Heat-treatment of plasma pro-teins at 60~C for ten hours has proved suitable for virus inactivation. An inactivation of this type can be carried out only if the protein is stable or can be stabilized.
~ 2 - 1 3 4 0 2 1 7 However, it ;s known that prot-eins, and in particular inhibitors of enzymes, may be unstable to heat.
. .
Hence the object of the present invention was to develop a process for the preparation, on the large scale indus-trially, of anantiplasmin concentrate which is pasteurized where appropriate.
It has been found, surprisingly, that this object can be achieved by modification of known purification steps and removal of contaminating proteins by precipitation in one dialysis step and heat-treatment with the addition of stabilizers, without essential loss of antiplasmin activity.
The invention relates to a process for the purification and, where appropriate, isolation and, where appropriate, pasteurization of a solution of antiplasmin.
For the purification, dialysis against an aqueous solu-tion of low ionic strength is carried out, and the result-ing sediment is removed.
The dialysis is preferably carried out against a low-concentration salt solution of pH 6 to 9, preferably 6.5 to 8.5, and of a conductivity equal to or less than 5, pre-ferably 1 to 2, mSi (20~C) until a corresponding conduc-tivity is reached in the dialyzed solution.
The precipitated impurities can be removed by filtration or centrifugation.
This dialysis brings about an improvement in the purity such as can be achieved only with the aid of several other purification steps which, by reason of their elaborateness and the loss of yield, have proven unfavorable.
The antiplasmin can be precipitated from the dialyzed solution using a neutral salt, for example using 0.1 to . ._.
13 40 2 ~ 7 134~217 0.77 kg/l, preferably 0.18 to~0.56 kg/l, ammonium sulfate.
However, the dialyzed solution can also be contacted with an anion exchanger and, by stepped gradient elution, the S antiplasmin separated from concomitant proteins and iso-lated.
For this purpose, the antiplasmin solution can be con-tacted with an anion exchanger, for example DEAE-Sephade the anion exchanger which is loaded with antiplasmin can be washed with, for example, a buffer containing 0.02 mol/l phosphate and O.OS mol/l NaCl, pH 6 to 9, preferably 6 to 8, and the antiplasmin can be eluted with a buffer containing, for example, 0.02 mol/l phosphate, pH 6 to 9, and 0.1 to 1 mol/l NaCl, preferably 0.2 to 1 mol/l NaCl.
To isolate a particularly pure antiplasmin, it is possi-ble to make use of its known property of binding to plasminogen. For this purpose it is possible to contact an antiplasmin-containing solution, which ought to be low in plasminogen, preferably plasma which is low in plas-minogen, with a plasminogen which is bound to a solid phase, to wash the solid phase ~hich is loaded with anti-plasmin, to elute the antiplasmin and, where appropriate,to precipitate the antiplasmin from the solution using a neutral salt. The latter measure can be carried out before or after the dialysis which is described above.
For the removal of plasminogen from the antiplasmin-containing solution, for example of 60 to 80% of the plasminogen present in plasma or a plasma fraction, it is possible to adsorb the plasminogen on a lysine-resin, for example on lysine-Sepharose~ It is then possible to con-tact the antiplasmin-containing solution with carri~-bound plasminogen, preferably plasminogen-Sepharose~ at 4~C to 30~C, for 10 minutes to 24 hours, to remove impurities from the loaded affinity material by washing with a buffer, for example a O.OS mol/l phosphate solution 1 3 ~ O 2 1 7 ~340~17 of pH 6 to 9, and to elute the antiplasmin with a buffer, for example a phosphate buffer containing NaCl, pH 6 to 9, preferably 7 to 8, and a conductivity of 20 to 60 mSi (20~C) s The invention also relates to a process for inactivating viruses in an antiplasmin solution, which comprises ad-justing the antiplasmin solution to a pH of 6 to 9.5, preferably 7 to 8.5, and heating it in the presence of a monosaccharide or disaccharide, sugar alcohol or an or-ganic dicarboxylic or tricarboxylic acid and, where appro-priate, an amino acid, for at least one hour, preferably 8 to 20 hours, at a temperature of 40 to 90~C, preferably 50 to 70~C.
It is preferable to add 0.5 to 2 kg/l sucrose or 0.5 to 2 kg/l sorbitol or 0.1 to 4 mol/l potassium citrate and 0.2 to 1.5 kg/l sucrose and, where appropriate, a water-soluble amino acid, preferably 1 to 114 g/l glycine, and to heat at 60~C for 10 hours. It is also possible to use mixtures of the said additives.
A purified and, where appropriate, pasteurized antiplasmin solution can then, where appropriate, be concentrated, sterilized and freeze-dried. ~efore the lyophilization it is possible to add stabilizers, for example glycine, albumin, a sugar or gelatin.
A product prepared by the process described is distinguished by the yield being two to five times that of products pre-pared according to the state of the art. Furthermore, a product of this type is very stable and has high activity.
The invention is to be illustrated in detail by the examples which follow.
Example 1 10 l of Cohn fraction I supernatant were dialyzed against , ,,.. . . , _, ~ .. .... . . . .. ...... .
1 3 ~,O 2 17 1340~17 - water at 4~C for 15 hours until~the conductivity of the dialysate was 0.5 mSi (20~C). The precipitate which had formed was removed by centrifugation.
This dialysis brought about a tripling of the specific activity and a loss of 2% of the yield.
Example 2 10 l of Cohn fractig~ I supernatant were mixed with 1 kg of lysine-SepnaroseW (Pharmacia, Sweden) and stirred at room temperature for 80 minutes. The supernatant which was low in plasminogen was mixed with 0.4 kg of plasmino-gen-Sepharose~ and stirred at 4~C for 2 hours. The affinity material was then washed with 0.05 mol/l phos-phate, pH 7, and the antiplasmin was eluted with a solu-tion containing 0.05 mol/l phosphate and 0.5 mol/l NaCl, pH 7. The eluate was mixed with 0.5 kg/l ammonium sul-fate, and the resulting precipitate was taken up in 0.05 mol/l phosphate, pH 7, and dialyzed against 0.02 mol/l phosphate and 0.05 moL/l NaCL, pH 7, at 4~C for 15~ ours.
The solution was mixed with 0.035 kg DEAE-Sephadex~ The anion exchanger was separated off, washed with a solution containing 0.02 mol/l phosphate and 0.05 mol/l NaCl, pH 7, and the antiplasmin eluted with a buffer containing 0.02 mol/l phosphate and 0.2 mol/l NaCl, pH 7.
The eluate was concentrated, sterilized by filtration and freeze-dried.
Example 3 A phosphate-buffered (0.02 mol/l) antiplasmin solution obtained as in Example 1 or 2 was mixed with 1 kg/l sucrose and 37 g/l glycine, adjusted to pH 8, and heated at 60~C for 10 hours. 83~ of the initial activity remained.
- Example 4 An antiplasmin solution was adjusted to pH 7.5 with 0.02 mol/l phosphate and 0.15 mol/l NaCl, and 0.5 mol/l glycine and 1.5 kg/l sucrose were added, and the mix-ture was heated at 60~C for 10 hours. The initial ac-tivity remained after the heating. The activity like-wise remained when, in place of sucrose, use was made of the same concentration of sorbitol or, in place of glycine and 1.5 kg/l sucrose, use was made of 4 mol/l potassium citrate and 0.5 kg/l sucrose.
Example S
A buffered antiplasmin solution was mixed with 1 kg/l sucrose and 37 g/l glycine, adjusted to pH 6.5, 7, 7.5, 8, 8.5, 9 or 9.5 and heated at 60~C for 10 hours.
The following results were found after the heating:
Activity before heating: 100%
Activity after heating: pH 6.5 46.8%
pH 7.0 51.1X
pH 7.5 71.1X
pH 8.0 83.3X
pH 8.5 63.8X
pH 9.0 56.4%
pH 9.5 56.9%
Example 6 An ~2-antiplasmin solution was adjusted to pH 7.5 with a buffer containing 0.02 mol/l phosphate and 0.15 mol/l NaCL, and 0.5 mol/l glycine and various amounts of sucrose, sorbitol or potassium citrate were added, and the mixture was heated at 60~C for 10 hours.
The following results were found:
Activity after heating in %
Additive (100% before heating) 5 0.1 g/ml sucrose 0%
0.25 g/mL " 0%
0.5 g/ml " 0%
0.75 g/ml " 35%
1.0 g/ml " 63%
1.5 g/ml " 100%
0.1 g/ml sorbitol 0%
0.25 g/ml " 0%
0.5 g/ml " 20%
0.75 g/ml " 45%
1.0 g/ml " 60%
1.5 g/ml " 100%
0.1 mol/l potassium citrate + 1 mg/ml sucrose 30%
20 0.5 mol/l " " 1 mg/ml " 33X
1 mol/l " " 1 mg/ml " 34X
2 mol/l " " 0.5 mg/ml " 50%
3 mol/l " " 0.5 mg/ml " 82%
4 mol/l " " 0.5 mg/ml " 100X
Claims (7)
1. A process for the pasteurization of an undialyzed antiplasmin solution, which comprises adjusting the antiplasmin solution to a pH of 6 to 9.5 and heating it in the presence of a monosaccharide or disaccharide, sugar alcohol or an organic dicarboxylic or tricarboxylic acid for at least one hour at a temperature of 40 to 90°C.
2. The process as claimed in claim 1, wherein the pH of the antiplasmin solution is adjusted to pH 7 to 8.5, and wherein the heating step continues for 8 to 20 hours at a temperature of 50 to 70°C.
3. The process as claimed in claim 1, wherein, for the pasteurization of an antiplasmin solution, 0.5 to 2 kg/l sucrose or 0.5 to 2 kg/l sorbitol or 0.1 to 4 ml/l potassium citrate and 0.2 to 1.5 kg/l sucrose are added and heating is carried out at 40 to 90°C for 8 to 20 hours.
4. The process as claimed in claim 2, wherein, for the pasteurization of an antiplasmin solution, 0.5 to 2 kg/l sucrose or 0.5 to 2 kg/l sorbitol or 0.1 to 4 ml/l potassium citrate and 0.2 to 1.5 kg/l sucrose are added and heating is carried out at 40 to 90°C for 8 to 20 hours.
5. The process as claimed in claim 1, wherein the antiplasmin solution is heated in the presence of a monosaccharide or disaccharide, sugar alcohol or organic dicarboxylic or tricarboxylic acid and an amino acid.
6. The process as claimed in claim 3 or 4, wherein a water-soluble amino acid is added.
7. The process as claimed in claim 5 or 6, wherein 1 to 114 g/l glycine are added.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19863602688 DE3602688A1 (en) | 1986-01-30 | 1986-01-30 | METHOD FOR OBTAINING AND PRODUCING A PASTEURIZED PREPARATE OF (ALPHA) (ARROW DOWN) 2 (ARROW DOWN) -ANTIPLASMINE |
| DE3602688.3 | 1986-01-30 | ||
| CA000528511A CA1307373C (en) | 1986-01-30 | 1987-01-29 | Process for the isolation and preparation of a pasteurized alpha_-antiplasmin product |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000528511A Division CA1307373C (en) | 1986-01-30 | 1987-01-29 | Process for the isolation and preparation of a pasteurized alpha_-antiplasmin product |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1340217C true CA1340217C (en) | 1998-12-15 |
Family
ID=25671219
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000616377A Expired - Fee Related CA1340217C (en) | 1986-01-30 | 1992-05-12 | Process for the isolation and preparation of pasteurized alpha2-antiplasmin product |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1340217C (en) |
-
1992
- 1992-05-12 CA CA000616377A patent/CA1340217C/en not_active Expired - Fee Related
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