CA1307373C - Process for the isolation and preparation of a pasteurized alpha_-antiplasmin product - Google Patents
Process for the isolation and preparation of a pasteurized alpha_-antiplasmin productInfo
- Publication number
- CA1307373C CA1307373C CA000528511A CA528511A CA1307373C CA 1307373 C CA1307373 C CA 1307373C CA 000528511 A CA000528511 A CA 000528511A CA 528511 A CA528511 A CA 528511A CA 1307373 C CA1307373 C CA 1307373C
- Authority
- CA
- Canada
- Prior art keywords
- antiplasmin
- solution
- buffer
- pasteurized
- plasminogen
- 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 - Lifetime
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/81—Protease inhibitors
- C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
- C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
- C07K14/8121—Serpins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/55—Protease inhibitors
- A61K38/57—Protease inhibitors from animals; from humans
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Gastroenterology & Hepatology (AREA)
- Animal Behavior & Ethology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Pharmacology & Pharmacy (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Diabetes (AREA)
- Zoology (AREA)
- Epidemiology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Immunology (AREA)
- Hematology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
86/B 005 - Ma 539 Dr. Ha/Sd.
Abstract of the disclosure:
A process for the purification and, where appropriate, isolation and, where appropriate, pasturization of a solution of alpha2-antiplasmin is described, in which, for the purification, dialysis is carried out against an aqueous solution of Low ionic strength, and the resulting sediment is removed. For the isolation of a particularly pure antiplasmin it is furthermore possible to purify the solution by chromatography using plasminogen bound to a solid phase. Furthermore, a process for the inactivation of viruses in an antiplasmin solution by heating in the presence of stabilizers is described.
Plasmin prepared in the manner described can be used for therapeutic purposes.
Abstract of the disclosure:
A process for the purification and, where appropriate, isolation and, where appropriate, pasturization of a solution of alpha2-antiplasmin is described, in which, for the purification, dialysis is carried out against an aqueous solution of Low ionic strength, and the resulting sediment is removed. For the isolation of a particularly pure antiplasmin it is furthermore possible to purify the solution by chromatography using plasminogen bound to a solid phase. Furthermore, a process for the inactivation of viruses in an antiplasmin solution by heating in the presence of stabilizers is described.
Plasmin prepared in the manner described can be used for therapeutic purposes.
Description
1307~73 8EHRINGWERKE AKTIENGESELLSCHAFT 86/B 005 - Ma 539 Dr. Ha/Sd~
A process for the isola~ion and preparation of a pasteurized alpha2-an.iplasmin product The invention relates to a process for the isolation of alpha2-antiplasmin from a body fluid, a tissue or a cell cùlture, and for the preparation of a pasteurized product thereof. Preparations of this type can be used for therapeutic purposes.
Alpha2-antiplasmin 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 with a molecular weight 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 congen;tal and acquired disease states, in particular of hyperfibrinolysis. The low 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, however, already been described (Biochem. J. 159 (1976) 545-553; Eur. J. 8iochem. 78 (1977) 19-26;
J.B.C. 251, No. 19 (1976) 5956-5965). These processes require process steps which cannot be used on the large scale industrially, such as the use of concanavalin A-Sepharos ~ or gel filtration.
The therapeutic use of proteins from body flu;ds, tissues or cell cultures is associated with the risk of trans-mission of viral diseases. ~eat-treatment of plasma pro-teins at 60C 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.
However, it is known that proteins, 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 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 ant;plasm;n solution can be con-tacted with an anion exchanger, for example DEAE-Sephade 1û 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 ~, 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 û.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 which 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-conta;ning solution, for example of 6û 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 4C to 30C, for 10 minutes to 24 hours, to remove impurities from the loaded affinity material by washing with a buffer, for example a 0.05 mol/l phosphate solution 13073~3 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 8r and a conductivity of 20 to 60 mSi (20C) 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 4n to 90C, preferably 50 to 70C.
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 60C for 10 hours. It is also possible to use mixtures of the said add;tives.
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.
_xample 1 10 l of Cohn fraction I supernatant were dialyzed against ~307373 water at 4C for 15 hours until the conductivity of the d;alysate was û.5 mSi (2ûC). 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 1û 10 l of Cohn fracti I supernatant were mixed with 1 kg of lysine-Sepnarose~ (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 4C 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 U.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 4C for 15,~hours.
The solution was mixed with 0.035 kg DEAE-Sepnadex~ The anion exchanger was separated off, washed with a solution containing O.û2 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 60C for 10 hours. 83% of the initial activity remained.
1307~73 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 60C 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 5 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 60C 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 51.1%
pH 7.5 71.1%
pH 8.0 83.3%
pH 8.5 63.8%
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 60C for 10 hours.
The following results were found:
~307373 . ., 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%
10 1.5 g/ml " 100%
0.1 g/ml sorbitol0%
0.25 g/ml " 0%
0.5 g/ml " 20%
15 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 " 33%
1 mol/l " " 1 mg/ml " 34%
2 mol/l " " 0.5 mg/ml " 50%
A process for the isola~ion and preparation of a pasteurized alpha2-an.iplasmin product The invention relates to a process for the isolation of alpha2-antiplasmin from a body fluid, a tissue or a cell cùlture, and for the preparation of a pasteurized product thereof. Preparations of this type can be used for therapeutic purposes.
Alpha2-antiplasmin 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 with a molecular weight 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 congen;tal and acquired disease states, in particular of hyperfibrinolysis. The low 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, however, already been described (Biochem. J. 159 (1976) 545-553; Eur. J. 8iochem. 78 (1977) 19-26;
J.B.C. 251, No. 19 (1976) 5956-5965). These processes require process steps which cannot be used on the large scale industrially, such as the use of concanavalin A-Sepharos ~ or gel filtration.
The therapeutic use of proteins from body flu;ds, tissues or cell cultures is associated with the risk of trans-mission of viral diseases. ~eat-treatment of plasma pro-teins at 60C 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.
However, it is known that proteins, 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 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 ant;plasm;n solution can be con-tacted with an anion exchanger, for example DEAE-Sephade 1û 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 ~, 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 û.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 which 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-conta;ning solution, for example of 6û 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 4C to 30C, for 10 minutes to 24 hours, to remove impurities from the loaded affinity material by washing with a buffer, for example a 0.05 mol/l phosphate solution 13073~3 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 8r and a conductivity of 20 to 60 mSi (20C) 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 4n to 90C, preferably 50 to 70C.
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 60C for 10 hours. It is also possible to use mixtures of the said add;tives.
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.
_xample 1 10 l of Cohn fraction I supernatant were dialyzed against ~307373 water at 4C for 15 hours until the conductivity of the d;alysate was û.5 mSi (2ûC). 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 1û 10 l of Cohn fracti I supernatant were mixed with 1 kg of lysine-Sepnarose~ (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 4C 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 U.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 4C for 15,~hours.
The solution was mixed with 0.035 kg DEAE-Sepnadex~ The anion exchanger was separated off, washed with a solution containing O.û2 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 60C for 10 hours. 83% of the initial activity remained.
1307~73 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 60C 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 5 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 60C 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 51.1%
pH 7.5 71.1%
pH 8.0 83.3%
pH 8.5 63.8%
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 60C for 10 hours.
The following results were found:
~307373 . ., 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%
10 1.5 g/ml " 100%
0.1 g/ml sorbitol0%
0.25 g/ml " 0%
0.5 g/ml " 20%
15 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 " 33%
1 mol/l " " 1 mg/ml " 34%
2 mol/l " " 0.5 mg/ml " 50%
3 mol/l " " 0.5 mg/ml " 82%
4 mol/l " " 0.5 mg/ml " 100%
Claims (28)
1. A process for the purification of a solution of antiplasmin, which comprises dialysis of a solution of antiplasmin against an aqueous solution of low ionic strength and removal of the resulting sediment and obtaining a solution of antiplasmin.
2. The process as claimed in claim 1, wherein dialysis is carried out against a Low-concentration salt solution of pH 6 to 9 and of a conductivity equal to or less than 5 mSi (20°C) until a corresponding conductivity is reacted in the dialyzed solution.
3. The process as claimed in claim 1, wherein dialysis is carried out against a low-concentration salt solution of pH 6.5 to 8.5 and of a conductivity equal to or less than 1 to 2 mSi (20°C) until a corresponding conductivity is reached in the dialyzed solution.
4. The process as claimed in claim 1, wherein a solution of antiplasmin which is low in plasminogen is used.
5. The process as claimed in claim 2, wherein a solution of antiplasmin which is low in plasminogen is used.
6. The process as claimed in claim 3, wherein a solution of antiplasmin which is low in plasminogen is used.
7. The process as claimed in claim 1 which comprises purifying the solution of antiplasmin.
8. The process as claimed in any one of claims 1 to 5 wherein the solution of antiplasmin is further purified by contacting the solution of antiplasmin with an anion exchanger, and the antiplasmin being separated from concomitant proteins and isolated by a stepped gradient elution.
9. The process as claimed in any one of claims 1 to 6, wherein the solution of antiplasmin is further purified by contacting with an anion exchanger, the anion exchanger which is loaded with antiplasmin is washed with a buffer of pH 6 to 9, and the antiplasmin is eluted with a buffer of pH 6 to 9, containing 0.1 to 1 mol/l of a neutral salt.
10. The process as claimed in any one of claims 1 to 6, wherein the solution of antiplasmin is further purified by contacting the solution of the antiplasmin with an anion exchanger, the anion exchanger which is loaded with antiplasmin is washed with a buffer of pH 6 to 9, and the antiplasmin is eluted with a buffer of pH 6 to 9, containing 0.2 to 1 mol/l of a neutral salt.
11. The process as claimed in any one of claims 4 to 6, wherein the solution of antiplasmin is further purified by contacting the solution of antiplasmin with plasminogen which is bound to a solid phase, washing the solid phase which is loaded with antiplasmin, eluting the antiplasmin, precipitating the antiplasmin from the solution using a neutral salt, dialyzing against a low-concentration salt solution, and removing the sediment.
12. The process as claimed in any one of claims 4 to 6, wherein before dialysis the solution of antiplasmin is purified by contacting the solution of antiplasmin with plasminogen which is bound to a solid phase, washing the solid phase which is loaded with antiplasmin, eluting the antiplasmin, precipitating the antiplasmin from the solution using a neutral salt, dialyzing against a low-concentration salt solution, and removing the sediment.
13. The process as claimed in any one of claims 4 to 6, wherein the solution of antiplasmin is further purified by contacting the solution of antiplasmin with plasminogen which is bound to a solid phase, impurities are removed from the loaded affinity material by washing with a buffer pH 6 to 9, and the antiplasmin is eluted with a buffer of pH 6 to 9 and of a conductivity of 20 to 60 mSi (20°C).
14. The process as claimed in any one of claims 4 to 6, wherein before dialysis the solution of antiplasmin is purified by contacting the solution of antiplasmin with plasminogen which is bound to a solid phase, impurities are removed from the loaded affinity material by washing with a buffer pH 6 to 9, and the antiplasmin is eluted with a buffer of pH 6 to 9 and of a conductivity of 20 to 60 mSi (20°C).
15. The process as claimed in any one of claims 4 to 6, wherein the solution of antiplasmin is further purified by contacting the solution of antiplasmin with plasminogen which is bound to a solid phase, impurities are removed from the loaded affinity material by washing with a buffer pH 6 to 9, and the antiplasmin is eluted with a buffer of pH 7 to 8 and of a conductivity of 20 to 60 mSi (20°C).
16. The process as claimed in any one of claims 4 to 6, wherein before dialysis the solution of antiplasmin is purified by contacting the solution of antiplasmin with plasminogen which is bound to a solid phase, impurities are removed from the loaded affinity material by washing with a buffer pH 6 to 9, and the antiplasmin is eluted with a buffer of pH 7 to 8 and of a conductivity of 20 to 60 mSi (20°C).
17. The process as claimed in claim 1 wherein the solution of antiplasmin is pasteurized.
18. The process as claimed in any one of claims 1 to 7, wherein the solution of antiplasmin is pasteurized by adjusting the antiplasmin solution to a pH 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.
19. The process as claimed in any one of claims 1 to 7, wherein the solution of antiplasmin is pasteurized by adjusting the solution of antiplasmin to a pH 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 an amino acid for at least one hour at a temperature of 40 to 90°C.
20. The process as claimed in any one of claims 1 to 7 wherein the solution of antiplasmin is pasteurized by adjusting the solution of antiplasmin to a pH of 7 to 8.5, heating for 8 to 20 hours at a temperature of 50 to 70°C in the presence of a monosaccharide or disaccharide, sugar alcohol or an organic dicarboxylic or tricarboxylic acid.
21. The process as claimed in any one of claims 1 to 7 wherein the solution of antiplasmin is pasteurized by adjusting the solution of antiplasmin to a pH of 7 to 8.5, heating for 8 to 20 hours at a temperature of 50 to 70°C in the presence of a monosaccharide or disaccharide, sugar alcohol or an organic dicarboxylic or tricarboxylic acid and an amino acid.
22. The process as claimed in any one of claims 1 to 7 wherein the solution of antiplasmin is pasteurized by adjusting the solution of antiplasmin to a pH of 6 to 9.5 and heating it at 40 to 90°C for 8 to 20 hours in the presence of 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 and a water-soluble amino acid.
23. The process as claimed in any one of claims 1 to 7 wherein the solution of antiplasmin is pasteurized by adjusting the solution of antiplasmin to a pH of 6 to 9.5 and heating it at 40 to 90°C for 8 to 20 hours in the presence of 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 and 1 to 114 g/l glycine.
24. The process as claimed in any one of claims 1 to 7, wherein the solution of antiplasmin is pasteurized by adjusting the solution of antiplasmin to a pH of 7 to 8.5 and heating it for 8 to 20 hours at a temperature of 50 to 70°C in the presence of 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 and a water-soluble amino acid.
25. The process as claimed in any one of claims 1 to 7, wherein the solution of antiplasmin is pasteurized by adjusting the solution of antiplasmin to a pH of 7 to 8.5 and heating it for 8 to 20 hours at a temperature of 50 to 70°C in the presence of 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 and 1 to 114 g/l glycine.
26. The process as claimed in claim 1, 7 or 17, wherein the solution of antiplasmin is dried.
27. The process as claimed in claim 1, 7 or 17, wherein stabilizers are added to the solution of antiplasmin and wherein the solution of antiplasmin is dried.
28. The process as claimed in claim 1, 7 or 17, wherein a stabilizer selected from glycine, albumin, a sugar and gelatin is added to the solution of antiplasmin, and wherein the solution of antiplasmin is dried by lyophilization.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000616377A CA1340217C (en) | 1986-01-30 | 1992-05-12 | Process for the isolation and preparation of pasteurized alpha2-antiplasmin product |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DEP3602688.3 | 1986-01-30 | ||
| 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 |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000616377A Division CA1340217C (en) | 1986-01-30 | 1992-05-12 | Process for the isolation and preparation of pasteurized alpha2-antiplasmin product |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1307373C true CA1307373C (en) | 1992-09-08 |
Family
ID=6292902
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000528511A Expired - Lifetime CA1307373C (en) | 1986-01-30 | 1987-01-29 | Process for the isolation and preparation of a pasteurized alpha_-antiplasmin product |
Country Status (7)
| Country | Link |
|---|---|
| EP (1) | EP0230956B1 (en) |
| JP (1) | JPH0819159B2 (en) |
| AT (1) | ATE109354T1 (en) |
| AU (1) | AU6810087A (en) |
| CA (1) | CA1307373C (en) |
| DE (2) | DE3602688A1 (en) |
| ES (1) | ES2058066T3 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU783155B2 (en) * | 2000-05-08 | 2005-09-29 | Csl Behring Gmbh | Stabilized protein preparation and process for its preparation |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| BE651649A (en) * | 1963-08-09 | |||
| DE2726886A1 (en) * | 1977-06-15 | 1979-01-18 | Behringwerke Ag | NEW GLYCOPROTEIN AND METHOD FOR THE PRODUCTION THEREOF |
| DE2916711A1 (en) * | 1979-04-25 | 1980-11-06 | Behringwerke Ag | Blood coagulation factors and process for their manufacture |
| EP0035204B2 (en) * | 1980-03-05 | 1991-05-22 | Miles Inc. | Pasteurized therapeutically active protein compositions |
| DE3330770A1 (en) * | 1983-08-26 | 1985-03-14 | Behringwerke Ag, 3550 Marburg | METHOD FOR PASTEURIZING HUMAN PLASMA |
| DE3336631A1 (en) * | 1983-10-08 | 1985-04-18 | Behringwerke Ag, 3550 Marburg | METHOD FOR THE PASTEURIZATION OF PLASMA OR CONCENTRATES OF THE BLOOD COAGINING FACTORS II, VII, IX AND X |
-
1986
- 1986-01-30 DE DE19863602688 patent/DE3602688A1/en not_active Ceased
-
1987
- 1987-01-20 ES ES87100685T patent/ES2058066T3/en not_active Expired - Lifetime
- 1987-01-20 EP EP87100685A patent/EP0230956B1/en not_active Expired - Lifetime
- 1987-01-20 DE DE3750298T patent/DE3750298D1/en not_active Expired - Fee Related
- 1987-01-20 AT AT87100685T patent/ATE109354T1/en not_active IP Right Cessation
- 1987-01-29 AU AU68100/87A patent/AU6810087A/en not_active Abandoned
- 1987-01-29 CA CA000528511A patent/CA1307373C/en not_active Expired - Lifetime
- 1987-01-29 JP JP62017425A patent/JPH0819159B2/en not_active Expired - Fee Related
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU783155B2 (en) * | 2000-05-08 | 2005-09-29 | Csl Behring Gmbh | Stabilized protein preparation and process for its preparation |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0819159B2 (en) | 1996-02-28 |
| ES2058066T3 (en) | 1994-11-01 |
| EP0230956A2 (en) | 1987-08-05 |
| EP0230956B1 (en) | 1994-08-03 |
| DE3750298D1 (en) | 1994-09-08 |
| JPS62209099A (en) | 1987-09-14 |
| DE3602688A1 (en) | 1987-08-06 |
| EP0230956A3 (en) | 1989-03-22 |
| AU6810087A (en) | 1987-08-06 |
| ATE109354T1 (en) | 1994-08-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US4230697A (en) | Virus-inactivated HGI-glycoprotein capable of stimulating proliferation and differentiation of human granulocyte, process for preparing same and leukopenia curative containing same | |
| US5516675A (en) | Separation of lactoperoxidase, secretory component and lactoferrin from milk or whey with a cation exchange resin | |
| EP0253395B2 (en) | A process for producing bovine lactoferrin in high purity | |
| US4275056A (en) | HGI-Glycoprotein capable of stimulating proliferation and differentiation of human granulocyte, process for preparing same and leukopenia curative containing same | |
| JPH07103044B2 (en) | Method for preparing medicament containing C1 inactivating agent | |
| JP3438735B2 (en) | Method for isolating human albumin from supernatant IV, especially IV-4 or corn fraction V or a similar supernatant or fraction | |
| EP1519944B1 (en) | Processes for the preparation of fibrinogen | |
| JP2532535B2 (en) | Medicine containing tissue protein PP4 | |
| JPH0580455B2 (en) | ||
| JPH07116236B2 (en) | Antithrombin preparation and method for preparing the same | |
| US4510084A (en) | Method of producing an antithrombin III-heparin concentrate or antithrombin III-heparinoid concentrate | |
| JPH0366292B2 (en) | ||
| EP0011739B1 (en) | Process for obtaining blood coagulation factor xiii derived from human placenta | |
| CA1307373C (en) | Process for the isolation and preparation of a pasteurized alpha_-antiplasmin product | |
| JP2568151B2 (en) | Method for isolating angiogenin | |
| JP3143507B2 (en) | Method for preparing pasteurized iron-free human transferrin and its use | |
| JPS61189228A (en) | Production of blood coagulation factor viii | |
| CA1340217C (en) | Process for the isolation and preparation of pasteurized alpha2-antiplasmin product | |
| JP2594256B2 (en) | Sterilization method of urokinase | |
| JPH08225599A (en) | Process for the preparation of C1-esterase inhibitor concentrate (C1-INH), and the resulting concentrate for therapeutic use | |
| EP0071647B1 (en) | Process for recovering interferon | |
| EP0098123B1 (en) | A process for concentrating and purifying human urinary kallikrein | |
| US4694074A (en) | Process for the purification of HBsAg | |
| JPS6159610B2 (en) | ||
| Grogan et al. | Studies on Fractionation of Saline-Soluble Nucleolar Proteins on Diethylaminoethylcellulose |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| MKLA | Lapsed |