CA2175670C - Process for producing a virus-inactivated factor viii-containing fraction by chromatographic methods - Google Patents
Process for producing a virus-inactivated factor viii-containing fraction by chromatographic methods Download PDFInfo
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- CA2175670C CA2175670C CA002175670A CA2175670A CA2175670C CA 2175670 C CA2175670 C CA 2175670C CA 002175670 A CA002175670 A CA 002175670A CA 2175670 A CA2175670 A CA 2175670A CA 2175670 C CA2175670 C CA 2175670C
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- 229960000301 factor viii Drugs 0.000 title claims abstract description 43
- 238000000034 method Methods 0.000 title claims abstract description 33
- 238000004587 chromatography analysis Methods 0.000 title claims abstract description 9
- 108010054218 Factor VIII Proteins 0.000 claims abstract description 42
- 102000001690 Factor VIII Human genes 0.000 claims abstract description 42
- 241000700605 Viruses Species 0.000 claims abstract description 13
- 230000002779 inactivation Effects 0.000 claims abstract description 10
- 238000011140 membrane chromatography Methods 0.000 claims abstract description 9
- 238000000926 separation method Methods 0.000 claims abstract description 9
- 210000002381 plasma Anatomy 0.000 claims abstract description 6
- 238000009928 pasteurization Methods 0.000 claims abstract description 5
- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 claims abstract description 4
- 229910021502 aluminium hydroxide Inorganic materials 0.000 claims abstract description 3
- 238000004090 dissolution Methods 0.000 claims abstract description 3
- 239000000463 material Substances 0.000 claims description 31
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 18
- 239000012528 membrane Substances 0.000 claims description 16
- 238000010828 elution Methods 0.000 claims description 10
- 239000011780 sodium chloride Substances 0.000 claims description 9
- 239000003446 ligand Substances 0.000 claims description 8
- 230000002209 hydrophobic effect Effects 0.000 claims description 7
- 238000005342 ion exchange Methods 0.000 claims description 7
- -1 poly(glycidyl methacrylates) Polymers 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 claims description 7
- 230000003993 interaction Effects 0.000 claims description 5
- 239000003599 detergent Substances 0.000 claims description 4
- 229910019142 PO4 Inorganic materials 0.000 claims description 3
- 239000004793 Polystyrene Substances 0.000 claims description 3
- 238000005349 anion exchange Methods 0.000 claims description 3
- 229920002223 polystyrene Polymers 0.000 claims description 3
- 229920001477 hydrophilic polymer Polymers 0.000 claims description 2
- 239000002736 nonionic surfactant Substances 0.000 claims description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 2
- 239000010452 phosphate Substances 0.000 claims description 2
- 239000002904 solvent Substances 0.000 claims 1
- 125000006850 spacer group Chemical group 0.000 description 8
- 238000000746 purification Methods 0.000 description 7
- 239000000243 solution Substances 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 206010011878 Deafness Diseases 0.000 description 5
- 238000013375 chromatographic separation Methods 0.000 description 5
- 239000007864 aqueous solution Substances 0.000 description 4
- 239000012876 carrier material Substances 0.000 description 3
- 239000001913 cellulose Substances 0.000 description 3
- 229920002678 cellulose Polymers 0.000 description 3
- 239000012266 salt solution Substances 0.000 description 3
- 239000003381 stabilizer Substances 0.000 description 3
- STCOOQWBFONSKY-UHFFFAOYSA-N tributyl phosphate Chemical compound CCCCOP(=O)(OCCCC)OCCCC STCOOQWBFONSKY-UHFFFAOYSA-N 0.000 description 3
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 150000001412 amines Chemical group 0.000 description 2
- 230000023555 blood coagulation Effects 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
- 235000014633 carbohydrates Nutrition 0.000 description 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- 230000003196 chaotropic effect Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- MSWZFWKMSRAUBD-IVMDWMLBSA-N glucosamine group Chemical group OC1[C@H](N)[C@@H](O)[C@H](O)[C@H](O1)CO MSWZFWKMSRAUBD-IVMDWMLBSA-N 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 235000021317 phosphate Nutrition 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 102000009027 Albumins Human genes 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- LSNNMFCWUKXFEE-UHFFFAOYSA-M Bisulfite Chemical compound OS([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-M 0.000 description 1
- 102100026735 Coagulation factor VIII Human genes 0.000 description 1
- 206010053567 Coagulopathies Diseases 0.000 description 1
- BRLQWZUYTZBJKN-UHFFFAOYSA-N Epichlorohydrin Chemical compound ClCC1CO1 BRLQWZUYTZBJKN-UHFFFAOYSA-N 0.000 description 1
- 102000008946 Fibrinogen Human genes 0.000 description 1
- 108010049003 Fibrinogen Proteins 0.000 description 1
- 102000006395 Globulins Human genes 0.000 description 1
- 108010044091 Globulins Proteins 0.000 description 1
- 101000911390 Homo sapiens Coagulation factor VIII Proteins 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical group OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- GSEJCLTVZPLZKY-UHFFFAOYSA-N Triethanolamine Chemical compound OCCN(CCO)CCO GSEJCLTVZPLZKY-UHFFFAOYSA-N 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 125000002015 acyclic group Chemical group 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 230000001476 alcoholic effect Effects 0.000 description 1
- 125000000217 alkyl group Chemical group 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 238000005571 anion exchange chromatography Methods 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- 239000003125 aqueous solvent Substances 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 238000010923 batch production Methods 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 238000005277 cation exchange chromatography Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000011210 chromatographic step Methods 0.000 description 1
- 238000011097 chromatography purification Methods 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 125000004093 cyano group Chemical group *C#N 0.000 description 1
- 125000006165 cyclic alkyl group Chemical group 0.000 description 1
- 230000003297 denaturating effect Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000003795 desorption Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000012869 ethanol precipitation Methods 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 229940012952 fibrinogen Drugs 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000004191 hydrophobic interaction chromatography Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- QRXWMOHMRWLFEY-UHFFFAOYSA-N isoniazide Chemical compound NNC(=O)C1=CC=NC=C1 QRXWMOHMRWLFEY-UHFFFAOYSA-N 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 229920002454 poly(glycidyl methacrylate) polymer Polymers 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 150000003856 quaternary ammonium compounds Chemical class 0.000 description 1
- NRHMKIHPTBHXPF-TUJRSCDTSA-M sodium cholate Chemical compound [Na+].C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC([O-])=O)C)[C@@]2(C)[C@@H](O)C1 NRHMKIHPTBHXPF-TUJRSCDTSA-M 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- 125000002088 tosyl group Chemical group [H]C1=C([H])C(=C([H])C([H])=C1C([H])([H])[H])S(*)(=O)=O 0.000 description 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
-
- 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
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/36—Blood coagulation or fibrinolysis factors
- A61K38/37—Factors VIII
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/745—Blood coagulation or fibrinolysis factors
- C07K14/755—Factors VIII, e.g. factor VIII C (AHF), factor VIII Ag (VWF)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Toxicology (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Analytical Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
A process for producing a virus-inactivated, factor VIII-containing fraction by chromatographic methods in which, starting with cryoprecipitate or blood plasma, possibly followed by a treatment with aluminium hydroxide, at least one separation operating is performed by membrane chromatography after the dissolution of the cryoprecipitate. The fractions are subjected to virus inactivation and preferably an additional pasteurisation step.
Description
A Process for the Preparation of a Virus Inactivated Fraction Containing Factor VIII by means of Chromatographic Methods This inventions is related to a process for the preparation of a virus inactivated fraction containing factor VIII by means of chromatographic methods.
Factor VIII is a vital material that plays an important role in blood clotting. Blood clotting disorders can thus be treated by administration of factor VIII. Therefore, a large need exists for administrable factor VIII preparations.
Numerous attempts have been made to isolate factor VIII in a highly enriched form from natural sources. Thus, chromatographic methods for the purification of factor VIII
from cryoprecipitates are already known. The latter is a fraction which is available by treatment of plasma in the cold. EP 367 840 B1 pertains to a chromatographic method for the isolation of factor VIII from blood plasma without a preliminary cryoprecipitation. The fraction containing factor VIII is separated by chromatographic separation on a hydrophilic chromatographic material modified by ion-exchanging groups . EP 0 238 701 pertains to a method for the preparation of an ultra-pure, infectious antihemophilic factor, where the pretreated fractions are a cryoprecipitate liberated from fibrinogen, globulin, albumins, and other interfering ingredients by means of ethanol precipitation.
EP-A-0 343 275 describes chromatographic separation with ion exchangers after virus inactivation of the cryoprecipitate fraction. EP 0 173 242 A describes a method for obtaining factor VIII preparations by chromatography on anion-exchanging materials which are solely based on carbohydrates, the carbohydrate matrix being modified by DEAF groups. In particular, DEAF sepharose and DEAF cellulose are described as being useful. In GB-A-1,178,958, a purification of factor VIII
using ECTEOLA cellulose columns is described. The modified cellulose contains basic substituents introduced by reaction of epichlorohydrin and triethanolamine. The above mentioned prior art makes use either of chromatographic separation in the form of batch processes or of column chromatography.
Although fairly good results are obtained with these methods, it still remains desirable for economic as well as ethical reasons to increase the yield of biologically valuable factor VIII.
Hence, the technical problem underlying this invention consists in providing a method which, proceeding from the prior art, allows for an improved preparation of factor VIII
with respect to yield and biological activity.
Surprisingly, the problem has been solved by a process proceeding from a cryoprecipitate after dissolution thereof or blood plasma, optionally followed by an aluminum hydroxide treatment, and employing a separation step using membrane chromatography on membranes or compact disks of the porous hydrophilic polymers poly(glycidyl methacrylates) or hydrophilized polystyrene.
The process of the invention can be performed with commercial cryoprecipitate or blood plasma. Preferably, the thawed cryoprecipitate is treated with aluminium hydroxide for further pre-purification of the sample in order to preconcentrate factor VIII.
Preferably, a virus inactivation is carried out prior to the actual chromatographic purification on materials arranged within or on membranes. This virus inactivation is performed according to the method described in EP 131 740 A1 by treatment with biocompatible organic solvents (detergents), Triton~ X-100/TNBP, preferably Tween~/TNBP (tri-n-butyl phosphate). Good results will also be obtained with sodium cholate/TNPB. Preferably, detergents are employed in amounts of up to 15o by weight. Wherein a virus inactivation is performed by treatment of said fraction with a di- or trialkyl phosphate and a non-ionic surfactant.
The chromatographic separation step for the purification of factor VIII in the sample may be carried out either on base materials modified by ion-exchanging groups, particularly anion exchangers, or on materials modified by immunoaffinity ligands. It is critical that the mentioned materials are arranged in membranes. Particularly useful are membranes as well as compact disks made of porous poly(glycidyl methacrylates) and/or hydrophilized polystyrene.
A membrane suitable for separation consists of compact disks made of polymer carriers. The base materials of the membranes or disks are provided with corresponding anion-exchanging groups or immunoaffinity ligands. Particularly anion-exchanging groups such as quaternary amines or diethylaminoethyl groups are considered as ion-exchanging groups. Suitable ca n on exchangers are in principle weakly and strongly acidic ration exchangers, such as materials which are modified by sulfonic acid or phosphoric acid groups.
The ion-exchanging groups can be bound to the base material fibers with or without a so-called spacer.
Materials provided with spacers are also referred to as tentacle materials. In DE 42 04 694 corresponding spacers and ligands are mentioned. For example, a glucosamine residue can serve as a spacer as well. Anion-exchanging groups such as DEAF or quaternary amines may also be bound at the membranes made of porous poly(glycidyl methacrylate) or the other materials mentioned. The binding of the anion-exchanging groups takes place either directly to the material forming the membrane or else through a spacer as well, e.g. a glucosamine residue.
In another embodiment of the process of the invention affinity membrane chromatography with immobilized substances having a high affinity for factor VIII is used.
The substances having affinity for factor VIII are immobilized at the carrier by means of chemically reactive groups. Preferably, the reactive group will attack at the end of a spacer rather than directly at the carrier material. Immobilization of the substances for factor VIII
takes place through binding at reactive groups such as tosyl, tresyl, hydrazid and others. Corresponding procedures are known from T. M. Phillips "Affinity chromatography" in "Chromatography" (E. Heftmann, ed.), 5th ed. Elsevier, Amsterdam 1992.
In another preferred embodiment materials are employed for the separation of factor VIII that can ensure a hydrophobic interaction. Hydrophobic materials which are employed are acyclic and/or cyclic alkyl chains, for instance C1 to C18 alkyl chains, and aromatic substances.
Suitable materials providing hydrophobic interaction preferably include those having graded hydrophobicity.
Hydrophobicity can be graded by introduction of polar groups, such as protic-polar or aprotic-polar groups, for example hydroxy, amino, cyano groups. Preferably, it is adapted corresponding to the respective separation conditions.
Virus inactivation may also take place by heat treatment. Preferably, the eluated sample containing factor VIII is subjected to a pasteurization step following a first membrane chromatographic step. A corresponding procedure is proposed in DE-A-4 318 435. Therein, fraction enriched with factor VIII are contacted with di- or trialkyl phosphates and optionally wetting agents in the presence of stabilizing agents and simultaneously or subsequently treated for a period of 5 hours to 30 hours at elevated temperatures ranging from 55°C to 67°C. It may be of advantage to combine the two methods of virus inactivation, the treatment with detergents and with heat.
In order to remove the chemicals employed in the pasteurization step, a second membrane chromatography may follow. Preferably, the separation of the added stabilizing agents takes place by means of a membrane modified by DEAF or quaternary ammonium compounds that are arranged on the surface of the chromatographic carrier material through a spacer. In addition, it is possible to arrange the corresponding ligands on the surface of the carrier material without a spacer. The stabilizing agents are not retarded by this anion exchange material under the conditions chosen, whereas factor VIII is adsorbed on the chromatographic material.
Thereafter, factor VIII is eluated with an aqueous solvent system exhibiting gradually increasing salt concentrations.
Factor VIII is a vital material that plays an important role in blood clotting. Blood clotting disorders can thus be treated by administration of factor VIII. Therefore, a large need exists for administrable factor VIII preparations.
Numerous attempts have been made to isolate factor VIII in a highly enriched form from natural sources. Thus, chromatographic methods for the purification of factor VIII
from cryoprecipitates are already known. The latter is a fraction which is available by treatment of plasma in the cold. EP 367 840 B1 pertains to a chromatographic method for the isolation of factor VIII from blood plasma without a preliminary cryoprecipitation. The fraction containing factor VIII is separated by chromatographic separation on a hydrophilic chromatographic material modified by ion-exchanging groups . EP 0 238 701 pertains to a method for the preparation of an ultra-pure, infectious antihemophilic factor, where the pretreated fractions are a cryoprecipitate liberated from fibrinogen, globulin, albumins, and other interfering ingredients by means of ethanol precipitation.
EP-A-0 343 275 describes chromatographic separation with ion exchangers after virus inactivation of the cryoprecipitate fraction. EP 0 173 242 A describes a method for obtaining factor VIII preparations by chromatography on anion-exchanging materials which are solely based on carbohydrates, the carbohydrate matrix being modified by DEAF groups. In particular, DEAF sepharose and DEAF cellulose are described as being useful. In GB-A-1,178,958, a purification of factor VIII
using ECTEOLA cellulose columns is described. The modified cellulose contains basic substituents introduced by reaction of epichlorohydrin and triethanolamine. The above mentioned prior art makes use either of chromatographic separation in the form of batch processes or of column chromatography.
Although fairly good results are obtained with these methods, it still remains desirable for economic as well as ethical reasons to increase the yield of biologically valuable factor VIII.
Hence, the technical problem underlying this invention consists in providing a method which, proceeding from the prior art, allows for an improved preparation of factor VIII
with respect to yield and biological activity.
Surprisingly, the problem has been solved by a process proceeding from a cryoprecipitate after dissolution thereof or blood plasma, optionally followed by an aluminum hydroxide treatment, and employing a separation step using membrane chromatography on membranes or compact disks of the porous hydrophilic polymers poly(glycidyl methacrylates) or hydrophilized polystyrene.
The process of the invention can be performed with commercial cryoprecipitate or blood plasma. Preferably, the thawed cryoprecipitate is treated with aluminium hydroxide for further pre-purification of the sample in order to preconcentrate factor VIII.
Preferably, a virus inactivation is carried out prior to the actual chromatographic purification on materials arranged within or on membranes. This virus inactivation is performed according to the method described in EP 131 740 A1 by treatment with biocompatible organic solvents (detergents), Triton~ X-100/TNBP, preferably Tween~/TNBP (tri-n-butyl phosphate). Good results will also be obtained with sodium cholate/TNPB. Preferably, detergents are employed in amounts of up to 15o by weight. Wherein a virus inactivation is performed by treatment of said fraction with a di- or trialkyl phosphate and a non-ionic surfactant.
The chromatographic separation step for the purification of factor VIII in the sample may be carried out either on base materials modified by ion-exchanging groups, particularly anion exchangers, or on materials modified by immunoaffinity ligands. It is critical that the mentioned materials are arranged in membranes. Particularly useful are membranes as well as compact disks made of porous poly(glycidyl methacrylates) and/or hydrophilized polystyrene.
A membrane suitable for separation consists of compact disks made of polymer carriers. The base materials of the membranes or disks are provided with corresponding anion-exchanging groups or immunoaffinity ligands. Particularly anion-exchanging groups such as quaternary amines or diethylaminoethyl groups are considered as ion-exchanging groups. Suitable ca n on exchangers are in principle weakly and strongly acidic ration exchangers, such as materials which are modified by sulfonic acid or phosphoric acid groups.
The ion-exchanging groups can be bound to the base material fibers with or without a so-called spacer.
Materials provided with spacers are also referred to as tentacle materials. In DE 42 04 694 corresponding spacers and ligands are mentioned. For example, a glucosamine residue can serve as a spacer as well. Anion-exchanging groups such as DEAF or quaternary amines may also be bound at the membranes made of porous poly(glycidyl methacrylate) or the other materials mentioned. The binding of the anion-exchanging groups takes place either directly to the material forming the membrane or else through a spacer as well, e.g. a glucosamine residue.
In another embodiment of the process of the invention affinity membrane chromatography with immobilized substances having a high affinity for factor VIII is used.
The substances having affinity for factor VIII are immobilized at the carrier by means of chemically reactive groups. Preferably, the reactive group will attack at the end of a spacer rather than directly at the carrier material. Immobilization of the substances for factor VIII
takes place through binding at reactive groups such as tosyl, tresyl, hydrazid and others. Corresponding procedures are known from T. M. Phillips "Affinity chromatography" in "Chromatography" (E. Heftmann, ed.), 5th ed. Elsevier, Amsterdam 1992.
In another preferred embodiment materials are employed for the separation of factor VIII that can ensure a hydrophobic interaction. Hydrophobic materials which are employed are acyclic and/or cyclic alkyl chains, for instance C1 to C18 alkyl chains, and aromatic substances.
Suitable materials providing hydrophobic interaction preferably include those having graded hydrophobicity.
Hydrophobicity can be graded by introduction of polar groups, such as protic-polar or aprotic-polar groups, for example hydroxy, amino, cyano groups. Preferably, it is adapted corresponding to the respective separation conditions.
Virus inactivation may also take place by heat treatment. Preferably, the eluated sample containing factor VIII is subjected to a pasteurization step following a first membrane chromatographic step. A corresponding procedure is proposed in DE-A-4 318 435. Therein, fraction enriched with factor VIII are contacted with di- or trialkyl phosphates and optionally wetting agents in the presence of stabilizing agents and simultaneously or subsequently treated for a period of 5 hours to 30 hours at elevated temperatures ranging from 55°C to 67°C. It may be of advantage to combine the two methods of virus inactivation, the treatment with detergents and with heat.
In order to remove the chemicals employed in the pasteurization step, a second membrane chromatography may follow. Preferably, the separation of the added stabilizing agents takes place by means of a membrane modified by DEAF or quaternary ammonium compounds that are arranged on the surface of the chromatographic carrier material through a spacer. In addition, it is possible to arrange the corresponding ligands on the surface of the carrier material without a spacer. The stabilizing agents are not retarded by this anion exchange material under the conditions chosen, whereas factor VIII is adsorbed on the chromatographic material.
Thereafter, factor VIII is eluated with an aqueous solvent system exhibiting gradually increasing salt concentrations.
The fraction containing factor VIII thus obtained is concentrated, filled and optionally lyophilized by conventional methods. Preferably, factor VIII is applied from a solution with low ionic strength in the first membrane chromatographic separation step.
Preferably, the aqueous system has an ionic strength corresponding to a 0 to 150 mM sodium chloride solution. At such ionic strengths, factor VIII is still adsorbed on the chromatographic material, whereas more weakly binding impurities can be washed out with aqueous systems of the same ionic strength.
In another embodiment of the process according to the invention, purification of the adsorbed material may be performed with an aqueous system having an ionic strength corresponding to a 200 to 400 mM sodium chloride solution.
Desorption of factor VIII and elution of this fraction then are carried out with an aqueous system having an ionic strength corresponding to a 500 to 1500 mm sodium chloride solution, while the pH value is maintained within a range of 4 to 9. If cation-exchange chromatography is performed, it preferably takes place at a pH value < 6, whereas anion-exchange chromatography is rather carried out at higher pH
values of above 6.
If purification of factor VIII is performed by immunoaffinity membrane chromatography, then, unlike with the above mentioned method using anion-exchange materials, elution is carried out with chaotropic reagents or highly concentrated salt solutions. Preferably, elution takes place with concentrations of chaotropic reagents or salts which are sufficient to disrupt the binding between the substance having high affinity to factor VIII and factor VIII itself.
The concentrations of the mentioned materials in the respective elution systems depends on the strength of the affinity between factor VIII and the corresponding binding component. As a result, elution can take place with aqueous solutions with lower denaturating potency. Preferably, aqueous solutions having concentrations of from 1 to 6 M of urea, especially from 2 to 4 M of urea, or correspondingly highly concentrated salt solutions are employed for elution of factor VIII from the immunoaffinity membrane.
In hydrophobic interaction chromatography, the sample is applied in an aqueous solution with very high ionic strength, such as, for example, highly concentrated ammonium sulfate (up to a concentration of 4 M) or sodium chloride (up to a concentration of up to 5 M). In particular, elution is carried out gradually or continuously with salt solutions of lower ionic strengths. An aqueous solution with organic solvents, particularly a diluted alcoholic solution, may also be used as solutions with lower ionic strengths for the elution of the samples in hydrophobic interaction membrane chromatography.
The procedure according to the invention surprisingly ensures a quick and uncomplicated purification of factor VIII which is obtained at the same time in high purity and high yields. In addition, the specific activity of the factor VIII obtained in this way is quite high which may be accounted for by the low denaturation of the active factor in the process according to the invention.
Preferably, the aqueous system has an ionic strength corresponding to a 0 to 150 mM sodium chloride solution. At such ionic strengths, factor VIII is still adsorbed on the chromatographic material, whereas more weakly binding impurities can be washed out with aqueous systems of the same ionic strength.
In another embodiment of the process according to the invention, purification of the adsorbed material may be performed with an aqueous system having an ionic strength corresponding to a 200 to 400 mM sodium chloride solution.
Desorption of factor VIII and elution of this fraction then are carried out with an aqueous system having an ionic strength corresponding to a 500 to 1500 mm sodium chloride solution, while the pH value is maintained within a range of 4 to 9. If cation-exchange chromatography is performed, it preferably takes place at a pH value < 6, whereas anion-exchange chromatography is rather carried out at higher pH
values of above 6.
If purification of factor VIII is performed by immunoaffinity membrane chromatography, then, unlike with the above mentioned method using anion-exchange materials, elution is carried out with chaotropic reagents or highly concentrated salt solutions. Preferably, elution takes place with concentrations of chaotropic reagents or salts which are sufficient to disrupt the binding between the substance having high affinity to factor VIII and factor VIII itself.
The concentrations of the mentioned materials in the respective elution systems depends on the strength of the affinity between factor VIII and the corresponding binding component. As a result, elution can take place with aqueous solutions with lower denaturating potency. Preferably, aqueous solutions having concentrations of from 1 to 6 M of urea, especially from 2 to 4 M of urea, or correspondingly highly concentrated salt solutions are employed for elution of factor VIII from the immunoaffinity membrane.
In hydrophobic interaction chromatography, the sample is applied in an aqueous solution with very high ionic strength, such as, for example, highly concentrated ammonium sulfate (up to a concentration of 4 M) or sodium chloride (up to a concentration of up to 5 M). In particular, elution is carried out gradually or continuously with salt solutions of lower ionic strengths. An aqueous solution with organic solvents, particularly a diluted alcoholic solution, may also be used as solutions with lower ionic strengths for the elution of the samples in hydrophobic interaction membrane chromatography.
The procedure according to the invention surprisingly ensures a quick and uncomplicated purification of factor VIII which is obtained at the same time in high purity and high yields. In addition, the specific activity of the factor VIII obtained in this way is quite high which may be accounted for by the low denaturation of the active factor in the process according to the invention.
Claims (14)
1. A process for the preparation of a virus inactivated fraction containing factor VIII by means of chromatographic methods, wherein - proceeding from a cryoprecipitate after dissolution thereof or blood plasma - followed by aluminium hydroxide treatment, a virus inactivation is performed by treatment of said fraction with a di- or trialkyl phosphate and a non-ionic surfactant, followed by at least one separation step using membrane chromatography on membranes or compact disks of the porous hydrophilic polymers poly(glycidyl methacrylates) or hydrophilized polystyrene.
2. The process according to claim 1, wherein said separation step takes place on an ion-exchange material, provided within or on a membrane.
3. The process according to claim 2, wherein the ion-exchange material is an anion-exchange material.
4. The process according to claim 1 or 2, wherein the virus inactivation is preceded by a pasteurization step.
5. The process according to claim 4, wherein the pasteurization step is followed by an additional separation step using membrane chromatography.
6. The process according to at least one of claims 1 or 4, wherein said membrane chromatography takes place on a material having a high affinity for factor VIII.
7. The process according to at least one of claims 1 or 6, wherein said material having a high affinity for factor VIII is modified by ligands having high or low molecular weights.
8. The process according to claim 6 or 7, wherein the modified material having a high affinity for factor VIII
bears immobilized ligands having a high affinity for factor VIII.
bears immobilized ligands having a high affinity for factor VIII.
9. The process according to any one of claims 1 to 5, wherein said chromatographic material allows for hydrophobic interaction with the factor VIII to be separated or bears appropriate ligands mediating hydrophobic interaction.
10. The process according to at least one of claims 1 to 5, wherein the sample to be purified is applied to the membrane containing ion-exchange material in an aqueous system having a low ionic strength, corresponding to 0 to 150 mM sodium chloride, optionally washed with an aqueous system having a higher ionic strength corresponding to a 200 to 400 mM sodium chloride solution, followed by elution with an aqueous system having a high ionic strength corresponding to a 500 to 1,500 mM sodium chloride solution, while the pH value is maintained at from 4 to 9.
11. The process according to claim 10, wherein the sample is washed with an aqueous system having higher ionic strength, corresponding to a 200 to 400 mM sodium chloride solution, followed by elution with an aqueous system having high ionic strength, corresponding to a 500 to 1,500 mM sodium chloride solution, while the pH value is maintained at from 4 to 9.
12. The process according to at least one of claims 1, 5 or 9, wherein the sample to be purified is applied from a solution having a very high ionic strength to a membrane bearing hydrophobic ligands on its surface, and is eluted with solvent systems having lower ionic strengths.
13. The process according to at least one of claims 1 to 11, wherein the eluted fraction containing factor VIII is concentrated, filled into appropriate containers or lyophilized.
14. The process according to at least one of claims 1 to 12, wherein said virus inactivation is performed by treatment with up to 15% by weight of a detergent.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE4337573A DE4337573C1 (en) | 1993-11-04 | 1993-11-04 | Process for the preparation of a virus-inactivated factor VIII-containing fraction by means of chromatographic methods |
| DEP4337573.1 | 1993-11-04 | ||
| PCT/EP1994/003258 WO1995012609A1 (en) | 1993-11-04 | 1994-09-30 | Process for producing a virus-inactivated factor viii-containing fraction by chromatographic methods |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2175670A1 CA2175670A1 (en) | 1995-05-11 |
| CA2175670C true CA2175670C (en) | 2005-08-02 |
Family
ID=6501733
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002175670A Expired - Fee Related CA2175670C (en) | 1993-11-04 | 1994-09-30 | Process for producing a virus-inactivated factor viii-containing fraction by chromatographic methods |
Country Status (21)
| Country | Link |
|---|---|
| EP (1) | EP0726907B1 (en) |
| JP (1) | JP3739788B2 (en) |
| KR (1) | KR960705841A (en) |
| CN (1) | CN1109045C (en) |
| AT (1) | ATE228533T1 (en) |
| AU (1) | AU687451B2 (en) |
| CA (1) | CA2175670C (en) |
| CZ (1) | CZ290186B6 (en) |
| DE (2) | DE4337573C1 (en) |
| ES (1) | ES2182846T3 (en) |
| FI (1) | FI116569B (en) |
| HU (1) | HU222087B1 (en) |
| IL (1) | IL111263A (en) |
| MY (1) | MY113294A (en) |
| NO (1) | NO317112B1 (en) |
| PL (1) | PL181725B1 (en) |
| RU (1) | RU2148411C1 (en) |
| SK (1) | SK284215B6 (en) |
| UA (1) | UA43855C2 (en) |
| WO (1) | WO1995012609A1 (en) |
| ZA (1) | ZA948667B (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE19618851C1 (en) * | 1996-05-10 | 1997-07-24 | Octapharma Ag | Testing factor VIII containing preparations |
| EP0885394B1 (en) * | 1996-03-08 | 2000-08-16 | Octapharma AG | Process for testing suitability of protein fractions containing factor viii |
| ES2137878B1 (en) * | 1997-12-01 | 2000-10-01 | Grifols Grupo Sa | PROCEDURE FOR OBTAINING AN ATTENUATED HUMAN PLASMA OF VIRUSES FOR THERAPEUTIC USE. |
| RU2253475C1 (en) * | 2004-02-02 | 2005-06-10 | Общество с ограниченной ответственностью "Гемоцентр" | Method of preparing factor viii preparation |
| RU2324495C1 (en) * | 2006-08-31 | 2008-05-20 | Федеральное государственное унитарное предприятие "Научно-производственное объединение по медицинским иммунобиологическим препаратам "Микроген" Министерства здравоохранения Российской Федерации | Method of making preparation of human blood coagulaton factor viii |
| AU2009264282B2 (en) * | 2008-06-24 | 2013-04-18 | Octapharma Ag | A process of purifying coagulation factor VIII |
| JP6088435B2 (en) | 2010-12-15 | 2017-03-01 | バクスアルタ ゲーエムベーハー | Eluate collection using conductivity gradient |
Family Cites Families (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4540573A (en) * | 1983-07-14 | 1985-09-10 | New York Blood Center, Inc. | Undenatured virus-free biologically active protein derivatives |
| DE3432083A1 (en) * | 1984-08-31 | 1986-03-06 | Behringwerke Ag, 3550 Marburg | PASTEURIZED, ISOAGGLUTININ-FREE FACTOR VIII PREPARATION AND METHOD FOR THE PRODUCTION THEREOF |
| US5043428A (en) * | 1984-08-31 | 1991-08-27 | Behringwerke Aktiengesellschaft | Pasteurized, isoagglutinin-free factor VIII preparation and a process for its production |
| JPS62191042A (en) * | 1986-02-17 | 1987-08-21 | Kanegafuchi Chem Ind Co Ltd | Blood coagulation factor viii adsorbent and method for purifying blood coagulation factor viii using same |
| CA1339946C (en) * | 1987-03-31 | 1998-07-07 | Michael J. Griffith | Ultrapurification process for polypeptides |
| US4774323A (en) * | 1987-06-29 | 1988-09-27 | Rorer Pharmaceutical Corporation | Purification of von Willebrand Factor solutions using gel permeation chromatography |
| US4795806A (en) * | 1987-07-16 | 1989-01-03 | Miles Laboratories, Inc. | Phospholipid affinity purification of Factor VIII:C |
| CA2001720C (en) * | 1988-10-31 | 2001-10-02 | Randal A. Goffe | Membrane affinity apparatus and purification methods related thereto |
| EP0367840B1 (en) * | 1988-11-05 | 1993-02-03 | Octapharma AG | Process for producing by chromatography a non-infectious antihemophilic factor with high purity |
| CH678155A5 (en) * | 1989-08-09 | 1991-08-15 | Fischer Ag Georg | |
| AT399818B (en) * | 1992-04-24 | 1995-07-25 | Immuno Ag | METHOD FOR PRODUCING A HIGH PURIFIED VIRUS-SAFE FACTOR VIII PREPARATION |
-
1993
- 1993-11-04 DE DE4337573A patent/DE4337573C1/en not_active Revoked
-
1994
- 1994-09-30 WO PCT/EP1994/003258 patent/WO1995012609A1/en not_active Ceased
- 1994-09-30 DE DE59410213T patent/DE59410213D1/en not_active Expired - Lifetime
- 1994-09-30 ES ES94928846T patent/ES2182846T3/en not_active Expired - Lifetime
- 1994-09-30 AT AT94928846T patent/ATE228533T1/en active
- 1994-09-30 CA CA002175670A patent/CA2175670C/en not_active Expired - Fee Related
- 1994-09-30 KR KR1019960702337A patent/KR960705841A/en not_active Ceased
- 1994-09-30 RU RU96110890A patent/RU2148411C1/en not_active IP Right Cessation
- 1994-09-30 EP EP94928846A patent/EP0726907B1/en not_active Expired - Lifetime
- 1994-09-30 PL PL94314185A patent/PL181725B1/en not_active IP Right Cessation
- 1994-09-30 JP JP51297795A patent/JP3739788B2/en not_active Expired - Fee Related
- 1994-09-30 SK SK566-96A patent/SK284215B6/en not_active IP Right Cessation
- 1994-09-30 UA UA96041756A patent/UA43855C2/en unknown
- 1994-09-30 AU AU78109/94A patent/AU687451B2/en not_active Expired
- 1994-09-30 CZ CZ19961187A patent/CZ290186B6/en not_active IP Right Cessation
- 1994-09-30 HU HU9601192A patent/HU222087B1/en not_active IP Right Cessation
- 1994-09-30 CN CN94194020A patent/CN1109045C/en not_active Expired - Fee Related
- 1994-10-12 IL IL11126394A patent/IL111263A/en not_active IP Right Cessation
- 1994-11-03 MY MYPI94002921A patent/MY113294A/en unknown
- 1994-11-03 ZA ZA948667A patent/ZA948667B/en unknown
-
1996
- 1996-05-03 FI FI961900A patent/FI116569B/en active IP Right Grant
- 1996-05-03 NO NO19961816A patent/NO317112B1/en not_active IP Right Cessation
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