DE4318435C2 - Method for inactivating viruses that are not provided with lipid envelopes - Google Patents
Method for inactivating viruses that are not provided with lipid envelopesInfo
- Publication number
- DE4318435C2 DE4318435C2 DE4318435A DE4318435A DE4318435C2 DE 4318435 C2 DE4318435 C2 DE 4318435C2 DE 4318435 A DE4318435 A DE 4318435A DE 4318435 A DE4318435 A DE 4318435A DE 4318435 C2 DE4318435 C2 DE 4318435C2
- Authority
- DE
- Germany
- Prior art keywords
- treatment
- elevated temperature
- hours
- protein
- viruses
- 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.)
- Revoked
Links
- 238000000034 method Methods 0.000 title claims description 23
- 241000700605 Viruses Species 0.000 title claims description 22
- 210000004779 membrane envelope Anatomy 0.000 title claims description 6
- 230000000415 inactivating effect Effects 0.000 title claims description 5
- 108090000623 proteins and genes Proteins 0.000 claims description 17
- 229910019142 PO4 Inorganic materials 0.000 claims description 16
- 235000021317 phosphate Nutrition 0.000 claims description 16
- 102000004169 proteins and genes Human genes 0.000 claims description 15
- 210000002381 plasma Anatomy 0.000 claims description 14
- 239000000203 mixture Substances 0.000 claims description 11
- 150000003013 phosphoric acid derivatives Chemical class 0.000 claims description 11
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 10
- 210000004369 blood Anatomy 0.000 claims description 8
- 239000008280 blood Substances 0.000 claims description 8
- 238000010438 heat treatment Methods 0.000 claims description 6
- 239000004471 Glycine Substances 0.000 claims description 5
- 229930006000 Sucrose Natural products 0.000 claims description 5
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 5
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 claims description 4
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims description 4
- 239000004472 Lysine Substances 0.000 claims description 4
- 150000001413 amino acids Chemical class 0.000 claims description 4
- 229910001424 calcium ion Inorganic materials 0.000 claims description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 4
- 239000010452 phosphate Substances 0.000 claims description 4
- 239000005720 sucrose Substances 0.000 claims description 4
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 claims description 3
- 239000000600 sorbitol Substances 0.000 claims description 3
- 239000004475 Arginine Substances 0.000 claims description 2
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 claims description 2
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims description 2
- 238000001556 precipitation Methods 0.000 claims description 2
- 229960004793 sucrose Drugs 0.000 claims 2
- IIDJRNMFWXDHID-UHFFFAOYSA-N Risedronic acid Chemical compound OP(=O)(O)C(P(O)(O)=O)(O)CC1=CC=CN=C1 IIDJRNMFWXDHID-UHFFFAOYSA-N 0.000 claims 1
- 239000000654 additive Substances 0.000 claims 1
- 230000000996 additive effect Effects 0.000 claims 1
- 239000003795 chemical substances by application Substances 0.000 claims 1
- 238000011097 chromatography purification Methods 0.000 claims 1
- 235000013681 dietary sucrose Nutrition 0.000 claims 1
- 230000007935 neutral effect Effects 0.000 claims 1
- 150000002632 lipids Chemical class 0.000 description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 230000002779 inactivation Effects 0.000 description 5
- 108010054218 Factor VIII Proteins 0.000 description 4
- 102000001690 Factor VIII Human genes 0.000 description 4
- -1 alkyl phosphates Chemical class 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 229960000301 factor viii Drugs 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 239000000080 wetting agent Substances 0.000 description 4
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 3
- 239000001110 calcium chloride Substances 0.000 description 3
- 229910001628 calcium chloride Inorganic materials 0.000 description 3
- 239000013592 cell lysate Substances 0.000 description 3
- 239000003599 detergent Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 239000001509 sodium citrate Substances 0.000 description 3
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 3
- STCOOQWBFONSKY-UHFFFAOYSA-N tributyl phosphate Chemical compound CCCCOP(=O)(OCCCC)OCCCC STCOOQWBFONSKY-UHFFFAOYSA-N 0.000 description 3
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 description 2
- 241000991587 Enterovirus C Species 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 125000004432 carbon atom Chemical group C* 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 238000005194 fractionation Methods 0.000 description 2
- 208000005252 hepatitis A Diseases 0.000 description 2
- 238000009928 pasteurization Methods 0.000 description 2
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 2
- 229920000136 polysorbate Polymers 0.000 description 2
- 229920000053 polysorbate 80 Polymers 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 102100022641 Coagulation factor IX Human genes 0.000 description 1
- 108010076282 Factor IX Proteins 0.000 description 1
- 108010049003 Fibrinogen Proteins 0.000 description 1
- 102000008946 Fibrinogen Human genes 0.000 description 1
- 239000012541 Fractogel® Substances 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- GTVWRXDRKAHEAD-UHFFFAOYSA-N Tris(2-ethylhexyl) phosphate Chemical compound CCCCC(CC)COP(=O)(OCC(CC)CCCC)OCC(CC)CCCC GTVWRXDRKAHEAD-UHFFFAOYSA-N 0.000 description 1
- 125000000217 alkyl group Chemical group 0.000 description 1
- 238000005571 anion exchange chromatography Methods 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- 230000000603 anti-haemophilic effect Effects 0.000 description 1
- 230000000712 assembly Effects 0.000 description 1
- 238000000429 assembly Methods 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229940043430 calcium compound Drugs 0.000 description 1
- 150000001674 calcium compounds Chemical class 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 229960004222 factor ix Drugs 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 229940012952 fibrinogen Drugs 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 108010074605 gamma-Globulins Proteins 0.000 description 1
- 230000010196 hermaphroditism Effects 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 125000004108 n-butyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- IQFXJRXOTKFGPN-UHFFFAOYSA-N n-ethenyl-n-ethylethanamine Chemical group CCN(CC)C=C IQFXJRXOTKFGPN-UHFFFAOYSA-N 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000036470 plasma concentration Effects 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000000244 polyoxyethylene sorbitan monooleate Substances 0.000 description 1
- 229940068968 polysorbate 80 Drugs 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 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
- 239000000243 solution Substances 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000003319 supportive effect Effects 0.000 description 1
- SFENPMLASUEABX-UHFFFAOYSA-N trihexyl phosphate Chemical compound CCCCCCOP(=O)(OCCCCCC)OCCCCCC SFENPMLASUEABX-UHFFFAOYSA-N 0.000 description 1
- KUHPLTBUBAGTDV-UHFFFAOYSA-N tris-decyl phosphate Chemical compound CCCCCCCCCCOP(=O)(OCCCCCCCCCC)OCCCCCCCCCC KUHPLTBUBAGTDV-UHFFFAOYSA-N 0.000 description 1
- CQXYINNETWHZTR-UHFFFAOYSA-N tritert-butyl phosphate Chemical compound CC(C)(C)OP(=O)(OC(C)(C)C)OC(C)(C)C CQXYINNETWHZTR-UHFFFAOYSA-N 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
- 238000009736 wetting Methods 0.000 description 1
Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
- A61L2/0005—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts
- A61L2/0082—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts using chemical substances
- A61L2/0088—Liquid substances
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
- A61L2/0005—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts
- A61L2/0011—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts using physical methods
- A61L2/0023—Heat
-
- 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
-
- 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)
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Toxicology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Biophysics (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Hematology (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
Description
Gegenstand der vorliegenden Anmeldung ist ein Verfahren zur Inaktivierung von Viren, die nicht mit einer Lipidhülle versehen sind, in Protein enthaltenden Zusammenstellungen aus Blut, Blutplasma oder ähnlichen natürlichen Quellen.The subject of the present application is a method for Inactivating viruses that do not have a lipid envelope are provided, in protein-containing compositions from blood, blood plasma or similar natural sources.
Die EP 0 131 740 B1 beschreibt ein Verfahren zur Inaktivie rung von Viren in labile Proteine enthaltenden Zusammen setzungen. Die zu inaktivierenden Viren enthalten Lipide und können insbesondere mit einer Lipidhülle versehen sein. Die von Lipid enthaltenden Viren zu befreiende Zusammen setzung stammt aus einer natürlichen Quelle, die ausgewählt ist aus der Gruppe Vollblut, Blutplasma, Plasmakonzen trat, Präzipitat irgendeiner Fraktionierung solchen Plas mas, Überstand aus irgendeiner Fraktionierung solchen Plas mas, Serum, Kryopräzipitat, Zell-Lysat, in Blutzellen indu zierte Proteine, Produkt einer nicht Blut entstammenden normalen oder Krebszelle oder Produkt eines Gen-Splicing- Vorgangs. Das in der EP 0 131 740 B1 beschriebene Verfahren besteht aus einem Inkontaktbringen der labiles Protein enthaltenden Zusammensetzung mit einer wirksamen Menge eines Di- oder Trialkylphosphats für eine Zeitdauer, die ausreichend ist, um die labiles Protein enthaltende Zusam mensetzung frei von Lipid enthaltenden Viren zu machen, ohne eine wesentliche Proteindenaturierung herbeizuführen. Dabei kann das beschriebene Verfahren zur Inaktivierung von Lipid enthaltenden Viren mit einer Wärmebehandlung bei 50 bis 70°C und einer Zeitdauer von mindestens 5 Stunden kom biniert werden. EP 0 131 740 B1 describes a method for inactivation tion of viruses in labile protein-containing assemblies settlements. The viruses to be inactivated contain lipids and can in particular be provided with a lipid envelope. The virus-containing virus to be rid of Settlement comes from a natural source that is selected is from the group whole blood, blood plasma, plasma concentrations precipitated any fractionation of such plas mas, supernatant from any fractionation of such plas mas, serum, cryoprecipitate, cell lysate, induce in blood cells graced proteins, product of a non blood derived normal or cancer cell or product of a gene splicing Operation. The method described in EP 0 131 740 B1 consists of contacting the labile protein containing composition with an effective amount a di- or trialkyl phosphate for a period of time is sufficient to get the labile protein-containing composition make the formulation free of lipid-containing viruses, without causing substantial protein denaturation. The described method for inactivating Viruses containing lipid with a heat treatment at 50 up to 70 ° C and a duration of at least 5 hours com be binated.
Bei Präparationen der eingangs genannten Art ist es jedoch zu nehmend von Bedeutung, auch solche Viren zu inaktivieren, die kein Lipid enthalten und keine Lipidhülle aufweisen. Zur Gruppe der keine Lipidhülle aufweisenden Viren, die als nicht Lipid enthaltende Viren im Sinne der EP 0 131 740 B1 aufzufassen sind, zählen insbesondere Hepatitis-A-, Parvo- wie Parvorirus B 19, Polioviren. Diese Viren können als Krankheitserreger in Blut, Plasma, Serum, Kryopräzipitat, Zell-Lysat und ähnlichen natür lichen Quellen vorkommen.With preparations of the type mentioned at the beginning, however, it is too important to also inactivate viruses that no lipid and no lipid shell. To the group of the non-lipid envelope viruses that are considered non-lipid containing viruses within the meaning of EP 0 131 740 B1, include in particular hepatitis A, parvo and parvorirus B 19, Poliovirus. These viruses can act as pathogens in blood, Plasma, serum, cryoprecipitate, cell lysate and similar natural sources.
Ein Ziel der vorliegenden Erfindung ist es, ein Verfahren be reitzustellen, das es erlaubt, Viren, die keine Lipidhülle oder nur wenige Lipide enthalten, in bestimmten Präparationen zu inaktivieren. Zu solchen Präparationen sollen insbesondere labile Proteine enthaltende Zusammensetzungen aus Vollblut, Blutplasma, Plasmakonzentrat, Präzipitat irgendeiner Fraktio nierungsstufe solchen Plasmas, Überstand aus irgendeiner Frak tionierung solchen Plasmas, Serum, Kryopräzipitat, Zell-Lysat oder ähnlichen natürlichen Quellen gerechnet werden.An object of the present invention is to provide a method which allows viruses that do not have a lipid coating or Contain only a few lipids in certain preparations inactivate. Such preparations should in particular whole blood compositions containing labile proteins, Blood plasma, plasma concentrate, precipitate of any fraction level of such plasma, supernatant from some frak tion of such plasma, serum, cryoprecipitate, cell lysate or similar natural sources.
Nowak et al. berichten in "Virological Safety Aspects of Plasma Derivatives" über eine Inaktivierung von HIV, HBV, HCV ver wandten Viren und anderen Viren in Derivaten humanen Plasmas mittels Pasteurisation. Hierbei werden die betreffenden Frak tionen in stabilisierter Lösung bei 60°C für eine Zeitdauer von 10 Stunden inaktiviert.Nowak et al. report in "Virological Safety Aspects of Plasma Derivatives "via an inactivation of HIV, HBV, HCV ver used viruses and other viruses in derivatives of human plasma using pasteurization. The relevant frak ions in stabilized solution at 60 ° C for a period of Inactivated for 10 hours.
Überraschenderweise wird dieses Ziel durch ein Verfahren, das Viren, die nicht mit einer Lipidhülle versehen sind, in Protein enthaltenden Zusammenstellungen aus Blut, Blutplasma oder ähnli chen natürlichen Quellen, erreicht, wobei eine Behandlung der Quelle mit einer wirksamen Menge von Di- oder Trialkylphosphaten und Benetzungsmitteln, gleichzeitig oder aufein anderfolgend, bei einer erhöhten Temperatur im Bereich von 55°C bis 67°C für eine Zeitdauer von 5 bis 30 Stunden. Surprisingly, this goal is achieved through a process that Viruses that do not have a lipid envelope in protein containing compositions from blood, blood plasma or the like Chen natural sources, achieved treatment of Source with an effective amount of di- or trialkyl phosphates and wetting agents, simultaneously or together otherwise, at an elevated temperature in the range of 55 ° C up to 67 ° C for a period of 5 to 30 hours.
Die Mengen an Di- oder Trialkylphosphat beträgt vorzugs weise zwischen 0,001% und 1%. Zur Durchführung des Ver fahrens werden vorzugsweise Temperaturen zwischen 60°C und 65°C eingestellt. Die Zeitdauer der Wärmebehandlung liegt vorzugsweise bei mindestens 10 Stunden.The amount of di- or trialkyl phosphate is preferred between 0.001% and 1%. To carry out the Ver temperatures between 60 ° C and 65 ° C set. The duration of the heat treatment is preferably at least 10 hours.
Die Proteinfraktion, in der die keine Lipide enthaltenden Viren inaktiviert werden sollen, stammt insbesondere aus natürlichen Quellen der eingangs genannten Art und kann insbesondere durch Präzipitations- oder chromatographische Verfahren vor der Inaktivierungsreaktion mit dem entspre chenden Protein angereichert worden sein.The protein fraction in which the lipids do not contain Viruses to be inactivated come in particular from natural sources of the type mentioned at the beginning and can especially by precipitation or chromatographic Procedure before the inactivation reaction with the corresponding protein.
Insbesondere hat sich dabei eine Anreicherung der Protein fraktion mit chromatographischen Methoden, wie sie in der EP 0 367 840 A1 beschrieben sind, bewährt. Dabei wird davon ausgegangen, daß die natürliche Quelle, die das Protein liefert, einer Anionenaustauscherchromatographie unterwor fen wird. Insbesondere haben sich mit Diethylaminoethylen- Gruppen modifizierte chromatographische Trägermaterialien bewährt.In particular, there has been an enrichment of the protein fraction using chromatographic methods as described in the EP 0 367 840 A1 are proven. In doing so assumed that the natural source of the protein provides an anion exchange chromatography will. In particular, diethylaminoethylene Groups of modified chromatographic substrates proven.
Die natürliche Quelle oder die aus der natürlichen Quelle angereicherte Fraktion wird dann dem oben beschriebenen Virusinaktivierungsverfahren durch Wärmebehandlung und Behandlung mit Di- oder Trialkylphosphaten unterworfen.The natural source or from the natural source The enriched fraction then becomes that described above Virus inactivation method by heat treatment and Subjected to treatment with di- or trialkyl phosphates.
Es hat sich als vorteilhaft erwiesen, die Behandlung mit Di- oder Trialkylphosphaten in Gegenwart von Benetzungs mitteln durchzuführen. Als Alkylphosphate kommen insbeson dere die in der EP 0 131 740 B1 genannten Dialkylphosphate oder Trialkylphosphate mit Alkylgruppen, die 1 bis 10 Kohlenstoffatome enthalten, inbesondere 2 bis 10 Kohlenstoffatome in Betracht. Insbesondere kommen Trialkylphosphate wie Tri-(n-butyl)phosphat, Tri-(t-butyl) phosphat, Tri-(n-hexyl)phosphat, Tri-(2-ethylhexyl)phosphat, Tri-(n-decyl)phosphat in Betracht. Auch gemische Trialkyl phosphate sind geeignet. In ähnlicher Weise können auch die entsprechend substituierten Dialkylphosphate oder Mischun gen entsprechender Di- oder Trialkylphosphate eingesetzt werden.It has proven beneficial to be treated with Di- or trialkyl phosphates in the presence of wetting to carry out means. In particular come as alkyl phosphates the others mentioned in EP 0 131 740 B1 Dialkylphosphate or Trialkylphosphate with Alkyl groups containing 1 to 10 carbon atoms, in particular 2 to 10 carbon atoms. Come in particular Trialkyl phosphates such as tri- (n-butyl) phosphate, tri- (t-butyl) phosphate, tri- (n-hexyl) phosphate, tri- (2-ethylhexyl) phosphate, Tri- (n-decyl) phosphate. Mixtures of trialkyl phosphates are suitable. Similarly, the appropriately substituted dialkyl phosphates or mixtures gene used corresponding di- or trialkyl phosphates will.
Als Benetzungsmittel kommen insbesondere nicht toxische Detergenzien in Betracht. Als nicht ionische Detergenzien, die in Mengen von mindestens 0,1 Gew. -% vorliegen sollten, sind die folgenden Derivate zu nennen: Polyoxyethylenderi vate von Fettsäuren, Partialester von Sorbitolanhydrid, z. B. Produkte unter dem Handelsnamen Tween 80, Tween 20 und Polysorbat 80 bekannt sind sowie nicht inonische, Öl lösli che Benetzungsmittel, insbesondere solche, die unter dem Handelsnamen TritonX100 (ethoxylierte Alkylphenole) bekannt sind. Auch Zwitterreagenzien, beispielsweise Sulfobetaine wie n-Dodecyl-N, n-dimethyl-2-ammonio-1-ethansulfonat oder Derivate davon oder nicht ionische Detergenzien wie Octyl- β-D-glucopyranoside kommen in Betracht. Die Menge des Be netzungsmittels beträgt vorzugsweise 0,01% bis 10%.In particular, non-toxic wetting agents are used Detergents into consideration. As non-ionic detergents, which should be present in quantities of at least 0.1% by weight, the following derivatives may be mentioned: polyoxyethylene di vate of fatty acids, partial esters of sorbitol anhydride, e.g. B. Products under the trade names Tween 80, Tween 20 and Polysorbate 80 are known and non-ionic, oil soluble che wetting agents, especially those under the Trade names TritonX100 (ethoxylated alkylphenols) known are. Hermaphrodite reagents, for example sulfobetaines such as n-dodecyl-N, n-dimethyl-2-ammonio-1-ethanesulfonate or Derivatives thereof or non-ionic detergents such as octyl β-D-glucopyranosides are possible. The amount of loading wetting agent is preferably 0.01% to 10%.
Insbesondere bevorzugt sind Kombinationen von Tri(n-butyl) phosphat und Tween oder Natriumcholat/TNBP (Tri-(n-butyl) phosphat).Combinations of tri (n-butyl) are particularly preferred. phosphate and tween or sodium cholate / TNBP (tri- (n-butyl) phosphate).
Es hat sich als vorteilhaft erwiesen, die Behandlung mit erhöhter Temperatur in Gegenwart von unterstützenden Sub stanzen wie Saccharose, Sorbitol oder kurzkettigen neutra len Aminosäuren durchzuführen. Grundsätzlich können die in der EP 0 018 561 und/oder DE 40 01 451 A1 genannten Stabi lisierungsfaktoren in den angegebenen Mengenbereichen ver wendet werden. Dabei kann die Konzentration der unterstüt zenden Substanzen (Stabilisierungsfaktoren) sehr hoch sein, vorzugsweise beträgt beispielsweise die Saccharosekonzen tration bis zu 200 Gew.-%. Als kurzkettige Aminosäuren kommen insbesondere Glycin, Lysin und/oder Arginin in Fra ge.It has proven beneficial to be treated with elevated temperature in the presence of supportive sub punch like sucrose, sorbitol or short chain neutra len amino acids. Basically, the in of the stabilizers mentioned in EP 0 018 561 and / or DE 40 01 451 A1 verification factors in the specified quantity ranges be applied. The concentration of the support substances (stabilizing factors) are very high, the sucrose concentration is preferably, for example tration up to 200 wt .-%. As short chain amino acids especially glycine, lysine and / or arginine come into Fra ge.
Es hat sich überraschenderweise herausgestellt, daß die Behandlung der Protein enthaltenden Zusammenstellungen aus Blut, Blutplasma oder ähnlichen natürlichen Quellen bei einer erhöhten Temperatur auch ohne Zusatz von Calciumionen erfolgen kann. Die Zugabe von Calciumionen wird in der EP 0 106 269 als unbedingt erforderlich angesehen. So offen bart die EP 0 106 269, das Ca2+ Fraktionen des antihämophi len Kryopräzipitats, in dem dort beschriebenen Pasteurisie rungsverfahren stabilisiert. Erfindungsgemäß ist dies nicht erforderlich.It has surprisingly been found that the protein-containing compositions from blood, blood plasma or similar natural sources can be treated at an elevated temperature without the addition of calcium ions. The addition of calcium ions is considered to be absolutely necessary in EP 0 106 269. EP 0 106 269 shows that the Ca 2+ fractions of the antihemophilic cryoprecipitate are stabilized in the pasteurization process described there. According to the invention, this is not necessary.
An die Behandlung mit Di- oder Trialkylphosphaten, auch in Gegenwart erhöhter Temperatur, kann sich eine chromatogra phische Reinigung anschließen. Dieser chromatographische Reinigungsschritt wird vorzugsweise an Anionenaustauscher materialien wie DEAE modifizierten Ionenaustauscherharzen durchgeführt. Der pH-Wert sollte im Bereich von 6 bis 8,5 liegen.Treatment with di- or trialkyl phosphates, also in In the presence of elevated temperature, a chromatograph can Connect phical cleaning. This chromatographic Cleaning step is preferably on anion exchangers materials such as DEAE modified ion exchange resins carried out. The pH should range from 6 to 8.5 lie.
Die auf diese Weise angereicherten oder erhaltenen pharma kologisch bedeutsamen Proteine wie Faktor VIII, Faktor IX, Fibrinogen, Gamma-Globulin usw., weisen keine aktiven Viren der Art Hepatitis-A-, Parvo- wie Parvorirus B 19, Polioviren auf.The pharma enriched or obtained in this way ecologically significant proteins such as factor VIII, factor IX, Fibrinogen, gamma globulin, etc. have no active ones Viruses of the type hepatitis A, parvo and parvorirus B 19, Polioviruses on.
Das erfindungsgemäße Verfahren wird anhand des folgenden Beispiels zur Inaktivierung lipidfreier Viren in einer Faktor VIII Präparation näher beschrieben.The method according to the invention is illustrated by the following Example of inactivating lipid-free viruses in one Factor VIII preparation described in more detail.
Handelsübliches Kryopräziptat wird auf eine mit Fractogel®- DEAE-Harz gefüllte Säule aufgetragen. Das Effluat wird aufgenommen und auf Faktor VIII-Aktivität untersucht. Da nach wird die Säule mit einem Puffer mit 110 mM Natrium chlorid, 10 mM Natriumcitrat × 5 H₂O, 120 mM Glycin, 1 mM Calciumchlorid × 2 H₂O, pH-Wert 6,9 bis 7,0 (mit 1 M HCl einzustellen) gewaschen. Danach wird die Säule mit einem Puffer behandelt, der die folgende Zusammensetzung auf weist: 250 mM Natriumchlorid, 20 mM Natriumcitrat × 5 H₂O, 80 mM Glycin, 16 mM Lysin, 2,5 mM Calciumchlorid × 2 H₂O bei einem pH-Wert von 6,9 bis 7,0.Commercial cryoprecipitate is placed on a Fractogel® DEAE resin filled column applied. The effluat will recorded and examined for factor VIII activity. There after the column with a buffer with 110 mM sodium chloride, 10 mM sodium citrate × 5 H₂O, 120 mM glycine, 1 mM Calcium chloride × 2 H₂O, pH 6.9 to 7.0 (with 1 M HCl adjust) washed. Then the column with a Buffer treats the following composition has: 250 mM sodium chloride, 20 mM sodium citrate × 5 H₂O, 80 mM glycine, 16 mM lysine, 2.5 mM calcium chloride × 2 H₂O at a pH of 6.9 to 7.0.
Die so erhaltene Fraktion wird mit Saccharose versetzt. Danach wird die Fraktion mit Tri-(n-butyl)phosphat/Tween 0,1% / 0,3% 12 Stunden bei 64°C gehalten. Dann wird er neut eine Ionenaustauscherchromatographie an TSK-Fracto gel®-DEAE oder EMD-Fractogel®-TMAE durchgeführt.The fraction thus obtained is mixed with sucrose. Then the fraction with tri (n-butyl) phosphate / Tween 0.1% / 0.3% held at 64 ° C for 12 hours. Then he will again ion exchange chromatography on TSK-Fracto gel®-DEAE or EMD-Fractogel®-TMAE.
Die Faktor VIII aktive Fraktion wird mit einem Puffer, der 250 mM Natriumchlorid, 20 mM Natriumcitrat × 5 H₂O, 80 mM Glycin, 16 mM Lysin, 2,5 mM Calciumchlorid × 2 H₂O enthält, eluiert.The factor VIII active fraction is mixed with a buffer that 250 mM sodium chloride, 20 mM sodium citrate × 5 H₂O, 80 mM Contains glycine, 16 mM lysine, 2.5 mM calcium chloride × 2 H₂O, eluted.
Die Virusinaktivierung ohne Anwesenheit von Calciumionen wird durchgeführt, indem, wie in Beispiel 1 beschrieben, das handelsübliche Kryopräzipitat behandelt wird, wobei den Puffern (Waschpuffer und Elutionspuffer) vor der Wärmebe handlung keine Calciumverbindungen zugesetzt worden sind. Die Wärmebehandlung wird danach ohne Zugabe von Calciumio nen durchgeführt und dann, wie in Beispiel 1 beschrieben, weiter aufgearbeitet.Virus inactivation without the presence of calcium ions is carried out by, as described in Example 1, the commercial cryoprecipitate is treated, the Buffer (wash buffer and elution buffer) before heating action no calcium compounds have been added. The heat treatment is then carried out without the addition of Calciumio and then, as described in Example 1, worked up further.
Claims (11)
Priority Applications (13)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE4318435A DE4318435C2 (en) | 1993-02-09 | 1993-06-03 | Method for inactivating viruses that are not provided with lipid envelopes |
| EP94906906A EP0683678B1 (en) | 1993-02-09 | 1994-02-05 | Method for inactivating viruses devoid of lipid envelopes |
| JP6517642A JPH08506498A (en) | 1993-02-09 | 1994-02-05 | Method for inactivating non-lipid coated virus |
| DE59406456T DE59406456D1 (en) | 1993-02-09 | 1994-02-05 | METHOD FOR INACTIVATING VIRUSES THAT HAVE NO LIPID CASES |
| CN94191261A CN1064552C (en) | 1993-02-09 | 1994-02-05 | Method for inactivating viruses devoid of lipid envelopes |
| KR1019950703292A KR100292912B1 (en) | 1993-02-09 | 1994-02-05 | How to inactivate an uncovered virus |
| ES94906906T ES2118386T3 (en) | 1993-02-09 | 1994-02-05 | PROCEDURE FOR THE INACTIVATION OF VIRUSES THAT ARE NOT PROVIDED WITH LIPID ENVELOPES. |
| FI953756A FI953756L (en) | 1993-02-09 | 1994-02-05 | Method for inactivating non-lipid enveloped viruses |
| AT94906906T ATE168274T1 (en) | 1993-02-09 | 1994-02-05 | METHOD FOR INACTIVATION OF VIRUSES THAT ARE NOT PROVIDED WITH LIPID COATS |
| CA002155337A CA2155337A1 (en) | 1993-02-09 | 1994-02-05 | Method for inactivating viruses devoid of lipid envelopes |
| PCT/EP1994/000328 WO1994017834A1 (en) | 1993-02-09 | 1994-02-05 | Method for inactivating viruses devoid of lipid envelopes |
| AU60389/94A AU676011B2 (en) | 1993-02-09 | 1994-02-05 | Method for inactivating viruses devoid of lipid envelopes |
| US08/501,034 US6528246B2 (en) | 1993-02-09 | 1995-02-05 | Method for the inactivation of non-lipid-coated viruses |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE4303609 | 1993-02-09 | ||
| DE4318435A DE4318435C2 (en) | 1993-02-09 | 1993-06-03 | Method for inactivating viruses that are not provided with lipid envelopes |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| DE4318435A1 DE4318435A1 (en) | 1994-08-11 |
| DE4318435C2 true DE4318435C2 (en) | 1997-01-23 |
Family
ID=6479891
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| DE4318435A Revoked DE4318435C2 (en) | 1993-02-09 | 1993-06-03 | Method for inactivating viruses that are not provided with lipid envelopes |
| DE59406456T Revoked DE59406456D1 (en) | 1993-02-09 | 1994-02-05 | METHOD FOR INACTIVATING VIRUSES THAT HAVE NO LIPID CASES |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| DE59406456T Revoked DE59406456D1 (en) | 1993-02-09 | 1994-02-05 | METHOD FOR INACTIVATING VIRUSES THAT HAVE NO LIPID CASES |
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| Country | Link |
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| KR (1) | KR100292912B1 (en) |
| DE (2) | DE4318435C2 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE19528221C2 (en) * | 1995-08-01 | 1998-10-22 | Blutspendedienst Der Drk Lande | Process for the production of a virus-safe, therapeutic preparation from human plasma |
Family Cites Families (1)
| 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 |
-
1993
- 1993-06-03 DE DE4318435A patent/DE4318435C2/en not_active Revoked
-
1994
- 1994-02-05 DE DE59406456T patent/DE59406456D1/en not_active Revoked
- 1994-02-05 KR KR1019950703292A patent/KR100292912B1/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| DE59406456D1 (en) | 1998-08-20 |
| DE4318435A1 (en) | 1994-08-11 |
| KR100292912B1 (en) | 2002-11-07 |
| KR960700765A (en) | 1996-02-24 |
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