NO317112B1 - A process for preparing a virus-activated fraction containing factor VIII by chromatographic methods - Google Patents
A process for preparing a virus-activated fraction containing factor VIII by chromatographic methods Download PDFInfo
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- NO317112B1 NO317112B1 NO19961816A NO961816A NO317112B1 NO 317112 B1 NO317112 B1 NO 317112B1 NO 19961816 A NO19961816 A NO 19961816A NO 961816 A NO961816 A NO 961816A NO 317112 B1 NO317112 B1 NO 317112B1
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- 108010054218 Factor VIII Proteins 0.000 title claims abstract description 39
- 102000001690 Factor VIII Human genes 0.000 title claims abstract description 39
- 229960000301 factor viii Drugs 0.000 title claims abstract description 39
- 241000700605 Viruses Species 0.000 title claims abstract description 11
- 238000004587 chromatography analysis Methods 0.000 title claims abstract description 10
- 238000004519 manufacturing process Methods 0.000 title claims description 6
- 238000000034 method Methods 0.000 claims abstract description 28
- 230000002779 inactivation Effects 0.000 claims abstract description 10
- 238000011140 membrane chromatography Methods 0.000 claims abstract description 10
- 210000002381 plasma Anatomy 0.000 claims abstract description 6
- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 claims abstract description 4
- 238000009928 pasteurization Methods 0.000 claims abstract description 4
- 238000004090 dissolution Methods 0.000 claims abstract description 3
- 239000000463 material Substances 0.000 claims description 30
- 239000012528 membrane Substances 0.000 claims description 16
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 14
- 239000003446 ligand Substances 0.000 claims description 8
- 238000000926 separation method Methods 0.000 claims description 8
- 238000005349 anion exchange Methods 0.000 claims description 7
- 230000002209 hydrophobic effect Effects 0.000 claims description 7
- 238000005342 ion exchange Methods 0.000 claims description 7
- 239000011780 sodium chloride Substances 0.000 claims description 7
- 230000003993 interaction Effects 0.000 claims description 5
- 239000003599 detergent Substances 0.000 claims description 4
- -1 poly(glycidyl methacrylates) Polymers 0.000 claims description 4
- 229910019142 PO4 Inorganic materials 0.000 claims description 3
- 239000004793 Polystyrene Substances 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
- 239000010452 phosphate Substances 0.000 claims description 2
- 239000002904 solvent Substances 0.000 claims 1
- 229910021502 aluminium hydroxide Inorganic materials 0.000 abstract 1
- 238000000746 purification Methods 0.000 description 9
- 238000010828 elution Methods 0.000 description 8
- 125000006850 spacer group Chemical group 0.000 description 8
- 239000000126 substance Substances 0.000 description 8
- 239000000243 solution Substances 0.000 description 7
- 238000013375 chromatographic separation Methods 0.000 description 5
- STCOOQWBFONSKY-UHFFFAOYSA-N tributyl phosphate Chemical compound CCCCOP(=O)(OCCCC)OCCCC STCOOQWBFONSKY-UHFFFAOYSA-N 0.000 description 4
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical group CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 description 3
- 239000007864 aqueous solution Substances 0.000 description 3
- 229920002678 cellulose Polymers 0.000 description 3
- 239000001913 cellulose Substances 0.000 description 3
- 239000012266 salt solution Substances 0.000 description 3
- 239000003381 stabilizer Substances 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
- 150000001768 cations 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
- 238000010438 heat treatment Methods 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 235000021317 phosphate Nutrition 0.000 description 2
- 238000002360 preparation method Methods 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
- 238000007445 Chromatographic isolation Methods 0.000 description 1
- 102100023804 Coagulation factor VII Human genes 0.000 description 1
- 229920002271 DEAE-Sepharose Polymers 0.000 description 1
- GUBGYTABKSRVRQ-WFVLMXAXSA-N DEAE-cellulose Chemical compound OC1C(O)C(O)C(CO)O[C@H]1O[C@@H]1C(CO)OC(O)C(O)C1O GUBGYTABKSRVRQ-WFVLMXAXSA-N 0.000 description 1
- BRLQWZUYTZBJKN-UHFFFAOYSA-N Epichlorohydrin Chemical compound ClCC1CO1 BRLQWZUYTZBJKN-UHFFFAOYSA-N 0.000 description 1
- 108010023321 Factor VII Proteins 0.000 description 1
- 108010049003 Fibrinogen Proteins 0.000 description 1
- 102000008946 Fibrinogen Human genes 0.000 description 1
- 102000006395 Globulins Human genes 0.000 description 1
- 108010044091 Globulins Proteins 0.000 description 1
- 208000031220 Hemophilia Diseases 0.000 description 1
- 208000009292 Hemophilia A Diseases 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical group OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 1
- GSEJCLTVZPLZKY-UHFFFAOYSA-N Triethanolamine Chemical compound OCCN(CCO)CCO GSEJCLTVZPLZKY-UHFFFAOYSA-N 0.000 description 1
- 229920004890 Triton X-100 Polymers 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
- 239000001166 ammonium sulphate Substances 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
- 230000004071 biological effect Effects 0.000 description 1
- 239000012876 carrier material Substances 0.000 description 1
- 238000005277 cation exchange chromatography Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000011097 chromatography purification Methods 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000001816 cooling 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
- 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
- 229940012413 factor vii Drugs 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 229940012952 fibrinogen Drugs 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 238000004191 hydrophobic interaction chromatography Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 150000002734 metacrylic acid derivatives Chemical class 0.000 description 1
- 229920002454 poly(glycidyl methacrylate) polymer Polymers 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 238000004094 preconcentration Methods 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 150000003856 quaternary ammonium compounds Chemical group 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
- BDHFUVZGWQCTTF-UHFFFAOYSA-N sulfonic acid Chemical group OS(=O)=O BDHFUVZGWQCTTF-UHFFFAOYSA-N 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
- 239000000080 wetting agent Substances 0.000 description 1
Classifications
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- 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)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
Description
Oppfinnelsen angår en fremgangsmåte for fremstilling av en virusinaktivert fraksjon inneholdende faktor VIII, ved hjelp av kromatografiske metoder. The invention relates to a method for producing a virus-inactivated fraction containing factor VIII, using chromatographic methods.
Faktor VIII er et livsviktig stoff som spiller en betydelig rolle ved blodkoaguleringen. Forstyrrelser av blodkoaguleringen lar seg således behandle ved administrasjon av faktor VIII. Det er derfor et stort behov for administrerbare faktor VIIl-preparater. Det har ikke manglet på forsøk på isolering av faktor VIII i sterkt anriket form fra naturlige kilder. Således er det allerede kjent kromatografiske metoder for rensing av faktor VIII fra kryopresipitater. Sistnevnte er en fraksjon som kan oppnås ved kjølebehandling av plasma. EP 367 840 B1 vedrører en kromatografisk fremgangsmåte for isolering av faktor VIII fra blodplasma uten forutgående kryopresipitering. Fraksjonen som inneholder faktor VIII separeres herunder ved en kromatografisk separasjon på et hydrofilt kromatografimateriale, som er modifisert med ionebyttergrupper. EP 0 238 701 vedrører en fremgangsmåte for fremstilling av en høyrenset, infeksiøs anti-hemofilifaktor, hvor de forbehandlede fraksjonene er et kryopresipitat som ved etanolfelling er befridd for fibrinogen, globulin, albuminer og andre forstyrrende bestanddeler. I den europeiske patentsøknad EP 0 343 275 beskrives en kromatografisk separasjon med ionebyttere etter virusinaktivering av kryopresipitatfraksjonen. EP 0 173 242 A beskriver en fremgangsmåte for kromatografisk isolering av faktor VII l-preparater på ionebyttermaterialer som utelukkende er basert på karbohydrater. Den anvendte karbohydratmatriks er modifisert med DEAE-grupper. Spesielt beskrives DEAE-Sepharose samt DEAE-cellulose, som egnet. I GB-A-1.178.958 beskrives en rensing av faktor VIII med ECTEOLA-cellulosesøyler. Den modifiserte cellulose inneholder basiske substituenter som er innført ved omsetning med epiklorhydrin og trietanolamin. Teknikkens stand som beskrevet ovenfor, benytter enten kromatografisk separering i form av satsvis teknikk eller søylekromatografi. Ved hjelp av disse fremgangsmåter oppnås riktignok ganske gode resultater, men det er både av økonomiske og etiske grunner ønskelig å øke utbyttet av den biologisk verdifulle faktor VIII. Factor VIII is a vital substance that plays a significant role in blood coagulation. Disturbances of blood coagulation can thus be treated by administering factor VIII. There is therefore a great need for administrable factor VIIl preparations. There has been no shortage of attempts to isolate factor VIII in 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 that can be obtained by cooling plasma. EP 367 840 B1 relates to a chromatographic method for isolating factor VIII from blood plasma without prior cryoprecipitation. The fraction containing factor VIII is separated below by a chromatographic separation on a hydrophilic chromatography material, which is modified with ion exchange groups. EP 0 238 701 relates to a method for the production of a highly purified, infectious anti-haemophilia factor, where the pre-treated fractions are a cryoprecipitate which, by ethanol precipitation, is freed from fibrinogen, globulin, albumins and other interfering components. In the European patent application EP 0 343 275, a chromatographic separation with ion exchangers after virus inactivation of the cryoprecipitate fraction is described. EP 0 173 242 A describes a method for the chromatographic isolation of factor VII 1 preparations on ion exchange materials which are exclusively based on carbohydrates. The carbohydrate matrix used is modified with DEAE groups. In particular, DEAE-Sepharose and DEAE-cellulose are described as suitable. GB-A-1,178,958 describes a purification of factor VIII with ECTEOLA cellulose columns. The modified cellulose contains basic substituents introduced by reaction with epichlorohydrin and triethanolamine. The state of the art, as described above, uses either chromatographic separation in the form of a batch technique or column chromatography. With the help of these methods quite good results are indeed achieved, but it is desirable for both economic and ethical reasons to increase the yield of the biologically valuable factor VIII.
EP-A-0 286 323 omhandler en fremgangsmåte for rensing av polypeptider med immobiliserte antistoffer. Videre utføres ytterligere rensetrinn med anionbytter-modifisert cellulosemembran. Faktor VIII lar seg rense ved hjelp av den der beskrevne fremgangsmåten. EP-A-0 286 323 deals with a method for the purification of polypeptides with immobilized antibodies. Further purification steps are carried out with anion-exchange-modified cellulose membrane. Factor VIII can be purified using the method described there.
Det tekniske problem som har ligget til grunn for oppfinnelsen består altså i å frembringe en fremgangsmåte som med bakgrunn i teknikkens stand, muliggjør en forbedret fremstilling av faktor VIII med hensyn til utbytte og biologisk aktivitet. The technical problem which has formed the basis of the invention therefore consists in producing a method which, based on the state of the art, enables an improved production of factor VIII with regard to yield and biological activity.
Problemet løses ved hjelp av en fremgangsmåte for fremstilling av en virusinaktivert fraksjon inneholdende faktor VIII, ved hjelp av kromatografiske metoder, kjennetegnet ved at idet det med utgangspunkt i kryopresipitat etter oppløsning derav eller blodplasma, eventuelt etterfulgt av en behandling med aluminiumhydroksyd, foretas en virusinaktivering gjennom behandling av fraksjonen med et di- eller tri-alkylfosfat og et ikke-ionisk tensid, fulgt av minst ett separasjonstrinn ved bruk av membrankromatografi på membraner eller kompaktskiver av porøse hydrofile polymerer poly(glycidylmetakrylater) eller hydrofilisert polystyren. The problem is solved by means of a method for producing a virus-inactivated fraction containing factor VIII, by means of chromatographic methods, characterized by the fact that starting from cryoprecipitate after dissolution thereof or blood plasma, optionally followed by treatment with aluminum hydroxide, virus inactivation is carried out through treatment of the fraction with a di- or tri-alkyl phosphate and a nonionic surfactant, followed by at least one separation step using membrane chromatography on membranes or compact discs of porous hydrophilic polymers poly(glycidyl methacrylates) or hydrophilized polystyrene.
Fremgangsmåten ifølge oppfinnelsen lar seg også gjennomføre med kommersielt tilgjengelig kryopresipitat eller blodplasma. For å oppnå en forkonsentrering av faktor VIII, behandles det opptinte kryopresipitat fortrinnsvis med aluminiumhydroksyd for videre forutgående rensing av prøven. The method according to the invention can also be carried out with commercially available cryoprecipitate or blood plasma. To achieve a pre-concentration of factor VIII, the thawed cryoprecipitate is preferably treated with aluminum hydroxide for further preliminary purification of the sample.
Før den egentlige kromatografiske rensing på materialer som er anordnet i eller på membraner, foretas en virusinaktivering. Virusinaktiveringen skjer herunder ifølge metoden som er beskrevet i EP 131 740 A1 ved behandling med biokompatible organiske løsningsmidler (detergenter), Triton® X-100/TNBP, fortrinnsvis Tween®/TNBP (tri-n-butylfosfat). Det oppnås også gode resultater med natriumcholat/TNBP. Fortrinnsvis anvendes detergentmengder på opp til 15vekt%. Before the actual chromatographic purification of materials arranged in or on membranes, a virus inactivation is carried out. The virus inactivation takes place below 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 are also achieved with sodium cholate/TNBP. Detergent amounts of up to 15% by weight are preferably used.
Det kromatografiske separasjonstrinn for rensing av faktor VIII i prøven, kan skje på basismaterialer, spesielt anionbyttere, som er modifisert med ionebyttergrupper, eller på materialer som er modifisert med immunaffinitetsligander. Avgjørende er herunder at de nevnte materialer er anordnet i membraner. Membraner <p>g kompakte skiver av porøse potyglycidylmetakrylater i likhet med hydrofilisert polystyren, er egnet. The chromatographic separation step for purification of factor VIII in the sample can take place on base materials, especially anion exchangers, which have been modified with ion exchange groups, or on materials which have been modified with immunoaffinity ligands. It is crucial here that the mentioned materials are arranged in membranes. Membranes <p>g compact discs of porous potyglycidyl methacrylates such as hydrophilized polystyrene are suitable.
En membran som er egnet for separeringen består av kompakte skiver eller polymere bærere. Basismaterialene for membranene eller skivene er forsynt med tilsvarende anionbyttergrupper eller immunaffinitetsligander. Som ionebyttergrupper er det særlig tale om anionbyttergrupper som kvartære aminer eller dietylaminoetylgrupper. Som kationbyttere kommer prinsipielt svake og sterkt sure, kationbyttere bestående av materialer som er modifisert med sulfonsyre- eller fosforsyre-grupper, i betraktning. A membrane suitable for the separation consists of compact discs or polymeric supports. The base materials for the membranes or discs are provided with corresponding anion exchange groups or immunoaffinity ligands. As ion exchange groups, we are particularly talking about anion exchange groups such as quaternary amines or diethylaminoethyl groups. As cation exchangers, in principle weak and strongly acidic cation exchangers consisting of materials modified with sulphonic acid or phosphoric acid groups come into consideration.
lonebyttergruppene kan være bundet til basismaterialets fibere med eller uten en såkalt spacer. Materialer forsynt med spacer betegnes også tentakel-materialer. I DE 42 04 694 er det nevnt tilsvarende spacere og ligander. Som spacer kan eksempelvis også benyttes en glukosaminrest. Også til membranene av porøst polyglycidylmetakrylat eller de øvrige nevnte materialer, kan det være bundet anionbyttergrupper som DEAE eller kvartære aminer. Bindingen av anionbyttergruppen skjer herunder enten direkte til materialet som danner membranen, eller via en spacer, f.eks. en glukosaminrest. the ion exchange groups can be bound to the fibers of the base material 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 also be used as a spacer. Anion exchange groups such as DEAE or quaternary amines can also be attached to the membranes of porous polyglycidyl methacrylate or the other materials mentioned. The binding of the anion exchange group takes place below either directly to the material that forms the membrane, or via a spacer, e.g. a glucosamine residue.
I en annen utførelsesform av fremgangsmåten ifølge oppfinnelsen, benyttes affinitetsmembrankromatografi med immobiliserte substanser som oppviser en høy affinitet for faktor VIII. In another embodiment of the method according to the invention, affinity membrane chromatography is used with immobilized substances that exhibit a high affinity for factor VIII.
Substansene med affinitet for faktor VIII immobiliseres på bæreren ved hjelp av kjemisk virksomme grupper. Fortrinnsvis vil den aktive gruppe ikke angripe bærermaterialet direkte, men i enden av en spacer. Immobiliseringen av substansene for faktor VIII skjer ved binding til aktive grupper som tosyl, tresyl, hydrazid og andre. Tilsvarende fremgangsmåter er kjent fra T.M. Phillips "Affinity chromatography" i "Chromatography" (E. Heftmann, red.) 5.utg. Elsevier, Amsterdam, 1992. The substances with affinity for factor VIII are immobilized on the carrier by means of chemically active groups. Preferably, the active group will not attack the support material directly, but at the end of a spacer. The immobilization of the substances for factor VIII takes place by binding to active groups such as tosyl, tresyl, hydrazide and others. Corresponding methods are known from T.M. Phillips "Affinity chromatography" in "Chromatography" (E. Heftmann, ed.) 5th ed. Elsevier, Amsterdam, 1992.
I en annen foretrukket utførelsesform anvendes materialer til separasjon av faktor VIII, som kan sikre en hydrofob vekselvirkning. Som hydrofobe materialer anvendes acykliske og/eller cykliske alkylkjeder, som f.eks. Cr til Cis-alkylkjeder så vel som aromatiske substanser. Aktuelle materialer som kan bevirke hydrofob vekselvirkning, er fortrinnsvis også slike som har gradert hydrofobisitet. Hydrofobisiteten kan graderes ved innføring av polare grupper, som polar-protiske eller polar-aprotiske grupper, som hydroksy-, amino- eller cyano-grupper. Fortrinnsvis tilpasses denne de respektive separasjonsbetingelsene. In another preferred embodiment, materials are used for the separation of factor VIII, which can ensure a hydrophobic interaction. Acyclic and/or cyclic alkyl chains are used as hydrophobic materials, such as e.g. Cr to Cis alkyl chains as well as aromatic substances. Current materials that can cause hydrophobic interaction are preferably also those that have graded hydrophobicity. The hydrophobicity can be graded by introducing polar groups, such as polar-protic or polar-aprotic groups, such as hydroxy, amino or cyano groups. Preferably, this is adapted to the respective separation conditions.
Virusinaktiveringen kan også skje gjennom en varmebehandling. I den forbindelse underkastes den faktor Vlll-holdige eluerte prøve etter en første membrankromatografering, fortrinnsvis et pasteuriseringstrinn. En tilsvarende fremgangsmåte er foreslått i DE-A-43 18 435. Herunder bringes faktor VIII-anrikede fraksjoner, i nærvær av stabilisatorer, i kontakt med di- eller tri-alkyl-fosfater og eventuelt med fuktemidler og behandles samtidig eller suksessivt ved temperaturer i området fra 55°C til 67°C i 5 til 30 timer. Det kan være fordelaktig å kombinere de to virusinaktiveirngsmetodene, detergent- og varmebehandlingen. Virus inactivation can also take place through a heat treatment. In this connection, the factor Vlll-containing eluted sample is subjected to a first membrane chromatography, preferably a pasteurization step. A similar method is proposed in DE-A-43 18 435. Here, factor VIII-enriched fractions, in the presence of stabilizers, are brought into contact with di- or tri-alkyl phosphates and possibly with wetting agents and treated simultaneously or successively at temperatures in range from 55°C to 67°C for 5 to 30 hours. It can be advantageous to combine the two virus inactivation methods, detergent and heat treatment.
For å fjerne kjemikalier benyttet ved pasteuriseringstrinnet, kan det også legges inn en ytterligere membrankromatografering. Fortrinnsvis skjer separeringen av de tilsatte stabilisatorer med en membran som er modifisert med DEAE- eller kvartære ammonium-forbindelser som er anordnet på overflaten av det kromatografiske bærermaterialet via en spacer. Det er likeledes mulig å anordne de tilsvarende ligander på bærermaterialets overflate uten spacer. Stabilisatorene retarderes ikke av dette anionbyttermaterialet under de valgte betingelser, mens faktor VIII adsorberes på kromatografimaterialet. In order to remove chemicals used in the pasteurization step, a further membrane chromatography can also be added. Preferably, the separation of the added stabilizers takes place with a membrane modified with DEAE or quaternary ammonium compounds which are arranged on the surface of the chromatographic support material via a spacer. It is also possible to arrange the corresponding ligands on the surface of the carrier material without a spacer. The stabilizers are not retarded by this anion exchange material under the chosen conditions, while factor VIII is adsorbed on the chromatography material.
Deretter elueres faktor VIII med et vandig løsningsmiddelsystem med trinnvis tiltagende saltkonsentrasjoner. Factor VIII is then eluted with an aqueous solvent system with stepwise increasing salt concentrations.
Den således oppnådde faktor VIIl-holdige fraksjon konsentreres, tappes og underkastes eventuelt lyofilisering, ved hjelp av konvensjonelle metoder. The factor VIIl-containing fraction thus obtained is concentrated, bottled and optionally subjected to lyophilisation, using conventional methods.
Fortrinnsvis påsettes faktor VIII i det første membrankromatografiske separasjonstrinn som en løsning av lav ionestyrke. Fortrinnsvis oppviser det vandige system en ionestyrke som tilsvarer en 0 til 150 mM natriumkloridløsning. Ved denne ionestyrke er faktor VIII fremdeles adsorbert til det kromatografiske materiale, mens derimot svakere bindende forurensninger kan utvaskes med vandige systemer av samme ionestyrke. Preferably factor VIII is applied in the first membrane chromatographic separation step as a solution of low ionic strength. Preferably, the aqueous system exhibits an ionic strength corresponding to a 0 to 150 mM sodium chloride solution. At this ionic strength, factor VIII is still adsorbed to the chromatographic material, while, on the other hand, weaker binding contaminants can be washed out with aqueous systems of the same ionic strength.
En rensing av det adsorberte materialet kan i henhold til en annen utførelsesform av fremgangsmåten ifølge oppfinnelsen, skje med et vandig system med en ionestyrke som tilsvarer en 200 til 400 mM natriumkloridløsning. Desorbsjonen av faktor VIM og eluering av denne fraksjon skjer deretter med et vandig system med en ionestyrke som tilsvarer 500 til 1500 mM natriumklorid-løsning. Herunder holdes pH-verdien i området fra 4 til 9. Foretas en kation-bytterkromatografi, skjer dette fortrinnsvis ved en pH-verdi <6, mens anionbytter-kromatografien heller foretas ved høyere pH-verdier på mer enn 6. Purification of the adsorbed material can, according to another embodiment of the method according to the invention, take place with an aqueous system with an ionic strength corresponding to a 200 to 400 mM sodium chloride solution. The desorption of factor VIM and elution of this fraction then takes place with an aqueous system with an ionic strength corresponding to 500 to 1500 mM sodium chloride solution. Below this, the pH value is kept in the range from 4 to 9. If a cation-exchange chromatography is carried out, this preferably takes place at a pH value <6, while the anion-exchange chromatography is rather carried out at higher pH values of more than 6.
Utføres rensingen av faktor VIII ved immunaffinitetsmembrankromatografi skjer elueringen til forskjell fra ovennevnte metoder, med anionbyttermaterialer, med chaotrope reagenser eller høykonsentrerte saltløsninger. Elueringen skjer fortrinnsvis med konsentrasjoner av chaotrope reagenser eller salter som er tilstrekkelige til å løse bindingen mellom substansen med høy affinitet for faktor VIII og selve faktor VIII. Konsentrasjonen av de nevnte stoffer i de respektive elueringssystemer avhenger herunder av affinitetsstyrken til faktor VIII og den tilsvarende bindende komponent. Derved kan det skje en eluering med vandige løsninger av lavere denaturerende styrke. Fortrinnsvis benyttes vandige urealøsninger med en konsentrasjon på 1 til 6M, spesielt 2 til 4M, urea eller tilsvarende høyt konsentrerte saltløsninger, for eluering av faktor VIII fra immunaffinitetsmembranen. If the purification of factor VIII is carried out by immunoaffinity membrane chromatography, the elution takes place differently from the above-mentioned methods, with anion exchange materials, with chaotropic reagents or highly concentrated salt solutions. The elution preferably takes place with concentrations of chaotropic reagents or salts that are sufficient to dissolve the bond between the substance with a high affinity for factor VIII and factor VIII itself. The concentration of the mentioned substances in the respective elution systems depends on the affinity strength of factor VIII and the corresponding binding component. Thereby, an elution can take place with aqueous solutions of lower denaturing strength. Preferably, aqueous urea solutions with a concentration of 1 to 6M, especially 2 to 4M, urea or similarly highly concentrated salt solutions are used for elution of factor VIII from the immunoaffinity membrane.
Ved hydrofob interaksjonskromatografi påsettes prøven i en vandig løsning av meget høy ionestyrke, som eksempelvis høykonsentrert ammoniumsulfat (opp til en konsentrasjon på 4M) eller natriumklorid (opp til en konsentrasjon på 5M). Elueringen foretas fortrinnsvis trinnvis eller kontinuerlig med saltløsninger av lavere ionestyrke. Som løsninger med lavere ionestyrke for eluering av prøvene ved den hydrofobe interaksjonsmembrankromatografering, kan det også benyttes en vandig løsning av organiske løsningsmidler, spesielt en fortynnet alkoholisk løsning. In hydrophobic interaction chromatography, the sample is placed in an aqueous solution of very high ionic strength, such as highly concentrated ammonium sulphate (up to a concentration of 4M) or sodium chloride (up to a concentration of 5M). The elution is preferably carried out in stages or continuously with salt solutions of lower ionic strength. As solutions with lower ionic strength for elution of the samples in the hydrophobic interaction membrane chromatography, an aqueous solution of organic solvents, especially a dilute alcoholic solution, can also be used.
Fremgangsmåten ifølge oppfinnelsen sikrer på en overraskende måte en hurtig og ukomplisert rensing av faktor VIII som samtidig fører til høy renhet og høyt utbytte. Dessuten er den spesifikke aktivitet av den derved oppnådde faktor VIII ganske høy, hvilket lar seg forklare gjennom en liten grad av denaturering av den virksomme faktor. The method according to the invention surprisingly ensures a quick and uncomplicated purification of factor VIII, which at the same time leads to high purity and high yield. Moreover, the specific activity of the thereby obtained factor VIII is quite high, which can be explained by a small degree of denaturation of the active factor.
i in
Claims (11)
Applications Claiming Priority (2)
| 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 |
| PCT/EP1994/003258 WO1995012609A1 (en) | 1993-11-04 | 1994-09-30 | Process for producing a virus-inactivated factor viii-containing fraction by chromatographic methods |
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| Publication Number | Publication Date |
|---|---|
| NO961816D0 NO961816D0 (en) | 1996-05-03 |
| NO961816L NO961816L (en) | 1996-05-03 |
| NO317112B1 true NO317112B1 (en) | 2004-08-16 |
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| NO19961816A NO317112B1 (en) | 1993-11-04 | 1996-05-03 | A process for preparing a virus-activated fraction containing factor VIII by chromatographic methods |
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| 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) |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| DE19618851C1 (en) * | 1996-05-10 | 1997-07-24 | Octapharma Ag | Testing factor VIII containing preparations |
| US6057164A (en) * | 1996-03-08 | 2000-05-02 | 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. |
| ES2391613T3 (en) * | 2008-06-24 | 2012-11-28 | Octapharma Ag | A procedure to purify coagulation factor VIII |
| SG191186A1 (en) | 2010-12-15 | 2013-07-31 | Baxter Int | Eluate collection using conductivity gradient |
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| 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 |
| ES2053684T3 (en) * | 1988-11-05 | 1994-08-01 | Octapharma Ag | PROCEDURE TO PREPARE A HIGHLY PURE, NON-INFECTIOUS ANTIHEMOPHILIC FACTOR THROUGH CHROMATOGRAPHY. |
| 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 |
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1993
- 1993-11-04 DE DE4337573A patent/DE4337573C1/en not_active Revoked
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- 1994-09-30 UA UA96041756A patent/UA43855C2/en unknown
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- 1994-09-30 RU RU96110890A patent/RU2148411C1/en not_active IP Right Cessation
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