EP0000651B1 - Procédé pour l'isolement d'une préparation du facteur IX à partir du plasma sanguin - Google Patents

Procédé pour l'isolement d'une préparation du facteur IX à partir du plasma sanguin Download PDF

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Publication number
EP0000651B1
EP0000651B1 EP78300177A EP78300177A EP0000651B1 EP 0000651 B1 EP0000651 B1 EP 0000651B1 EP 78300177 A EP78300177 A EP 78300177A EP 78300177 A EP78300177 A EP 78300177A EP 0000651 B1 EP0000651 B1 EP 0000651B1
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EP
European Patent Office
Prior art keywords
plasma
factor
copolymer
factors
preparation
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired
Application number
EP78300177A
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German (de)
English (en)
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EP0000651A1 (fr
Inventor
Jacques Jean Joseph Delente
Richard Alan Schoenfeld
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Monsanto Co
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Monsanto Co
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Publication date
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Publication of EP0000651A1 publication Critical patent/EP0000651A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/644—Coagulation factor IXa (3.4.21.22)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21022—Coagulation factor IXa (3.4.21.22)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
    • Y10S530/827—Proteins from mammals or birds
    • Y10S530/829—Blood
    • Y10S530/83—Plasma; serum

Definitions

  • This invention relates to blood fractionation and more particularly to the separation of a blood coagulation Factor IX preparation from plasma by the selective adsorption of Factors II, IX and X.
  • the process of blood coagulation is a complicated physiological activity that involves the interaction of numerous substances found in normal whole blood. It is known that certain factors associated with the blood coagulation mechanism are absent or seriously deficient in certain individuals. In those patients suffering from classical hemophilia, antihemophilic factor A (AHF, Factor VIII) is deficient. In those patients afflicted with hemophilia B, plasma thromboplastin component (PTC, Factor IX) is missing from the blood.
  • AHF antihemophilic factor A
  • PTC Factor IX
  • Factors II, VII and X are factors which are important in the coagulation mechanism. As with Factors VIII and IX, these other factors also are deficient or absent in certain individuals. Factors II, VII and X are usually associated with Factor IX in the fractionation of blood plasma into various fractions, and a concentrate of these four factors has come to be known as the prothrombin complex.
  • hemo- philiac needing certain blood coagulation factors ideally should be given only those factors required or at least a purified concentrate of these factors containing a reduced level of unneeded factors.
  • Patent 3,920,625 further describe the use of DEAE-Sephadex specifically for the preparation of Factor IX concentrates.
  • Use of polyethylene glycol for the production of prothrombin complex is taught by Fekete and Shanbrom in U.S. Patents 3,560,475 and 3,682,881.
  • Aluminum hydroxide and other such gel materials also are known as useful in the concentration of prothrombin complex factors as seen from Bidwell, U.S. Patent 2,867,567.
  • Casillas et al. in Revista de la Associacion Bioquimica Argentina 40 (222-223), 147-157 (1975) describe techniques for obtaining clotting factors (or groups of factors which do not contaminate one another) for clinical use and characterisation studies.
  • preparation 3 entitled “Concentrado de fac- tores II-IX-X para uso clinico”
  • Casillas et al. describes the production of a concentrate of factors II, IX and X by fractionating plasma with cold ethanol (-5°C.) according to Cohn fractionation procedures followed by addition of moistened and equilibrated DEAE-cellulose to the Cohn I supernatant at 5-10°C. in an amount of 1 gram of the ion exchange resin (dry basis) for each 100 ml of the Cohn I supernatant.
  • the concentrate of factors II, IX and X is then recovered by further column chromatographic procedures.
  • Factor VIII is removed from plasma prior to Factors II, VII, IX and X by cryo- precipitation.
  • Factors II, IX and X are separated from the plasma without separation of Factors VII and VIII, which is substantially different from the Dike et al. method.
  • the method of the present invention is one of separating a Factor IX preparation from plasma, comprising contacting liquid blood plasma with a water-insoluble, cross-linked polyelectrolyte copolymer of ethylene and maleic anhydride containing pendant diloweralkylamino-loweralkyl functional groups linked to the copolymer via imide linkages, wherein "loweralkyl” has from 1 to 4 carbon atoms, characterised in that the contacting is carried out at a pH of from 7.4 to 8.5 and the amount of the copolymer is from 0.025% to 0.1% by weight of the plasma, whereby Factors II, IX and X are selectively adsorbed by the polyelectrolyte copolymer to the substantial exclusion of Factors VII and VIII which are unadsorbed and remain in the liquid plasma.
  • a Factor IX preparation containing Factors II, IX and X is separated from liquid plasma with a water-insoluble, cross-linked polyelectrolyte copolymer of ethylene and maleic anhydride containing pendant diloweralkylamino-loweralkyl functional groups.
  • a Factor IX preparation containing Factors II, IX and X surprisingly is selectively adsorbed by the polyelectrolyte copolymer to the substantial exclusion of Factors VII and VIII which are unadsorbed and remain in the liquid plasma.
  • the adsorbed Factor IX preparation can then be eluted from the polyelectrolyte by washing with an aqueous solution of a physiologically acceptable salt such as NaCl, for example a solution of about one to three molar NaCI.
  • a physiologically acceptable salt such as NaCl
  • the elution preferably is carried out at a pH of from about 5.5 to about 6.5 although higher pH's also can be used.
  • the starting plasma used in the fractionation method of this invention is generally obtained fresh frozen. This plasma should be thawed before fractionation with the polyelectrolyte copolymer, preferably by heating to a temperature of at least about 35°C.
  • the appropriate polyelectrolyte copolymer can then be admixed with the plasma at a concentration of from 0.025% to 0.1% and preferably 0.035% to 0.05%, and the pH adjusted to a range of from 7.4 to 8.5.
  • the mixture can be stirred for a suitable time, for example at least about 10 minutes, during which time the Factor IX preparation is selectively adsorbed by the polyelectrolyte copolymer and the remaining liquid plasma is made deficient in Factors II, IX and X.
  • the water-insoluble, cross-linked polyelectrolyte copolymers employed in this invention are copolymers of ethylene and maleic anhydride containing pendant diloweralkylaminoloweralkyl functional groups.
  • loweralkyl is meant an alkyl having from 1 to 4 carbon atoms.
  • the base copolymer of ethylene and maleic anhydride can be prepared, for example, by reacting ethylene and maleic anhydride in the presence of a peroxide catalyst in a suitable solvent.
  • the copolymer will preferably contain substantially equimolar quantities of the ethylene residue and the anhydride residue.
  • the base EMA copolymer can be reacted with a loweralkyliminobisloweralkylamine which has two primary amine groups and leads to a cross-linked EMA copolymer.
  • the desired pendant diloweralkylaminoloweralkyl functional groups can then be incorporated into the cross-linked copolymer by reaction of diloweralkyl- aminoloweralkylamine with part of all of the remaining anhydride groups of the EMA polymer.
  • the polyelectrolyte copolymer also desirably is converted to the HCI salt form to provide better handling characteristics. Further details on the preparation of these polyelectrolyte copolymers can be had by reference to the disclosure in U.S. Patent 3,554,985 which is incorporated herein by reference. Use of these polyelectrolyte copolymers in blood fractionation is described in U.S. Patent 3,555,001.
  • a preferred diloweralkylaminoloweralkyl functional group is dimethylaminopropyl and a preferred cross-linking agent is methyliminobispropylamine.
  • a preferred polyelectrolyte copolymer for use in this invention contains about five methyliminobispropylamine cross-linking groups and about 90 pendant dimethylaminopropylamine functional groups per 100 maleic anhydride units in the EMA copolymer.
  • cross-linking agents for example, divinylbenzene and ethylene diamine
  • other functional groups for example, dimethylaminoethyl and diethylaminobutyl
  • dimethylaminoethyl and diethylaminobutyl also can be used in the polyelectrolyte copolymers which are employed in the method of separating the Factor IX preparation herein.
  • the Factor VIII remaining in the plasma solution can be further concentrated and recovered by known techniques.
  • about 0.035% by weight of the polyelectrolyte copolymer containing about five methyliminobispropylamine cross-linking groups and about 90 dimethylaininopropylamine functional groups per 100 maleic anhydride units in the EMA copolymer is employed for selective adsorption of the Factor IX preparation at a pH of about 8.
  • the adsorbed Factor IX preparation is then eluted from the polyelectrolyte copolymer by washing with 1.7 molar NaCI at pH 6.
  • the eluant can then be dialyzed against 0.1 molar NaCI at 4°C and freeze dried for storage.
  • the polyelectrolyte copolymer consisted of the reaction product of substantially equimolar parts of ethylene and maleic anhydride (EMA) cross-linked with methyliminobispropylamine (MIBPA) and then further reacted with dimethylaminopropylamine (DMAPA) such as to provide about five MIBPA cross-linking groups and about 90 DMAPA pendant groups per 100 maleic anhydride units in the EMA copolymer and converted to the HCI salt form.
  • EMA ethylene and maleic anhydride
  • MIBPA methyliminobispropylamine
  • DMAPA dimethylaminopropylamine
  • One liter of normal human plasma was adjusted to pH 8 with 1 molar NaOH and 0.35 grams of the aforesaid polyelectrolyte copolymer was added thereto and the mixture was stirred for 20 minutes. The mixture was then filtered and the filtrate was retained as a Factor IX depleted plasma. The filter cake was washed
  • a Factor IX preparation containing Factors II, IX and X was then eluted from the polyelectrolyte copolymer by washing with 25 ml. of 1.7 molar NaCl at pH 6.0 (the pH being adjusted with 0.1 molar citric acid) for 20 minutes. The copolymer slurry was then filtered and the filtrate was retained as the desired Factor IX preparation. In a series of seven one-liter replicate fractionations using the above procedure, an average of 483 ⁇ 48 units of Factor IX per liter were obtained having a purification index of 178 ⁇ 33.
  • One unit of Factor IX is defined as the amount of said factor in one ml of pooled normal whole plasma.
  • the purification index is calculated as the ratio of the amount of total protein in the starting plasma to the amount of total protein in the final Factor IX preparation multiplied by the ratio of the units of Factor IX in the final Factor IX preparation to the units of Factor IX in the starting plasma.
  • the partial thromboplastin reagent contains crude cephalin obtained from rabbit brain which is known to clot normal plasma faster than its clots hemo- philic plasma.
  • Such reagents are well-known and described, for example, in U.S. Patents 3,395,210, 3,486,981 and 3,522,148.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)

Claims (6)

1. Procédé de séparation d'une préparation de facteur IX d'un plasma comprenant la mise en contact d'un plasma sanguin liquide avec un copolymère polyélectrolytique réticulé, insoluble dans l'eau, d'éthylène et d'anhydride maléique contenant des groupes fonctionnels dialkyl(inférieur)aminoalkyle inférieur pendants liés au copolymère par des liaisons imides, où le groupe "alkyle inférieur" a entre 1 et 4 atomes de carbone, caractérisé en ce que le contact est exécuté à un pH compris entre 7,4 et 8,5 et la quantité de copolymère est comprise entre 0,025% et 0,1% en poids de plasma, à la suite de quoi les facteurs II, IX et X sont sélectivement adsorbés par le copolymère à l'exclusion importante des facteurs VII et VIII qui ne sont pas adsorbés et restent dans le plasma liquide.
2. Procédé selon la revendication 1, caractérisé en ce que le groupe fonctionnel dialkyl-(inférieur)aminoalkyle inférieur est le diméthylaminopropyle.
3. Procédé selon l'une des revendications 1 ou 2, caractérisé en ce que le copolymère d'éthylène et d'anhydride maléique est réticulé avec la methyliminobispropylamine.
4. Procédé selon la revendication 1, caractérisé en ce que le copolymère polyélectrolytique contient environ 5 groupes de réticulation méthyliminobispropylamine et environ 90 groupes diméthylaminopropyle pendants par 100 groupes d'anhydride maléique.
5. Procédé selon l'une des revendications 1 à 4, caractérisé en ce que la préparation de facteur IX absorbée est soumise à une élution pour la séparer du copolymère polyélectrolytique par lavage avec une solution aqueuse de NaCI ayant une molarité comprise entre 1 et 3.
6. Procédé selon l'une des revendications 1 à 5, caractérisé en ce que la concentration du copolymère est comprise entre 0,035% et 0,05% en poids du plasma.
EP78300177A 1977-07-25 1978-07-21 Procédé pour l'isolement d'une préparation du facteur IX à partir du plasma sanguin Expired EP0000651B1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US818920 1977-07-25
US05/818,920 US4081432A (en) 1977-07-25 1977-07-25 Method of separating a Factor IX preparation from plasma using ethylene-maleic anhydride polymers

Publications (2)

Publication Number Publication Date
EP0000651A1 EP0000651A1 (fr) 1979-02-07
EP0000651B1 true EP0000651B1 (fr) 1982-01-27

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ID=25226755

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Application Number Title Priority Date Filing Date
EP78300177A Expired EP0000651B1 (fr) 1977-07-25 1978-07-21 Procédé pour l'isolement d'une préparation du facteur IX à partir du plasma sanguin

Country Status (15)

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US (1) US4081432A (fr)
EP (1) EP0000651B1 (fr)
JP (1) JPS5426322A (fr)
AT (1) AT359645B (fr)
AU (1) AU517885B2 (fr)
CA (1) CA1107649A (fr)
DE (1) DE2861573D1 (fr)
ES (1) ES471857A1 (fr)
HU (1) HU180882B (fr)
IL (1) IL55194A0 (fr)
IT (1) IT1097302B (fr)
MX (1) MX5405E (fr)
PT (1) PT68337B (fr)
RO (1) RO75338A (fr)
SU (1) SU841572A3 (fr)

Families Citing this family (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1603244A (en) * 1977-05-20 1981-11-18 Max Planck Gesellschaft Medicaments for the suppression of pathological processes
ES471858A1 (es) * 1977-07-25 1979-02-01 Monsanto Co Un metodo para separar el factor especifico de coagulacion de la sangre de una mezcla con otras proteinas de la sangre en un medio fluido
FR2472390A1 (fr) * 1979-05-04 1981-07-03 Merieux Inst Procede de preparation de concentres de complexe prothrombinique hautement purifies, et concentres obtenus
SE448945B (sv) * 1979-12-20 1987-03-30 Blombaeck E G B Forfarande for rening och /eller koncentrering av faktor viii-komplexet
AT368883B (de) 1980-07-22 1982-11-25 Immuno Ag Verfahren zur herstellung einer neuen blutgerinnungsfoerdernden praeparation auf basis von humanproteinen
DE3045153A1 (de) * 1980-11-29 1982-07-08 Behringwerke Ag, 3550 Marburg Verfahren zur herstellung von blutgerinnungsfaktoren und danach hergestellte praeparation der faktoren ix und x
US4447416A (en) * 1982-04-28 1984-05-08 American National Red Cross Plasma protein concentrates of reduced thrombogenicity and their use in clinical replacement therapy
US4397841A (en) * 1982-06-28 1983-08-09 Monsanto Company Production of blood coagulation factor VIII:C
US4382028A (en) * 1982-07-19 1983-05-03 Monsanto Company Separation of plasma proteins from cell culture systems
US5055557A (en) * 1983-03-04 1991-10-08 Scripps Clinic & Research Foundation Ultrapurification of factor IX and other vitamin K-dependent proteins
US5614500A (en) * 1983-03-04 1997-03-25 The Scripps Research Institute Compositions containing highly purified factor IX proteins prepared by immunoaffinity chromatography
US4786726A (en) * 1986-01-06 1988-11-22 Blood Systems, Inc. Factor IX therapeutic blood product, means and methods of preparing same
DE3627762A1 (de) * 1986-08-16 1988-02-18 Behringwerke Ag Verfahren zur herstellung einer prothrombin-armen praeparation vitamin k-abhaengiger gerinnungsfaktoren, sowie ein danach erhaeltliches mittel
ZA877535B (en) * 1986-10-09 1988-04-11 F. Hoffmann-La Roche & Co. Aktiengesellschaft Factor ix-peptides
KR100447685B1 (ko) * 1995-08-28 2004-12-03 세키스이 가가쿠 고교 가부시키가이샤 혈청또는혈장분리용조성물

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3554985A (en) * 1963-01-02 1971-01-12 Monsanto Co Cross-linked copolymer polyelectrolytes based on alpha,beta-ethylenically unsaturated acids
US3717708A (en) * 1968-10-24 1973-02-20 Cutter Lab Blood coagulation complex
US3555001A (en) * 1969-05-29 1971-01-12 Monsanto Co Process for the fractionation of plasma and serum using water-insoluble polyelectrolytes containing diloweralkylaminoloweralkylimide groups
US3682881A (en) * 1970-10-02 1972-08-08 Baxter Laboratories Inc Fractionation of plasma using glycine and polyethylene glycol
IL42254A (en) * 1973-05-13 1977-03-31 Yeda Res & Dev Polymeric carrier for biologically active proteins and biologically active substances based on these
US3920625A (en) * 1973-06-19 1975-11-18 Kabi Ab Isolation of coagulation factors from biological material using cross linked sulfated, sulfonated carbohydrates
DE2534603C2 (de) * 1975-08-02 1984-10-31 Basf Ag, 6700 Ludwigshafen Verfahren zur Herstellung von Copolymerisaten mit N-Dialkyl-N-amido-Gruppen

Also Published As

Publication number Publication date
PT68337A (en) 1978-08-01
ES471857A1 (es) 1979-02-01
PT68337B (pt) 1994-02-25
IL55194A0 (en) 1978-09-29
AU3824378A (en) 1980-01-24
EP0000651A1 (fr) 1979-02-07
CA1107649A (fr) 1981-08-25
AT359645B (de) 1980-11-25
AU517885B2 (en) 1981-09-03
ATA531778A (de) 1980-04-15
IT7826005A0 (it) 1978-07-21
JPS5426322A (en) 1979-02-27
IT1097302B (it) 1985-08-31
DE2861573D1 (en) 1982-03-11
JPS6154008B2 (fr) 1986-11-20
SU841572A3 (ru) 1981-06-23
HU180882B (en) 1983-05-30
MX5405E (es) 1983-07-13
US4081432A (en) 1978-03-28
RO75338A (fr) 1980-11-30

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