US3682881A - Fractionation of plasma using glycine and polyethylene glycol - Google Patents

Fractionation of plasma using glycine and polyethylene glycol Download PDF

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US3682881A
US3682881A US77491A US3682881DA US3682881A US 3682881 A US3682881 A US 3682881A US 77491 A US77491 A US 77491A US 3682881D A US3682881D A US 3682881DA US 3682881 A US3682881 A US 3682881A
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ahf
plasma
precipitate
glycine
polyethylene glycol
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Lajos F Fekete
Edward Shanbrom
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Baxter International Inc
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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)
    • C12N9/6429—Thrombin (3.4.21.5)
    • 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)
    • 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)
    • C12Y304/21005—Thrombin (3.4.21.5)
    • 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

  • Concentrates of antihemophilic factor A and prothrombin complex are prepared from citrated blood plasma by an initial fractionation with glycine followed by multiple fractionations of the AHF-containing precipitate and the prothrombin complex-containing supernate with polyethylene glycol, the AHF-containing fraction being given an additional fractionation with glycine and the prothrombin complex-containing fraction being given an in termediate adsorption with tribasic calcium phosphate.
  • the process of blood coagulation is a complicated physiological activity and involves the interaction of numerous substances found in normal whole blood. It is know that certain factors associated with the blood coagulation mechanism are absent or seriously deficient in certain individuals. In those patients suifering from classical hemophilia, antihemophilic factor A (AHF, Factor VIII) is deficient. In those patients afllicted with hemophilia B, plasma thromboplastin component (PTC, Factor IX) is missing from the blood.
  • AHF antihemophilic factor A
  • PTC Plasma thromboplastin component
  • the hemophiliac needing certain blood coagulation factors ideally is given only those factors required or at least a purified concentrate of those factors.
  • antihemophilic factor A fractions 20 to 35 times purified in terms of activity per miligram of protein and 10 times concentrated, have been prepared by glycine-precipitation of either fresh or frozen plasma by Webster et al., Amer. J.
  • citrated plasma could be used directly for the prepartion of separate concentrates of antihemophilic factor A and prothrombin complex would find much use in the field of blood coagulation therapy.
  • fresh or frozen citrated plasma is mixed with glycine, using a final glycine concentration of from about 1.3 to about 1.8 molar, to produce an AHF-containing precipitate and a prothrombin complex-containing plasma supernatant.
  • glycine glycine
  • concentration of from about 1.3 to about 1.8 molar
  • Each of the respective precipitate and supernate fractions are then subjected to multiple fractionations with polyethylene glycol, the AHF-containing fraction being given an additional precipitation with glycine and the prothrombin complex-containing faction being given an intermediate adsorption with tribasic calcium phosphate.
  • FIG. 1 is a schematic chart of a preferred embodiment of the method of preparing the separate concentrates of antihemophilic factor A and prothrombin complex from citrated blood plasma.
  • prothrombin complex refers to a concentrate of blood proteins which are active in the coagulation process comprising principally prothrombin (Factor II), proconvertin (Factor VII), antihemophilic factor B (Factor IX) and Stuart-Prower factor (Factor X).
  • the polyethylene glycol used as a precipitating agent in this inventtion is a high molecular weight polymer which is generally produced by reacting ethylene oxide with ethylene glycol or water and has the following structure:
  • the plasma supernatant from the glycine precipitation step is suspended in normal physiological saline (about 0.9% NaCl), preferably to a dilution of about 1:1, the pH is adjusted to from about 6.0 to about 7.2, and the resulting suspension is precipitated with polyethylene glycol to a final concentration of about 30% PEG.
  • the resulting plasma protein precipitate is then separated from the supernatant.
  • the supernatant, which contains the undesired citrate and glycine salts, is discarded.
  • the plasma protein precipitate is resuspended in normal physiological saline, the pH is ad justed to from about 6.8 to about 7.2, and tribasic calcium phosphate is thoroughly mixed with the suspension to adsorb the coagulation factors.
  • the resultant tribasic calcium phosphate adsorbed-protein precipitate is then thoroughly mixed with from about 0.05 M to about 0.2 M trisodium citrate followed by recovery of the resulting supernatant which contains the desired coagulation factors.
  • the supernatant is subjected to a succession of two polyethylene glycol precipitations, first at a pH of from about 6.8 to abut 8.0 and to a final concentration of from about to about polyethylene glycol with retention of the resulting supernatant, and then at a pH of from about 5.0 to about 5.4 and to a final concentration of at least about 20% polyethylene glycol by weight of said retained supernatant with retention of the resulting precipitate.
  • the precipitate which contains the active prothrombin complex, is then preferably suspended in citrated saline to a final volume of from about one twentyfifth to about one tenth the volume of the suspension of the starting blood plasma.
  • the adsorption of the coagulation factors is carried out by adjusting the pH of the suspension to within a range of from about 6.8 to about 7.2 followed by adding a small amount of tribasic calcium phosphate.
  • the tribasic calcium phosphate used in this invention is a polymeric type material which can be described by the formula 10CaO-3P O -H O and, alternatively, by the formula Ca (OH) (PO).
  • the use of from about 0.5% to about 2% by weight of tribasic calcium phosphate has been found to be suitable for the adsorption of the coagulation factors and a concentration of about 1% is preferred.
  • the tribasic calcium phosphate preferably is allowed to mix with the suspension for about to about 30 minutes in order to provide for substantially maximum adsorption of the coagulation factors.
  • the precipitate preferably is suspended in the trisodium citrate with constant stirring for about 15 to about 30 minutes in order to provide for substantially maximum elution of the respective contaminating proteins and the desired coagulation factors. Separation of these substances from the tricalcium phosphate particles is preferably carried out by centrifugation accompanied with constant stirring.
  • the resuspended polyethylene glycol precipitate which contains the active prothrombin complex, is preferably lyophilized or freeze-dried after the addition of an anticoagulant, for example, heparin, in an amount of from about one to about ten units per mil. (U.S. Pharmacopoeia units), adjustment of the pH to about 6.8, and filtering to remove any undesired particles and at the same time to obtain a sterile product without heating.
  • the dry, lyophilized product is stable and can be reconstituted with water prior to use for intravenous, subcutaneous or intramuscular administration.
  • the preferred alkaline reagent for pH adjustment is an aqueous solution of about 1 N sodium hydroxide.
  • Other conventional alkaline reagents for example, sodium bicarbonate, can be used in place of sodium hydroxide.
  • the preferred acid reagent for pH adjustment when required, is an aqueous solution of about 1 N hydrochloric acid.
  • Other conventional acidifying reagents for example, acetic acid, can be used in place of hydrochloric acid.
  • the AHE- containing precipitate is first redissolved and then the redissolved material is subjected to the above-described two successive precipitations with polyethylene glycol, followed by recovery and redissolution of the second precipitate.
  • the redissolved polyethylene glycol precipitated fraction is then subjected to precipitation with glycine.
  • Recovery of the polyethylene glycol and glycine precipitated fractions for use in this invention can be accomplished, for example, by centrifugation or filtration of the respective precipitates or by similar such procedures. Redissolution of the recovered precipitates can be achieved by warming and agitating in citrated saline solution. In the case of the redissolution of the AHF- containing starting precipitate, it is preferred to use a glycine citrated saline solution and to increase the volume of the mixture to about one twentieth the volume of the original plasma which the starting precipitate represents.
  • the polyethylene glycoland glycine-precipitated fractions are preferably redissolved with citrated saline solution to increase the volume of the fractions to about one two-hundredth the volume of plasma which the respective precipitated fractions represent.
  • each of the respective redissolved starting precipitate and polyethylene glycoland glycine-precipitated fractions by clarifying with additional centrifugation and/or filtration to remove any insoluble matter.
  • the above fractionation by successive precipitation with polyethylene glycol and glycine has been found to provide a highly soluble AHF concentrate of high potency which can be frozen and rendered stable, such as by lyophilization, and retained under ordinary refrigeration conditions for periods of a year or longer.
  • the potency of each batch of material prepared by the above fractionation method can be precisely determined so that the administering physician can know exactly how much AHF his patient receives.
  • the hemophiliac can be given a quantity of AHF which the heart could not otherwise tolerate. Even more importantly, the AHF activity in the above-prepared concentrate is contained in less than 2.5% the amount of protein present in plasma providing an equal amount of AHF activity. This lower protein content minimizes the likelihood of allergic reactions by the hemophiliac recipient and reduces the possibility of overloading the circulatory system.
  • the concentrate of AHF which has been prepared by successive precipitation with polyethylene glycol and glycine can be further purified by treatment with ECTEOLA cellulose resin.
  • This purification can be carried out either before or after the polyethylene glycol and glycine precipitation and may be done by column or batch techniques.
  • the concentrate purified by this method has the additional advantage in that it can also be administered intramuscularly as well as by intravenous administration methods generally used in the case of the AHF concentrate which has not been treated with the ECTEOLA cellulose resin.
  • the AHF concentrate purified with ECTEOLA cellulose resin has been found to be free of fibrinogen by the addition of thrombin and by immunoelectrophoresis.
  • ECTEOLA cellulose resin refers to a modified cellulose 'which contains active basic substituents introduced into the cellulose molecule by reaction with epichlorohydrin and triethanolamine.
  • Methods of preparation of ECTEOLA cellulose resins are described in general by Sober and Peterson, J. Am. Chem. Soc. vol. 76, pp. 1711-12 (1956); id., vol. 78, pp. 751- 55 (1956); vol. 78, pp. 756-63 (1956); and Peterson and Sober, Biochem Preparations, vol. 8, pp. 43-4 (1961).
  • ECTEOLA cellulose resins are available commercially. However, it has been found desirable to initially treat these resins by recycling them with caustic soda before use in the herein-defined purification procedure.
  • the resin preferably is first equilibrated with a chloride buffer solution having a concentration of about 0.8% NaCl and then poured into a chromatographic glass column.
  • the AHF concentrate which is desired to be purified is then applied to the column and finally eluted with a chloride buffer solution having a molarity of about 0.5.
  • Fresh citrated plasma (from ACD preserved blood) is mixed with an aqueous solution of glycine to a concentration of 1.8 molar glycine and the resulting suspension is centrifuged.
  • the precipitate which contains a concentrate of AHF, is retained for subsequent treatment in Examples 4 and 5, below.
  • the plasma supernatant (pH 6 7) is diluted with an equal volume of normal 6 physiological saline and then mixed with polyethylene glycol having a molecular weight of about 4000 (PEG 4000) to a concentration of 30% PEG.
  • PEG 4000 polyethylene glycol having a molecular weight of about 4000
  • the plasma protein precipitate is suspended in normal physiological saline to a concentration of 10% (weight/ volume) and the pH adjusted to 7.2 with 1 N NaOH.
  • 500 grams of tribasic calcium phosphate is then added to 50 liters of the plasma protein suspension and the mixture stirred for about 30 minutes.
  • the suspension is then centrifuged and the supernatant discarded.
  • the retained precipitate is suspended in 0.1 M trisodium citrate to a final volume of 5 liters.
  • the suspension is again centrifuged and the precipitate discarded.
  • the pH of the retained supernatant (about 5 liters) is then adjusted to 7.2 with l N HCl, polyethylene glycol 4000 is added to a final concentration of 5%, and the suspension stirred for about 30 minutes.
  • the suspension is clarified by centrifugation, with retention of the supernatant and discardal of the precipitate.
  • the pH of the retained supernatant is then adjusted to 5.2 with 1 N HCl, and polyethylene glycol 4000 is added to a final concentration of 20%.
  • the suspension is centrifuged and the precipitate that is recovered is dissolved in citrated saline (1 part 0.1 M trisodium citrate to 4 parts 0.9% sodium chloride) to a final volume of 2 to 5 liters, which is equivalent to one twenty fifth to one tenth the volume of the original plasma protein suspension. Heparin is added in an amount of one unit per ml., and the solution is clarified and sterilized by passage through a combination of graded pore sizes of membrane filters.
  • the solution is filled under aseptic conditions in 10 to 30 ml. sterile bottles, freeze dried and capped with stoppers.
  • the freeze-dried material can be reconstituted with sterile water and then administered intravenously, subcutaneously or intramuscularly to patients who are deficient in one or more of the abovementioned coagulation factors, particularly Factor IX.
  • the Factor IX activity of the reconstituted product is about 20 times as great as an equal volume of normal whole plasma and is contained in about one eighteenth the amount of protein in normal whole plasma.
  • Example 1 is repeated up to the point of suspending the plasma protein precipitate in normal physiological saline.
  • Tribasic calcium phosphate N.F. is added to the suspension to a concentration of 1%.
  • the resultant tribasic calcium phosphate precipitate is then suspended in 0.005 M trisodium citrate to a volume equal to one two-hundredth the original plasma volume.
  • the suspension is centrifuged to remove undesired contaminating materials in the resulting supernatant.
  • the retained precipitate is then suspended in 0.2 M trisodium citrate to a volume equal to one two-hundredth the original plasma volume.
  • the suspension is centrifuged to remove the tribasic calcium phosphate particles.
  • the pH is adjusted to 6.8 with 2 N acetic acid and PEG 4000 is added to provide a final concentration of 10%.
  • the precipitate that forms is discarded.
  • the supernatant is then adjusted to pH 5.2 and sufiicient PEG 4000 is added to provide a final concentration of 20%.
  • the suspension is centrifuged and the precipitate that is recovered is dissolved in citrated saline (one part of 0.1 M sodium citrate to 9 parts of a 5% sodium chloride solution) to a volume to provide 40 units of Factor IX per ml.
  • the pH is adjusted to 6.8 with 1 N sodium hydroxide.
  • Heparin in an amount of 3 units per ml. is added, and the solution is filtered through a series or combination of graded pore sizes of Millipore filters.
  • the solution is filled under aseptic conditions into sterile ml. glass bottles in units of 20 ml. of solution per bottle. After shell-freezing and drying from the frozen state under aseptic conditions, the bottles are closed with sterile stoppers under vacuum and capped.
  • the dry product prepared in accordance with this example can be used for intravenous or intramuscular injection after reconstitution with 20 ml. of sterile water per each unit of dry product.
  • Example 2 is repeated except that frozen plasma is used instead of fresh plasma.
  • the plasma is obtained from a plasma pool ranging in age from two weeks to two months and stored at -25 C. for various periods of time ranging up to nine weeks before processing in accordance with the procedure of Example 2.
  • the prothrombin complex prepared in this manner is assayed after reconstitution with sterile water as follows:
  • Factor II content The solution is assayed for prothrombin activity by the methods of Ware and Seegers, Am. J. Clin. PathoL, vol. 19, pp. 471-82 (1949) and Wagner et al., Blood Coagulation, Hemorrhage and Thrombosis, edited by Tocantins and Kazal, published by Grune and Stratton, New York, pp. 159-165 (1964).
  • Thrombin activity The presence of thrombin is tested for by determining the clotting time of recalcified normal plasma at various dilutions according to the procedure of Bidwell and Dike, Treatment of Hemophilia and Other Coagulation Disorders, edited by Biggs and MacFarlane, published by F. A. Davis Co., Philadelphia, pp. 6269 (1966).
  • Total protein content Total protein content is determined by ultraviolet absorption at a wavelength of 280 my.
  • the prothrombin complex of this example was found to be free of thrombin activity and to contain (on the average of many lots) about 8 mg. of protein per ml.
  • the average Factor IX activity of these lots was about 20 to 40 times that of an equivalent volume of normal whole plasma; the aver age Factor II activity of these lots was about to times that of an equivalent volume of normal whole plasma. Since normal whole plasma contains about 70 mg.
  • the Factor IX activity in the prothrombin complex of this example is contained in only about one two-hundredth to one four-hundredth to the amount of protein present in plasma providing an equal amount of Factor IX activity and the Factor II activity is contained in only one fiftieth to one hundred fiftieth to the amount of protein present in plasma providing an equal amount of Factor II activity.
  • the supernate is decanted and the precipitate is Washed in cold water (2 0.). Spin washing is then carried out for five minutes at 5000 r.p.m. at a temperature of 4 C. The supernate is decanted and the precipitate is redissolved in glycine citrated saline.
  • Millipore filter membrane used: 1.2 microns, 0.45 micron, and 0.3 micron.
  • This liquid product is then frozen by shell freezing (60 C.) and storing in a flash freezer (20 C. to 30 C.) for at least three hours.
  • the frozen product can then be retained under ordinary refrigeration conditions (4 0., preferably at 20 C. to -30 C.) without loss of its AHF activity for periods of time of one year and longer.
  • This product when reconstituted can be administered intravenously to hemophiliacs as required by conventional transfusion means.
  • the AHF concentrate prepared by the procedure of this example contains less than 0. 05% (generally as little as 0.01%) residual polyethylene glycol and is highly soluble in water.
  • Example 4 is repeated including the additional step of purification of the AHF fraction with ECTEOLA cellulose resin in the following manner:
  • Recycled commercial ECTEOLA cellulose resin (1) Mix 60 grams commercial ECTEOLA cellulose resin with 350 ml. 2 M NaCl.
  • the AHF fraction is first dissolved in chloride buffer.
  • the AHF fraction is first dialyzed against the chloride butfer for one hour to remove glycine and reduce its ionic strength. Purification of the buffered AHF fraction by column technique with the above-prepared ECTEOLA cellulose resin proceeds as follows:
  • chloride buffer is washed through the resin.
  • the eluting buffer is applied to the column.
  • the eluate is collected in ten ml. portions and analyzed for protein, fibrinogen and AHF activity.
  • the eluate portions having the most active AHF activity are retained and stabilized by the addition of 1% albumin.
  • the stabilized solution is then filtered using a 293 mm. Millipore filter as in Example 4.
  • a silver filter of the same size can also be used in place of the Millipore filter.
  • This final liquid product can then be frozen by shell freezing, followed by storage in a flash freezer for at least three hours, and then retained under ordinary refrigeration conditions in the manner of the final product of Example 4.
  • Human whole blood plasma, and bovine and porcine plasma and AHF-containing plasma fractions can be fractionated by the successive polyethylene glycol and glycine precipitation procedures described herein to produce AHF concentrates similar to those described above in Examples 4 and 5.
  • a method for the preparation of a prothrombin complex from citrated blood plasma comprising fractionating citrated blood plasma by mixing with glycine having a molarity of from about 1.3 to about 1.8 to produce an AHF-containing precipitate and a prothrombin complex-containing supernatant, fractionating said prothrombin complex-containing supernatant by mixing with polyethylene glycol having a molecular weight of from about 200 to about 20,000 to a concentration of about 30% by weight and then separating the resulting prothrombin complex-containing precipitate from the undesired citrate and glycine salt-containing supernatant, suspending said prothrombin complex-containing precipitate in normal physiological saline, adjusting the suspension to a pH of from about 6.8 to about 7.2, thoroughly mixing the supernatant with tribasic calcium phosphate to adsorb the coagulation factors, thoroughly mixing the tricalcium phosphate adsorbed protein precipitate with from about 0.05 M to about 0.2 M trisodium citrate followed by recovery
  • a method for the preparation of a concentrate of AHF from citrated blood plasma comprising fractionating citrated blood plasma by mixing with glycine having a molarity of from about 1.3 to about 1.8 to produce an AHF-containing precipitate and a prothrombin complexcontaining supernatant, fractionating said AHF-containing precipitate by mixing with polyethylene lycol having a molecular weight of from about 200 to about 20,000 to a concentration of from about 3% to about 4% by weight, mixing the AHF-containing supernatant with said polyethylene glycol to a concentration of about 10% by Weight, mixing the resulting AHF-containing precipitate with glycine having a molarity of about 1.8 and recoverlIlg the remaining precipitate as the active concentrate of AHF.

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Cited By (31)

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US3920625A (en) * 1973-06-19 1975-11-18 Kabi Ab Isolation of coagulation factors from biological material using cross linked sulfated, sulfonated carbohydrates
US3956259A (en) * 1973-01-30 1976-05-11 Baxter Laboratories, Inc. Fractionation of blood using block copolymer of ethylene oxide and polyoxypropylene polymer to recover fraction suitable for organ perfusate
US4025500A (en) * 1974-06-06 1977-05-24 Baxter Laboratories, Inc. Preparation of albumin by fractionation of blood plasma or serum
JPS52102410A (en) * 1976-01-30 1977-08-27 Rushierushiyu Ematorojiiku Sos Easy method of production of highly purified concentrated factorr8
US4069216A (en) * 1975-06-16 1978-01-17 Edward Shanbrom, Inc. Simplified methods for preparation of very high purity Factor VIII concentrate
US4073886A (en) * 1973-01-30 1978-02-14 Baxter Travenol Laboratories, Inc. Blood fractionation process using block copolymers of ethylene oxide and polyoxypropylene
US4081432A (en) * 1977-07-25 1978-03-28 Monsanto Company Method of separating a Factor IX preparation from plasma using ethylene-maleic anhydride polymers
US4081431A (en) * 1974-12-14 1978-03-28 Biotest-Serum-Institut Gmbh Blood fractionation
US4087415A (en) * 1976-06-09 1978-05-02 William L. Wilson Antithrombin III
USRE29698E (en) * 1972-05-17 1978-07-11 Baxter Travenol Laboratories, Inc. Stabilization of AHF using heparin
US4157431A (en) * 1977-07-25 1979-06-05 Monsanto Company Separation of blood coagulation factors with non-activating polyelectrolytes
US4170590A (en) * 1974-12-14 1979-10-09 Biotest-Serum-Institut Gmbh Ion exchanger treatment of citrate-stabilized plasma
US4203891A (en) * 1977-12-19 1980-05-20 Rock Gail A Method of collecting anti-hemophilic factor VIII from blood and blood plasma using heparin or sodium heparin
US4272523A (en) * 1979-01-20 1981-06-09 Biotest Serum Institut Gmbh Fractionating citrate-stabilized plasma
US4297344A (en) * 1979-04-25 1981-10-27 Behringwerke Aktiengesellschaft Blood coagulation factors and process for their manufacture
US4361509A (en) * 1981-12-14 1982-11-30 Scripps Clinic And Research Foundation Ultrapurification of factor VIII using monoclonal antibodies
US4361510A (en) * 1980-05-27 1982-11-30 Cutter Laboratories, Inc. Blood coagulation promoting product
US4364861A (en) * 1980-05-27 1982-12-21 Cutter Laboratories, Inc. Blood-coagulation-promoting products and methods of preparing them
US4391746A (en) * 1980-05-27 1983-07-05 Cutter Laboratories, Inc. Blood-coagulation-promoting products and methods of preparing them
US4404132A (en) * 1980-05-27 1983-09-13 Cutter Laboratories, Inc. Blood coagulation promoting product
US4404131A (en) * 1980-08-27 1983-09-13 Immuno Aktiengesellschaft Fur Chemisch-Medizinische Produkte Method of producing a factor-VIII(AHF)-high-concentrate
WO1984003628A1 (en) * 1983-05-09 1984-09-27 Nordisk Insulinlab A concentrate of the antihemophilic factor viii and a process for producing it
US4543210A (en) * 1984-10-04 1985-09-24 Miles Laboratories, Inc. Process for producing a high purity antihemophilic factor concentrate
USRE32011E (en) * 1981-12-14 1985-10-22 Scripps Clinic And Research Foundation Ultrapurification of factor VIII using monoclonal antibodies
US5288853A (en) * 1992-04-30 1994-02-22 Alpha Therapeutic Corporation Factor viii purification process
US5330974A (en) * 1993-03-01 1994-07-19 Fibratek, Inc. Therapeutic fibrinogen compositions
US5659017A (en) * 1995-11-07 1997-08-19 Alpha Therapeutic Corporation Anion exchange process for the purification of Factor VIII
US5770705A (en) * 1996-11-01 1998-06-23 Shanbrom Technologies Llc Method for recovering proteins from plasma using insoluble, water-absorbing material
EP0823476A3 (en) * 1996-08-09 1999-09-01 Juridical Foundation, The Chemo-Sero-Therapeutic Research Institute Method for activating prothrombin to thrombin
US20030129167A1 (en) * 2000-10-23 2003-07-10 Shanbrom Technologies, Llc Enhanced production of blood components, blood cells and plasma without freezing
US20050196393A1 (en) * 2000-10-23 2005-09-08 Shanbrom Technologies, Llc Enhanced production of blood clotting factors and fibrin fabric

Cited By (38)

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