WO1988005825A1 - A method for producing factor viii in high yield - Google Patents
A method for producing factor viii in high yield Download PDFInfo
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- WO1988005825A1 WO1988005825A1 PCT/US1988/000292 US8800292W WO8805825A1 WO 1988005825 A1 WO1988005825 A1 WO 1988005825A1 US 8800292 W US8800292 W US 8800292W WO 8805825 A1 WO8805825 A1 WO 8805825A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
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- 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)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- This invention relates to the production of factor VIII in high yields. More particularly, this invention relates to a method for increasing yield of factor VIII by growing factor VIII-producing cells in the presence of the von Willebrand factor.
- Factor VIII the procoagulant component of factor VIII/vWf, is initially synthesized as a single chain macromolecular precursor, which is later cleaved to yield the fragments which constitute "mature" factor VIII [see generally, W. J. Williams et al., Hematology, pp. 1085-90, McGraw-Hill, New York (1972)].
- Mature factor VIII is composed of two chains bridged by a calcium ion; an amino-terminal heavy chain of 740 amino acids, and a carboxy-terminal light chain of 684 amino acids.
- the primary translation product of factor VIII is a single chain in which the heavy chain of mature factor VIII is separated from the light chain by a "maturation polypeptide" of 908 amino acids.
- vWf Von Willebrand factor
- vWf is a large multimeric plasma protein, comprised of at least two subunits linked together by disulfide bonds. It is a protein important in the hemostatic process, and is associated with normal platelet aggregation and adhesive properties. It mediates the attachment of platelets to the basement membrane after vascular injury.
- the larger polymers of vWf function in platelet aggregation at the site of vascular injury [T. S.
- the mature vWf protein is synthesized in several steps.
- a 240,000-260,000 molecular weight prepro-vWf subunit of 2813 amino acids is synthesized in vascular endothelial cells and megakaryocytes [E. A. Jaffe et al., "Synthesis of Antihemophilic Factor Antigen By Cultured Human Endothelial Cells," J. Clin. Invest., 52, pp. 2757-64 (1973); R. Nachman et. al., "Synthesis of Factor VIII Antigen By Cultured Guinea Pig Megakaryocytes," J. Clin. Invest. , 60, pp. 914-21 (1977); C. L.
- factor VIII is synthesized and secreted by the hepatocyte [M. G. Zelechowska et al., "Ultra structural Localization of Factor VIII Procoagulant Antigen In Human Liver Hepatocytes, " Nature, 317, pp. 729-30 (1985); K. L. Wion et al., "Distribution of Factor VIII mRNA and Antigen in Human Liver and Other Tissues," Nature, 3T7, pp. 726-28 (1985)].
- the secreted factor VIII is endocytosed by the neighboring sinusoidal endothelial cell [H. V.
- Haemophilia A is a sex-linked hemorrhagic disease which is caused by a deficiency, either in amount or in biological activity, of factor VIII.
- the symptoms of acutely bleeding haemophilia patients are treated with factor VIII traditionally purified from normal sera.
- Various methods of purification have been described in the literature [see, Zimmerman et al., United States patent 4,361,509; Saundrey et al. United States patent 4,578,218; E. G. D. Tuddenham et al., "The Properties of Factor VIII Coagulant Activity Prepared By Immunoadsorbent Chromatography, Journal of Laboratory Clinical Medicine, 93, pp. 40-53 (1979); D. E. G. Austen, "The Chromatographic Separation of Factor VIII on
- the present invention solves the problems referred to above by providing a means of producing factor VIII in high yields. More specifically, it provides a method of increasing the yield of factor VIII by growing factor VIII-producing cells in the presence of human vWf. According to this method, 5 to 30 ⁇ g of vWf are present in the culture medium ' in which the factor VIII-producing cells are grown, either by supplementing the medium with vWf or by cotransforming recombinant factor VIII-producing cells with the DNA sequence encoding vWf. According to this invention, this method can be used to increase the yield of both native and recombinant factor VIII.
- Figure 1 °compares the results of several induction experiments with clone K using a culture fluid containing 10 ⁇ g/ml vWf to those obtained using a culture fluid containing no vWf.
- Figure 2 depicts the levels of factor VIII accumulated in the culture fluid when clone K is induced to secrete factor VIII into culture fluid containing 0, 5, 10, 20, or 30 ⁇ g/ml human vWf.
- Figure 3 depicts the factor VIII yield in two other clones tested, Aat2.10 and Aat2.27.
- Figure 4 is a schematic representation of plasmid RE.neo, which contains a (modified) factor VIII gene, under the transcriptional control * of the adenovirus-2 major late promoter.
- Factor VIII A polypeptide having a mole ⁇ cular weight of 265,000, and upon maturation and activation, being capable of functioning as cofactor for the factor IXa-dependent maturation of factor X in the blood coagulation cascade.
- factor VIII includes the glycoproteins also known as factor VIII:C, factor VIII procoagulant activity protein, or factor VIII-clotting activity [see W. J. Williams et al., Hematology, pp. 1056, 1074 and 1081] .
- factor VIII also refers to polypeptides characterized by a dele ⁇ tion of a major portion of the maturation polypeptide of factor VIII.
- factor VIII includes proteins that comprise the N-terminal mature heavy chain and the C-terminal mature light chain of factor VIII either linked together as a single chain or bridged by a calcium or other metal ion bridge. It also includes proteins having an amino terminal methionine, e.g., f-Met-factor VIII, and proteins that are characterized by other amino acid deletions, additions or substitutions so long as those proteins substantially retain the biological activity of factor VIII. It also includes polypep ⁇ tides having natural allelic variations that may exist and occur from individual to individual. Fur ⁇ thermore, it includes factor VIII polypeptides whose degree .and location of glycosylation, or other post- translation modifications, may vary depending on the cellular environment of the producing host or tissue.
- Von Willebrand Factor a polypeptide which is responsible for platelet adhesion to blood vessel walls and acts as a carrier for factor VIII in plasma.
- the present invention relates to a method for increasing the yield of factor VIII. More par ⁇ ticularly, it provides a method for the production of recombinant and native factor VIII produced in culture in high yields through the addition of vWf to the culture medium of the growing factor VIII- producing host or cell-line.
- vWf may be added to fresh culture fluid for host cells which constituitively produce factor VIII or, in the case of host cells which inducibly produce factor VIII, vWf may be added to culture fluid immediately prior to the induction of those cells.
- vWf may be co-synthesized with factor VIII in hosts which are transformed with DNA sequences encoding both vWf and factor VIII.
- vWf affinity-purified human vWF to demon ⁇ strate the increase in factor VIII yield.
- recombinant vWf may also be used.
- vWf present in the culture fluid may bind to factor VIII as it is released from the exocytosed Golgi vesicles of a mammalian host cell, thereby protecting the factor VIII molecule from extracellular proteolytic degradation.
- vWf may be endocytosed into either the endoplasmic reticulum or some compartment of the Golgi apparatus of the mammalian host, where it binds to newly synthesized factor VIII and facilitates its export, perhaps by protecting it from lysosomal proteolysis.
- the factor VIII produced according to the methods of this invention may be purified by a variety of conventional steps and strategies.
- Useful puri ⁇ fication steps include those used to purify natural and recombinant factor VIII [see, for example, L.-O. Andersson et al., PNAS (USA), 83, pp. 2979-83 (1986)].
- the intermediate factor Vlll-vWf complex formed according to the methods of this invention may be dissociated to form factor VIII according to Anderson's method.
- the factor VIII produced by the methods of this invention are useful in compo- sition and methods for treatment of haemophilia A and in a variety of agents useful in treating uncon ⁇ trolled bleeding.
- the factor VIII produced according to the methods of this invention may be formulated using known methods to prepare pharmaceutically useful compositions.
- Such compositions also will preferably include conventional pharmaceutically acceptable carriers and may include other medicinal agents, carriers, adjuvants, excipients, etc., e.g., human serum albumin or plasma preparations. See, e.g.,
- the resulting formulations will contain an amount of modified factor VIII effective in the recipient to treat uncontrolled bleeding.
- Administration of these polypeptides, or pharmaceutically acceptable deriva ⁇ tives thereof, may be via any of the conventional accepted modes of administration of factor VIII. These include parenteral, subcutaneous, or intra ⁇ venous administration.
- compositions of this invention used in the therapy of haemophilia A may also be in a variety of forms.
- the preferred form depends on the intended mode of administration and therapeutic application.
- the dosage and dose rate will depend on a variety of factors, for example, whether the treatment is given to an acutely bleeding patient or as a prophylactic treatment.
- the factor VIII level should be high enough to prevent hemorrhage and promote epithelialization [see discussion in Williams, Hematology, pp. 1335-43].
- EXAMPLE We describe in this example the increased production of a modified factor VIII using the method of this invention. First, we constructed cDNA sequences which encode a modified factor VIII molecule having a deletion of a major part or all of the maturation polypeptide of native factor VIII.
- RE.neo was constructed by insert ⁇ ing into RE a transcription unit directing the expres ⁇ sion of agpt in animal cells.
- Plasmid pSVneo2911 was con ⁇ structed as follows: first, the SV40 Hpall-BamHI restriction fragment containing the entire SV40 early region was inserted between Clal and BamHI into pBRd [F. A. M. Asselbergs et al. "A Recombinant Chinese Hamster Ovary Cell Line Containing A 300-fold Ampli ⁇ fied Tetramer of the Hepatitis B Genome Together With A Double Selection Marker Expresses High Levels of Viral Protein," J. Mol. Biol., 189, pp. 401-11 (1986)].
- the resultant plasmid, RE.neo contained two SV40 replication origins, one 5' to the adeno- virus-2 major late promoter and the other overlapping the SV40 early promoter which controls transcription of the aminoglycoside 3 '-phosphotransferase (G418/neo) gene.
- G418/neo aminoglycoside 3 '-phosphotransferase
- BSC40 cells which are BSC1 African green monkey kidney cells which have been adapted to grow at 40°C [W. W. Brockman and D. Nathans, PNAS (USA) , 71, pp. 942-46 (1974)].
- BSC40 cells which contain a SV40 origin of replication, a transcription unit for factor VIII, and a transcription unit for amino- glycoside phosphotransferase and (2) a 10-fold molar excess of plasmid LTRtsA58, containing a transcrip ⁇ tion unit for a temperature sensitive SV40 T antigen allele.
- the mutant tsA58 virus is a temperature- sensitive mutant of SV40 which does not produce progeny at 39°C.
- the large T antigen protein specified by the tsA58 mutant is much more labile at the non-per ⁇ missive temperature than wild type large T antigen protein [P. Tegtmeyer et al., Journal of Virology, 16, pp. 168-78 (1975)].
- Our preferred cell lines, clones K, Aat2.10, and Aat2.27 are BSC40 transfectants which inducibly synthesize and secrete recombinant factor VIII.
- Clone K [ATCC No. CRL 9206] was obtained by co-trans- fecting BSC40 with supercoiled RE.neo and supercoiled LTRtsA58 and selecting at 39.5°C for G418 resistance.
- Clones Aat2.10 and Aat2.27 were obtained by co-trans- fecting BSC40 with RE.neo linearized at the unique Aat2 site and supercoiled LTRtsA58 and selecting at 39.5°C for G418 resistance.
- We made our preferred cell lines by co- transfecting confluent cultures of BSC40 cells in 100 mm Petri dishes with 10 micrograms pLTRtsA58 and 1.5 micrograms RE.neo using the standard technique of calcium phosphate precipitation.
- Factor VIII assay using conditioned medium for each line at both temperatures. We selected clones exhibiting the highest levels of activity at 33°C for further study. No clone expressed factor VIII at 39.5°C at non-negligible levels.
- Figure 3 demonstrates that the increased yield of factor VIII activity in the presence of human vWf is not specific only to clone K. We also observed increases in factor VIII yield in clones Aat2.10 and Aat2.27.
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Abstract
The method comprises the addition of vWf to the medium in which factor VIII-producing cells are grown. According to this method, increased amounts of both recombinant factor VIII and native factor VIII are purified from cells producing factor VIII.
Description
A METHOD FOR PRODUCING FACTOR VIII IN HIGH YIELD
This invention relates to the production of factor VIII in high yields. More particularly, this invention relates to a method for increasing yield of factor VIII by growing factor VIII-producing cells in the presence of the von Willebrand factor.
BACKGROUND OF THE INVENTION In this application, we will use the nomen¬ clature proposed by V. J. Marder et al. in "Standard Nomenclature for Factor VIII and von Willebrand Factor: A Recommendation By The International Committee on Thrombosis and Hae ostasis, " Thrombosis and Haemostasis, 54, pp. 871-72 (1985).. "Factor VIII" will designate the procoagulant cofactor, and "von Willebrand factor" will designate the adhesive factor of the bimolecular complex, hereinafter desig¬ nated as "factor VIII/vWf" . In plasma, coagulant factor VIII circu¬ lates as a non-covalent complex with the adhesive von Willebrand factor ("vWf") [L. W. Hoyer, Blood, 58., pp. 1-13 (1981)].
Factor VIII, the procoagulant component of factor VIII/vWf, is initially synthesized as a single chain macromolecular precursor, which is later cleaved to yield the fragments which constitute "mature" factor VIII [see generally, W. J. Williams et al.,
Hematology, pp. 1085-90, McGraw-Hill, New York (1972)]. Mature factor VIII is composed of two chains bridged by a calcium ion; an amino-terminal heavy chain of 740 amino acids, and a carboxy-terminal light chain of 684 amino acids. The primary translation product of factor VIII is a single chain in which the heavy chain of mature factor VIII is separated from the light chain by a "maturation polypeptide" of 908 amino acids. The excision of this maturation polypep- tide is initiated by proteolytic cleavage of the primary translation product at the Arg 1648 - Glu 1649 peptide bond. The initial nick event begins a series of successive proteolytic cleavages which shorten the nascent heavy chain from its carboxy terminus. Eventually the mature heavy chain of 740 amino acids emerges and in combination with the light chain of 684 amino acids, comprises mature factor VIII [see L.-O. Andersson et al. "Isolation and Characterization of Human Factor VIII: Molecular Forms In Commercial Factor VIII Concentrate, Cryoprecipitate, and Plasma," PNAS(USA), 83, pp. 2979-83 (1986)]. This complex is then activated by thrombin by cleavage at the Arg 1689-Ser 1690 bond [D. Eaton et al., Biochemistry, 25, pp. 505-12 (1986)]. Von Willebrand factor ("vWf") is a large multimeric plasma protein, comprised of at least two subunits linked together by disulfide bonds. It is a protein important in the hemostatic process, and is associated with normal platelet aggregation and adhesive properties. It mediates the attachment of platelets to the basement membrane after vascular injury. The larger polymers of vWf function in platelet aggregation at the site of vascular injury [T. S. Zimmerman et al., "Factor VHI/von Willebrand Factor," Progress In Hematoloqy, 13, pp. 279-309 (1983), E. B. Brown, editor]. The vWf protein is missing in the bleeding disorder, von Willebrand
disease type IIA [Z. M. Ruggeri and T. S. Zimmerman, "Variant von Willebrand's Disease, J. Clin. Invest., 65., pp. 1318-25 (1980)] .
The mature vWf protein is synthesized in several steps. A 240,000-260,000 molecular weight prepro-vWf subunit of 2813 amino acids is synthesized in vascular endothelial cells and megakaryocytes [E. A. Jaffe et al., "Synthesis of Antihemophilic Factor Antigen By Cultured Human Endothelial Cells," J. Clin. Invest., 52, pp. 2757-64 (1973); R. Nachman et. al., "Synthesis of Factor VIII Antigen By Cultured Guinea Pig Megakaryocytes," J. Clin. Invest. , 60, pp. 914-21 (1977); C. L. Verweij et al., "Full-length von Willebrand factor (vWf) cDNA encodes a Highly Repetitive Protein Considerably Larger Than the Mature vWf Subunit," EMBO J. , 5_, pp. 1939-47 (1986)]. After carbohydrate processing, inter-subunit disulfide bond formation and precursor cleavage, the mature dimer is formed. Multimers of up to 50 subunits are assembled in specialized secretory vacuoles within endothelial cells, called the Weibel-Palade bodies, and secreted via a regulated pathway [L. A. Sporn et al., "Inducible Secretion Of Large, Biologically Patent von Willebrand Factor Multimers," Cell, 46, pp. 185-90 (1986)]. Release of vWf from endothelial cells is inducible by thrombin; its release causes the disappearance of the Weibel-Palade bodies.
The site(s) and mechanism of factor VIII/vWf complex formation are largely unknown. One common view is that factor VIII is synthesized and secreted by the hepatocyte [M. G. Zelechowska et al., "Ultra structural Localization of Factor VIII Procoagulant Antigen In Human Liver Hepatocytes, " Nature, 317, pp. 729-30 (1985); K. L. Wion et al., "Distribution of Factor VIII mRNA and Antigen in Human Liver and Other Tissues," Nature, 3T7, pp. 726-28 (1985)]. According to this view, the secreted factor VIII is
endocytosed by the neighboring sinusoidal endothelial cell [H. V. Stel et al., "Detection of Factor VIII/Coagulant Antigen In Human Liver Tissue," Nature, 303, pp. 530-32 (1982)] where it assembles into a complex with newly synthesized vWf; the complex is then exocytosed. Alternatively, the complex may be assembled in plasma. Once bound to vWf, factor VIII circulates with a 9-10 hour half-life until thrombin cleavage of the mature factor VIII light chain at arginine 1689 allows it to float free of vWf and attach to a platelet surface in the immediate vicinity where it then assembles into a ternary complex with Factor IXa and Factor X [K. Sewerin and L.-O. Andersson, "Binding of Native And Thrombin Activated Factor VIII To Platelets," Thrombosis
Research, 31, pp. 695-706 (1983); D. Eaton et al., "Proteolytic Processing of Human Factor VIII. Correlation of Specific Cleavages by Thrombin, Factor Xa, and Activated Protein C with Activation and Inactivation of Factor VIII Coagulant Activity," Biochemistry, 25, pp. 505-12 (1986)].
Haemophilia A is a sex-linked hemorrhagic disease which is caused by a deficiency, either in amount or in biological activity, of factor VIII. The symptoms of acutely bleeding haemophilia patients are treated with factor VIII traditionally purified from normal sera. Various methods of purification have been described in the literature [see, Zimmerman et al., United States patent 4,361,509; Saundrey et al. United States patent 4,578,218; E. G. D. Tuddenham et al., "The Properties of Factor VIII Coagulant Activity Prepared By Immunoadsorbent Chromatography, Journal of Laboratory Clinical Medicine, 93, pp. 40-53 (1979); D. E. G. Austen, "The Chromatographic Separation of Factor VIII on
Aminohexyl Sepharose," British Journal of Hematoloqy, 43, pp. 669-74 (1979); M. Weinstein et al., "Analysis
of Factor VIII Coagulant Antigen In Normal, Thrombin- treated, and Hemophilic Plasma," PNAS (USA), 7_8, pp. 5137-41 (1981); P. J. Fay et al., "Purification And Characterization Of A Highly Purified Human Factor VIII Consisting Of A Single Type Of Polypeptide Chain," PNAS (USA), 1±> PP- 7200-04 (1982); C. A. Fulcher and T. S. Zimmerman, "Characterization Of The Human Factor VIII Procoagulant Protein With A Ξeterologous Precipitating Antibody, " PNAS (USA), 79_, pp. 1648-52 (1982); F. Rotblat et al., Thromb.
Haemostasis, 50, p. 108 (1983); C. A. Fulcher et al., Blood, 61, pp. 807-11 (1983)]..
In view of its importance in the treatment of haemophilia, numerous attempts have been made to produce large quantities of factor VIII, for instance, by using recombinant DNA technology [See, for example, Genetics Institute, PCT application W085/01961; Genentech European Patent application 160,457; Chiron European Patent application 150,735; J. J. Toole et al., "Molecular Cloning Of a cDNA Encoding Human Antihaemophilic Factor" Nature, 312, pp. 342-47 (1984); and W. I. Wood et al., Nature, 312, pp. 330-37 (1984)]. However, due to difficulties in producing recombinant factor VIII in sufficiently high yields, a method for increased production of factor VIII continues to be needed.
SUMMARY OF THE INVENTION
The present invention solves the problems referred to above by providing a means of producing factor VIII in high yields. More specifically, it provides a method of increasing the yield of factor VIII by growing factor VIII-producing cells in the presence of human vWf. According to this method, 5 to 30 μg of vWf are present in the culture medium' in which the factor VIII-producing cells are grown, either by supplementing the medium with vWf or by
cotransforming recombinant factor VIII-producing cells with the DNA sequence encoding vWf. According to this invention, this method can be used to increase the yield of both native and recombinant factor VIII.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1°compares the results of several induction experiments with clone K using a culture fluid containing 10 μg/ml vWf to those obtained using a culture fluid containing no vWf. Figure 2 depicts the levels of factor VIII accumulated in the culture fluid when clone K is induced to secrete factor VIII into culture fluid containing 0, 5, 10, 20, or 30 μg/ml human vWf.
Figure 3 depicts the factor VIII yield in two other clones tested, Aat2.10 and Aat2.27.
Figure 4 is a schematic representation of plasmid RE.neo, which contains a (modified) factor VIII gene, under the transcriptional control *of the adenovirus-2 major late promoter.
DETAILED DESCRIPTION OF THE INVENTION
In order that the invention herein described may be more fully understood, the following detailed description is set forth.
In the description the following terms are employed:
Factor VIII —A polypeptide having a mole¬ cular weight of 265,000, and upon maturation and activation, being capable of functioning as cofactor for the factor IXa-dependent maturation of factor X in the blood coagulation cascade. As used in this application, factor VIII includes the glycoproteins also known as factor VIII:C, factor VIII procoagulant activity protein, or factor VIII-clotting activity [see W. J. Williams et al., Hematology, pp. 1056, 1074 and 1081] .
As used in this application, "factor VIII" also refers to polypeptides characterized by a dele¬ tion of a major portion of the maturation polypeptide of factor VIII. For example, where the entire matura- tion polypeptide has been deleted, "factor VIII" includes proteins that comprise the N-terminal mature heavy chain and the C-terminal mature light chain of factor VIII either linked together as a single chain or bridged by a calcium or other metal ion bridge. It also includes proteins having an amino terminal methionine, e.g., f-Met-factor VIII, and proteins that are characterized by other amino acid deletions, additions or substitutions so long as those proteins substantially retain the biological activity of factor VIII. It also includes polypep¬ tides having natural allelic variations that may exist and occur from individual to individual. Fur¬ thermore, it includes factor VIII polypeptides whose degree .and location of glycosylation, or other post- translation modifications, may vary depending on the cellular environment of the producing host or tissue.
Von Willebrand Factor — a polypeptide which is responsible for platelet adhesion to blood vessel walls and acts as a carrier for factor VIII in plasma.
The present invention relates to a method for increasing the yield of factor VIII. More par¬ ticularly, it provides a method for the production of recombinant and native factor VIII produced in culture in high yields through the addition of vWf to the culture medium of the growing factor VIII-
producing host or cell-line. According to the methods of this invention, vWf may be added to fresh culture fluid for host cells which constituitively produce factor VIII or, in the case of host cells which inducibly produce factor VIII, vWf may be added to culture fluid immediately prior to the induction of those cells. Alternatively, vWf may be co-synthesized with factor VIII in hosts which are transformed with DNA sequences encoding both vWf and factor VIII. Preferably, between 5 and 30 μg of vWf should be present per milliliter of the culture fluid during synthesis of factor VIII. More preferably, 10 μg per ml of culture is present.
-In the example of this invention, provided below, we used affinity-purified human vWF to demon¬ strate the increase in factor VIII yield. However, recombinant vWf may also be used. For example, one could introduce the vWf gene into a cell line expres¬ sing factor VIII which would result in a cell line ' which either (1) co-secretes factor VIII and vWf into the culture fluid to subsequently form the com¬ plex; or (2) assembles the factor VIII/vWf complex in either the endoplasmic reticulum or the Golgi apparatus prior to exocytosis [see, for example, D. T. Bonthron et al., "Structure of prepro- von Willebrand factor and Its Expression in Ξetero- logous Cells," Nature, 324, pp. 270-73 (1986)]. Most preferably, we produce recombinant factor VIII containing a deletion of a major part of the matura- tion polypeptide, in cells which have been cotrans- formed with the DNA sequence which encodes vWf, said sequence being operatively linked to an expression control sequence in a recombinant DNA molecule.
While not wishing to be bound by theory, we believe that the high yields of factor VIII ac¬ tivity which result from the methods of the present
invention can be explained by either of the follow¬ ing two models. First, vWf present in the culture fluid may bind to factor VIII as it is released from the exocytosed Golgi vesicles of a mammalian host cell, thereby protecting the factor VIII molecule from extracellular proteolytic degradation. Alter¬ natively, vWf may be endocytosed into either the endoplasmic reticulum or some compartment of the Golgi apparatus of the mammalian host, where it binds to newly synthesized factor VIII and facilitates its export, perhaps by protecting it from lysosomal proteolysis.
The factor VIII produced according to the methods of this invention may be purified by a variety of conventional steps and strategies. Useful puri¬ fication steps include those used to purify natural and recombinant factor VIII [see, for example, L.-O. Andersson et al., PNAS (USA), 83, pp. 2979-83 (1986)]. For example, the intermediate factor Vlll-vWf complex formed according to the methods of this invention may be dissociated to form factor VIII according to Anderson's method.
After purification, the factor VIII produced by the methods of this invention are useful in compo- sition and methods for treatment of haemophilia A and in a variety of agents useful in treating uncon¬ trolled bleeding.
The factor VIII produced according to the methods of this invention may be formulated using known methods to prepare pharmaceutically useful compositions. Such compositions also will preferably include conventional pharmaceutically acceptable carriers and may include other medicinal agents, carriers, adjuvants, excipients, etc., e.g., human serum albumin or plasma preparations. See, e.g.,
Remingto 's Pharmaceutical Sciences (E. W. Martin) . The resulting formulations will contain an amount of
modified factor VIII effective in the recipient to treat uncontrolled bleeding. Administration of these polypeptides, or pharmaceutically acceptable deriva¬ tives thereof, may be via any of the conventional accepted modes of administration of factor VIII. These include parenteral, subcutaneous, or intra¬ venous administration.
The compositions of this invention used in the therapy of haemophilia A may also be in a variety of forms. The preferred form depends on the intended mode of administration and therapeutic application. The dosage and dose rate will depend on a variety of factors, for example, whether the treatment is given to an acutely bleeding patient or as a prophylactic treatment. However, the factor VIII level should be high enough to prevent hemorrhage and promote epithelialization [see discussion in Williams, Hematology, pp. 1335-43].
In order that this invention may be better understood, the following example is set forth.
This example is for purposes of illustration only and is not to be construed as limiting the scope of the invention.
EXAMPLE We describe in this example the increased production of a modified factor VIII using the method of this invention. First, we constructed cDNA sequences which encode a modified factor VIII molecule having a deletion of a major part or all of the maturation polypeptide of native factor VIII.
Referring now to Figure 4, we have presented therein a restriction enzyme map of the RE.neo construction, indicating the position of the factor VIII cDNA with the RE deletion.
We constructed RE.neo in several steps. First we constructed the RE [ARG 740-GLU 1649] [ATCC No. 53517] deletion in two steps, as follows:
In the first step we ligated four fragments which resulted in an intermediate plasmid. These four fragments were:
(1) the 462 bp fragment, obtained by digesting our factor VIII expression plasmid for the full-length gene with HindiII between the codons for Arg 740 and Ser 741, removing the 5' AGCT with nuclease SI, and subsequently digesting with Kpnl which cleaves uniquely between the codons for Tyr 586 and Leu 587.
(2) the synthetic oligonucleotide duplex fragment
5'pGAA ATA ACT CGT ACT ACT CTT CAG TCA
CTT TAT TGA GCA TGA TGA GAA GTC AGT CTA Gp 5' Glu lie Thr Arg Thr Thr Leu Gin Ser Asp 1649 1657 (3) the 135 bp fragment obtained by digest¬ ing the expression plasmid for the full-length factor VIII gene first with Sau3A; we isolated the 411 bp fragment which resulted from Sau3A digestion between the codons for Ser 1657 and Asp 1658 and between the codons for Glu 1794 and Asp 1795. Then, we digested the 411 bp fragment with Pstl which cleaves between the codons for Ala 1702 and Val 1703, to obtain the 135 bp 5' fragment.
(4) pUC18 digested with Kpnl and Pstl.
We then isolated a 624 base-pair fragment encoding the RE fusion from this intermediate plasmid. To do this, we digested the intermediate plasmid generated in the four-fragment ligation with Asp718 and Pstl. The fragment encoding the RE fusion was used to replace the corresponding fragment in the expression plasmid for an earlier-created factor VIII
deletion, called the QD deletion. The QD deletion removes a major portion of DNA sequence coding on expression for the maturation polypeptide (amino acids 741-1648), and retains approximately 90 amino acids of the maturation polypeptide (four amino acids at the N-terminal end of the maturation polypeptide and 86 amino acids at its carboxy terminal end). To make QD, we had partially digested one aliquot of the expression plasmid for the full-length factor VIII gene with EcoRI, which cleaves between the codons for Gin 744 and Asn 745. We removed the 5'AATT over¬ hang with nuclease SI, and then digested the plasmid at the unique Pvul site within the ampicillin re¬ sistance gene. We partially digested another aliquot with BamHl, which cleaves between the codons for Leu 1562 and Asp 1563. We filled out the 5'GATC overhang with the Klenow fragment, and again digested the plasmid with Pvul within amp. We then combined the two mixtures of fragments- and ligated them with T4 DNA ligase. A BamHl site between the codons for Gin 744 and Asp 1563 was created in this fusion.
We completely digested the expression plasmid for the QD deletion at the unique Asp718 site, dephosphorylated the 5' GTAC overhang with calf intestinal phosphatase, and then partially digested the plasmid with Pstl, and then ligated the 624 bp fragment encoding the RE fusion to the result¬ ant mixture of fragments. The resultant plasmid encoding the expression of the RE fusion is named RE. Referring now to Figure 4, we depict therein a map of the RE.neo. RE.neo was constructed by insert¬ ing into RE a transcription unit directing the expres¬ sion of agpt in animal cells. We isolated the tran¬ scription unit from plasmid pSVneo2911, a gift of Fred A. M. Asselbergs. Plasmid pSVneo2911 was con¬ structed as follows: first, the SV40 Hpall-BamHI restriction fragment containing the entire SV40 early
region was inserted between Clal and BamHI into pBRd [F. A. M. Asselbergs et al. "A Recombinant Chinese Hamster Ovary Cell Line Containing A 300-fold Ampli¬ fied Tetramer of the Hepatitis B Genome Together With A Double Selection Marker Expresses High Levels of Viral Protein," J. Mol. Biol., 189, pp. 401-11 (1986)]. Next, the smaller HindiII - BamHI restric¬ tion fragment encoding T antigen, was replaced in this intermediate plasmid (pSV81) with the large HindiII - BamHI restriction fragment encoding agpt from pSV2neo [P. Southern and P. Berg, J. Mol. Appl. Genet., 1, pp. 327-41 (1982)] to form pSVneo2911. The EcoRI - BamHI restriction fragment from pSVneo2911, which contained the entire transcription unit (pro- oter - coding sequence - polyadenylation sequence) for agpt, was blunt-ended with Klenow enzyme and ligated into RE at the Klenow enzyme-treated Sail site.
The resultant plasmid, RE.neo, contained two SV40 replication origins, one 5' to the adeno- virus-2 major late promoter and the other overlapping the SV40 early promoter which controls transcription of the aminoglycoside 3 '-phosphotransferase (G418/neo) gene. We thus introduced a positive selection marker into our RE plasmid to create a stable cell line which grows on G418 medium [P. Southern and P. Berg, "Transformation of Mammalian Cells To Antibiotic Resistance With a Bacterial Gene Under Control of the SV40 Early Region Promoter, " J. Mol. Appl. Genetics, 1 , pp. 327-41 (1982); A. Jimenez and J. Davies, "Expression of a Transposable Resistance Element in Saccharomyces Cerevisiae, Nature, 287, pp. 869-71 (1980)].
Next, we obtained cell lines which inducibly synthesize and secrete factor VIII. We obtained our cell lines from BSC40 cells, which are BSC1 African green monkey kidney cells which have been adapted to
grow at 40°C [W. W. Brockman and D. Nathans, PNAS (USA) , 71, pp. 942-46 (1974)]. We co-transfected BSC40 cells with (1) plasmid RE.neo, which contains a SV40 origin of replication, a transcription unit for factor VIII, and a transcription unit for amino- glycoside phosphotransferase and (2) a 10-fold molar excess of plasmid LTRtsA58, containing a transcrip¬ tion unit for a temperature sensitive SV40 T antigen allele. The mutant tsA58 virus is a temperature- sensitive mutant of SV40 which does not produce progeny at 39°C. The large T antigen protein specified by the tsA58 mutant is much more labile at the non-per¬ missive temperature than wild type large T antigen protein [P. Tegtmeyer et al., Journal of Virology, 16, pp. 168-78 (1975)].
We then tested transfectants which survived selection in the aminoglycoside antibiotic G418 (Geneticin _D; G418 sulfate, Gibco Laboratories) for factor VIII expression. DNA sequences in these trans- fectants which are linked to a SV40 origin of replica¬ tion are rapidly amplified by shifting the culture to the temperature permissive for T antigen expression (33°C). When the SV40 T antigen is expressed, it initiates multiple rounds ("onion skin") of replica- tion at an SV40 origin of replication, which results in the amplification of DNA sequences in the neighbor¬ hood of the origin.
Our preferred cell lines, clones K, Aat2.10, and Aat2.27 are BSC40 transfectants which inducibly synthesize and secrete recombinant factor VIII.
Clone K [ATCC No. CRL 9206] was obtained by co-trans- fecting BSC40 with supercoiled RE.neo and supercoiled LTRtsA58 and selecting at 39.5°C for G418 resistance. Clones Aat2.10 and Aat2.27 were obtained by co-trans- fecting BSC40 with RE.neo linearized at the unique Aat2 site and supercoiled LTRtsA58 and selecting at 39.5°C for G418 resistance.
We made our preferred cell lines by co- transfecting confluent cultures of BSC40 cells in 100 mm Petri dishes with 10 micrograms pLTRtsA58 and 1.5 micrograms RE.neo using the standard technique of calcium phosphate precipitation. After 48 hours of incubation in drug-free medium at the nonpermissive temperature (39.5°C), the cells were replated at one-eighth the original density and cultured at 39.5°C in the presence of 700 μg/ml G418 (Gibco Laboratories). The medium was replaced every three days. We isolated clones with cloning rings 15 days after transfection and plated into duplicate 24-well dishes. Then we grew these primary cells lines in the presence of G418 at 39.5°C to confluence. For both sets of plates, we changed the culture fluid at confluence using complete medium with the antibiotic G418 but lacking phenol red (a pH indicator interfer¬ ing with the chromogenic factor VIII assay).
We then put one plate into a 33°C incubator, and four days later, performed KabiVitrum's Coatest ®
Factor VIII assay, using conditioned medium for each line at both temperatures. We selected clones exhibiting the highest levels of activity at 33°C for further study. No clone expressed factor VIII at 39.5°C at non-negligible levels.
We produced factor VIII with these clones, as described above, by growing cultures to confluence in a humidified, 5% carbon dioxide incubator at 39.5°C in T75 flasks. We used the following culture medium: DMEM - 10% fetal calf serum - 4 mM L-glutamine -20 mM HEPES (pH 7.2) - 720 μg/ml G418. At confluence, we replaced the culture fluid with 10 ml of fresh culture fluid and placed the T75 flask in a humidified, 5% carbon dioxide incubator at 33°C. The synthesis of factor VIII was induced and factor VIII activity accumulated over a period of days in the culture fluid.
We added von Willebrand factor to the fresh culture fluid used to feed the cells immediately prior to temperature shift. The concentration of von Willebrand factor in the culture fluid ranged from 5 to 30 μg/ml. We used mature human vWf
(2050 amino acids) which had been obtained as a side product obtained during the purification of plasma factor VIII (KabiVitrum AB). The first step in the purification of plasma factor VIII was affinity chromatography of the factor VIII/vWf complex on solid phase goat anti-human vWf [Andersson et al., PNAS, 8_3, pp. 2979-83 (1986)]. After washing the i munoabsorbent, the factor VIII was eluted with 0.6 M NaCl; the vWf remained attached to the antibody. Human vWf was subsequently eluted with a 0.1 M glycine (pH 2.2) and the solution was neutralized to pH 7.4 and frozen. Our analysis of this affinity-purified preparation on SDS polyaery1amide gel showed primarily the 220 K subunit; some preparations also contained an amino-terminal 130 K proteolytic fragment of the 220 K subunit.
We then quantitated factor VIII activity with KabiVitrum AB's Coatest® Factor VIII.
Referring to Figure 1, we depict therein our observation that factor VIII accumulates to a higher level in the presence of vWf. Comparing the level of factor VIII activity accumulated using culture fluid containing no human vWf to that accumu¬ lated using culture fluid supplemented with 10 μg/ml human vWf (the plasma concentration of human vWf is 10 μg/ml), we observed that the yield of factor VIII activity is increased by a factor of approximately three when secreted into culture fluid containing human vWf. The dose-response relationship between human vWf concentration and factor VIII activity is depicted in Figure 2. In experiments designed to
measure the level of factor VIII accumulating in culture fluid in the presence of vWf, we observed that even 5 μg/ml human vWf is sufficient to increase the yield of factor VIII activity by a factor of three.
Figure 3 demonstrates that the increased yield of factor VIII activity in the presence of human vWf is not specific only to clone K. We also observed increases in factor VIII yield in clones Aat2.10 and Aat2.27.
Finally, we also observed that when factor VIII is affinity-purified from culture fluid contain¬ ing no added human vWf, bovine vWf is copurified. We determined this through amino acid sequence analysis of the major contaminants of affinity-puri¬ fied factor VIII. Our observation suggests that bovine vWf is present in 10% fetal calf serum in a concentration sufficient to form a factor VHI/bvWf complex. Nevertheless, the addition of human vWf .to culture fluid still results in an increased yield. The reason for the increased yield may be that only a subset of multimers is able to bind to factor VIII and the concentration of this class of multimer is low in 10% fetal calf serum relative to that of the affinity-purified human vWf.
While we have hereinbefore presented a number of embodiments of this invention, it is apparent that our basic construction can be altered to provide other embodiments which utilize the pro- cesses and compositions of this invention. Therefore, it will be appreciated that the scope of this inven¬ tion is to be defined by the claims appended hereto rather than by the specific embodiments which have been presented hereinbefore by way of example.
Claims
1. A method of increasing the yield of factor VIII, comprising growing factor VIII- secreting cells in the presence of vWf.
2. The method according to claim 1, wherein the amount of vWf present comprises from 5 to 30 μg of vWf per ml of culture fluid.
3. The method according to claim 1, wherein the amount of vWf present is 10 μg per ml of culture fluid.
4. The method according to claim 1, wherein the cells are unicellular mammalian host cells which have been transformed with a recombinant DNA molecule characterized by a DNA sequence coding on expression for factor VIII, said DNA sequence being operatively linked to an expression control sequence in said DNA molecule.
5. The method according to claim 4, wherein the cells have been transformed with a recombinant DNA molecule which is additionally char¬ acterized by a DNA sequence encoding vWf, said DNA sequence being operatively linked to an expression control sequence in said DNA molecule.
6. The method according to claim 4, wherein the host is selected from BMT 10, BSC 1,
BSC 40, COS 1, COS 7, CHO cells and other mammalian and human cells in culture.
7. The methods according to claim 1, wherein the cells are a human cell line which secretes factor VIII.
8. A process for producing factor VIII in high yield comprising growing a host which secretes factor VIII in a culture medium which contains vWf, collecting the factor VIII/vWf complex and isolating factor VIII from the complex.
9. The factor VIII/vWf complex produced according to the method of any one of claims 1-8.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR1019880701229A KR890700671A (en) | 1987-01-30 | 1989-01-29 | How to prepare the factor 로 in high yield |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US923787A | 1987-01-30 | 1987-01-30 | |
US009,237 | 1987-01-30 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1988005825A1 true WO1988005825A1 (en) | 1988-08-11 |
Family
ID=21736435
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1988/000292 WO1988005825A1 (en) | 1987-01-30 | 1988-01-29 | A method for producing factor viii in high yield |
Country Status (6)
Country | Link |
---|---|
EP (1) | EP0302925A4 (en) |
JP (1) | JP2872255B2 (en) |
KR (1) | KR890700671A (en) |
AU (1) | AU606925B2 (en) |
NZ (1) | NZ223369A (en) |
WO (1) | WO1988005825A1 (en) |
Cited By (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0319944A2 (en) * | 1987-12-08 | 1989-06-14 | Zymogenetics, Inc. | Co-expression in eukaryotic cells |
EP0362218A1 (en) * | 1987-04-06 | 1990-04-11 | Genetics Inst | IMPROVED METHOD FOR PRODUCING FACTOR VIII:c-TYPE PROTEINS. |
US5198349A (en) * | 1986-01-03 | 1993-03-30 | Genetics Institute, Inc. | Method for producing factor VIII:C and analogs |
US5648254A (en) * | 1988-01-15 | 1997-07-15 | Zymogenetics, Inc. | Co-expression in eukaryotic cells |
EP0808901A2 (en) * | 1996-05-24 | 1997-11-26 | Immuno Ag | Pharmaceutical preparation with factor VIII procoagulant activity and vWF-binding activity |
US5914109A (en) * | 1990-06-15 | 1999-06-22 | New York University | Heterohybridomas producing human monoclonal antibodies to HIV-1 |
US7498146B2 (en) | 1996-03-15 | 2009-03-03 | Baxter Innovations Gmbh | Stable factor VIII/von willebrand factor complex |
US9394353B2 (en) | 2011-10-18 | 2016-07-19 | Csl Limited | Method for improving the stability of purified factor VIII after reconstitution |
EP2870250B1 (en) | 2012-07-06 | 2018-04-18 | Bioverativ Therapeutics Inc. | Cell line expressing single chain factor viii polypeptides and uses thereof |
EP2482841B1 (en) | 2009-10-02 | 2018-11-21 | The Children's Hospital Of Philadelphia | Compositions and methods for enhancing coagulation factor viii function |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE3720246A1 (en) * | 1987-06-19 | 1988-12-29 | Behringwerke Ag | FACTOR VIII: C-LIKE MOLECULE WITH COAGULATION ACTIVITY |
JPH0387173A (en) * | 1987-09-10 | 1991-04-11 | Teijin Ltd | Preparation of human active natural type factor viii c and transformant using the same |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0150735A2 (en) * | 1984-01-12 | 1985-08-07 | Chiron Corporation | Protein composition exhibiting coagulation activity and method for the preparation thereof |
EP0160457A1 (en) * | 1984-04-20 | 1985-11-06 | Genentech, Inc. | Human factor VIII, Compositions containing it, methods and materials for use in it production |
WO1987004187A1 (en) * | 1986-01-03 | 1987-07-16 | Genetics Institute, Inc. | METHOD FOR PRODUCING FACTOR VIII:c-TYPE PROTEINS |
EP0197592B1 (en) * | 1985-04-01 | 1993-02-17 | Stichting Vrienden Van De Stichting Dr. Karl Landsteiner | Preparation of the human von willebrand factor by recombinant dna |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0162067B1 (en) * | 1983-10-28 | 1992-07-15 | Genetics Institute, Inc. | Production of factor viii and related products |
GB8505882D0 (en) * | 1985-03-07 | 1985-04-11 | Central Blood Lab Authority | Purification of blood coagulation factor viii |
US4758657A (en) * | 1985-07-11 | 1988-07-19 | Armour Pharmaceutical Company | Method of purifying Factor VIII:C |
EP0251843A1 (en) * | 1986-06-06 | 1988-01-07 | Transgene S.A. | Process for the preparation of factor VIII from mammalian cells |
-
1988
- 1988-01-29 AU AU13409/88A patent/AU606925B2/en not_active Expired
- 1988-01-29 JP JP63501908A patent/JP2872255B2/en not_active Expired - Lifetime
- 1988-01-29 WO PCT/US1988/000292 patent/WO1988005825A1/en not_active Application Discontinuation
- 1988-01-29 EP EP19880902010 patent/EP0302925A4/en not_active Withdrawn
- 1988-02-01 NZ NZ223369A patent/NZ223369A/en unknown
-
1989
- 1989-01-29 KR KR1019880701229A patent/KR890700671A/en not_active Application Discontinuation
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
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EP0150735A2 (en) * | 1984-01-12 | 1985-08-07 | Chiron Corporation | Protein composition exhibiting coagulation activity and method for the preparation thereof |
EP0160457A1 (en) * | 1984-04-20 | 1985-11-06 | Genentech, Inc. | Human factor VIII, Compositions containing it, methods and materials for use in it production |
EP0197592B1 (en) * | 1985-04-01 | 1993-02-17 | Stichting Vrienden Van De Stichting Dr. Karl Landsteiner | Preparation of the human von willebrand factor by recombinant dna |
WO1987004187A1 (en) * | 1986-01-03 | 1987-07-16 | Genetics Institute, Inc. | METHOD FOR PRODUCING FACTOR VIII:c-TYPE PROTEINS |
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Title |
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E.BRAUNWALD, "Harrison's Principles of Internal Medicine", published 1986, by Mcgraw-Hill (New York, USA) see page 1473. * |
Journal of Biological Chemistry, Vol.258, 25 February 1983 (Baltimore, Maryland, USA), WAGNER et al, "Biosynthesis of von Willebrand Protein by Human Endothelial Cells", pages 2065-2067. * |
See also references of EP0302925A4 * |
Cited By (19)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5198349A (en) * | 1986-01-03 | 1993-03-30 | Genetics Institute, Inc. | Method for producing factor VIII:C and analogs |
EP0362218A1 (en) * | 1987-04-06 | 1990-04-11 | Genetics Inst | IMPROVED METHOD FOR PRODUCING FACTOR VIII:c-TYPE PROTEINS. |
EP0362218A4 (en) * | 1987-04-06 | 1990-09-19 | Genetics Institute, Inc. | Improved method for producing factor viii:c-type proteins |
EP0319944A2 (en) * | 1987-12-08 | 1989-06-14 | Zymogenetics, Inc. | Co-expression in eukaryotic cells |
EP0319944A3 (en) * | 1987-12-08 | 1990-05-16 | Zymogenetics Inc. | Co-expression in eukaryotic cells |
US5648254A (en) * | 1988-01-15 | 1997-07-15 | Zymogenetics, Inc. | Co-expression in eukaryotic cells |
US5914109A (en) * | 1990-06-15 | 1999-06-22 | New York University | Heterohybridomas producing human monoclonal antibodies to HIV-1 |
US7498146B2 (en) | 1996-03-15 | 2009-03-03 | Baxter Innovations Gmbh | Stable factor VIII/von willebrand factor complex |
EP0808901A3 (en) * | 1996-05-24 | 1999-05-06 | Immuno Ag | Pharmaceutical preparation with factor VIII procoagulant activity and vWF-binding activity |
EP0808901A2 (en) * | 1996-05-24 | 1997-11-26 | Immuno Ag | Pharmaceutical preparation with factor VIII procoagulant activity and vWF-binding activity |
EP2482841B1 (en) | 2009-10-02 | 2018-11-21 | The Children's Hospital Of Philadelphia | Compositions and methods for enhancing coagulation factor viii function |
EP2482841B2 (en) † | 2009-10-02 | 2022-09-28 | The Children's Hospital Of Philadelphia | Compositions and methods for enhancing coagulation factor viii function |
US9394353B2 (en) | 2011-10-18 | 2016-07-19 | Csl Limited | Method for improving the stability of purified factor VIII after reconstitution |
US9956269B2 (en) | 2011-10-18 | 2018-05-01 | Csl Limited | Method for improving the stability of purified factor VIII after reconstitution |
US10537616B2 (en) | 2011-10-18 | 2020-01-21 | Csl Limited | Method for improving the stability of purified factor VIII after reconstitution |
US10881717B2 (en) | 2011-10-18 | 2021-01-05 | Csl Limited | Method for improving the stability of purified Factor VIII after reconstitution |
US11510968B2 (en) | 2011-10-18 | 2022-11-29 | Csl Limited | Method for improving the stability of purified Factor VIII after reconstitution |
EP2870250B1 (en) | 2012-07-06 | 2018-04-18 | Bioverativ Therapeutics Inc. | Cell line expressing single chain factor viii polypeptides and uses thereof |
EP2870250B2 (en) † | 2012-07-06 | 2022-06-29 | Bioverativ Therapeutics Inc. | Cell line expressing single chain factor viii polypeptides and uses thereof |
Also Published As
Publication number | Publication date |
---|---|
JP2872255B2 (en) | 1999-03-17 |
JPH01501683A (en) | 1989-06-15 |
AU1340988A (en) | 1988-08-24 |
EP0302925A4 (en) | 1989-04-24 |
KR890700671A (en) | 1989-04-26 |
NZ223369A (en) | 1990-08-28 |
AU606925B2 (en) | 1991-02-21 |
EP0302925A1 (en) | 1989-02-15 |
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