AU606925B2 - A method for producing factor viii in high yield - Google Patents

Description

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PCT

AU-AI-13409/88 WORLD INTELLECTUAL PROPERTY QXGAN M ION nON inE TI AT COOPERATION TREAe

(PCT)

TION fiJ E TZ ATOT COOPERATION TREATY (PCT) INTERNATIONAL APPLICA (51) International Patent Classification 4 (11) International Publication Number: WO 88/ 05825 C'2P 21/00, C07K 15/06 Al (43) International Publication Date: 11 August 1988 (11.08.88) (21) International Application Number: PCT/US88/00292 (81) Designated States: AT (European patent), AU, BE (European patent), CH (European patent), DE (Euro- (22) International Fi!ing Date: 29 January 1988 (29.01.88) pean patent), FR (European patent), GB (European patent), IT (European patent), JP, KR, LU (European patent), NL (European patent), SE (European pa- (31) Priority Application Number: 009,237 tent).

(32) Priority Date: 30 January 1987 (30.01,87) Published (33) Priority Country: US With international search report.

(71) Applicant: BIOGENN-V. [NL/NL]; Willemstad,-Guragab-(-A-N) (71)(72) Applicant and Inventor: PASEK, Mark, P. [US/US]; 177 Lexington Street, Belmont, MA 02178 Jr 4 29 SEP 1988 (74) Agents: HALEY, James, Jr. et al.; Fish Neave, 875 Third Avenue, New York, NY 10022-6250 (US).

Ht0ss^cetsifc7' OZt O AUSTRALIAN 24 AIG 1988 This document contain, the amendments made u-idr PATENT OFFICE Section 49 and is courrect furprinting.

(54) Title: A METHOD FOR PRODUCING FACTOR VIII IN HIGH YIELD (57) Abstract The method comprises the addition of vWf t( 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, 1 ,j .L foil WO 88/05825 PCT/US88/00292 1 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 nomenclature 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 Haemostasis," 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 designated as "factor VIII/vWf".

In plasma, coagulant factor VIII circulates as a non-covalent complex with the adhesive von Willebrand factor 4 vWf") 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., WO 88/05825 PCT/US88/00292 -2- Hematology, pp. 1085-90, McGraw-Eill, 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 polypeptide 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 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 Eaton et al., Biochemistry, pp. 505-12 (1986)].

Von Willebrand factor 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 S. Zimmerman et al., "Factor VTII/von Willebrand Factor," Progress In Hematology, 13, pp. 279-309 (1983), E. B. Brown, editor]. The vWf protein is missing in the bleeding disorder, von Willebrand L, I :i i t SWO 88/05825 PCT/US88/00292 -3disease type IIA M. Ruggeri and T. S. Zimmerman, "Variant von Willebrand's Disease, J. Clin. Invest., 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 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., 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 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 A. Sporn et al., "Inducible Secretion Of Large, Biologically Potent 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 Weib=l-Palade bodies.

The site(s) and mechanism of factor VIII/vWf complex formation are largely unknown. One common view is that factor III is synthesized and secreted by the hepatocyte 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, 317, pp. 726-28 (1985)].

According to this view, the secreted factor VIII is ob i i WO 88/05825 PCT/US88/00292 endocytosed by the neighboring sinusoidal endothelial cell 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 Sewerin and 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 Hematology, 43, pp. 669-74 (1979); M. Weinstein et al., "Analysis i "I" WO 88/05825 PCT/US88/00292 of Factor VIII Coagulant Antigen In Normal, Thrombintreated, and Hemophilic Plasma," PNAS (USA), 78, 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), 79, pp. 7200-04 (1982); C. A.

Fulcher and T. S. Zimmerman, "Characterization Of The Human Factor VIII Procoagulant Protein With A Heterologous 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. Accorao ig to this method, to 30 pg 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 'e ~F -i i _L i r WO 88/05825 PCT/US88/00292 -6cotransforming 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 pg/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 pg/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 molecular 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.].

le WO 88/05825 PCT/US88/00292 -7- As used in this application, "factor VIII" also refers to polypeptides characterized by a deletion of a major portion of the maturation polypeptide of factor VIII. For example, where the entire maturation 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, 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 polypeptides having natural allelic variations that may exist and occur from individual to individual. Furthermore, it includes factor VIII polypeptides whose degree and location of glycosylation, or other posttranslation modifications, may vary depending on the cellular environment of the producing host or tissue.

Von Willebrand Factor a polypeptide -7hich 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 particularly, 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- 8 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 g of vWf should be present per milliliter of the culture fluid during synthesis of factor VIII. More preferably, 10 Ag per ml of culture is present. Among the host cells useful in this invention are BMT 10, BSC 1, BSC 40, COS 1, CHO 15 cells and other mammalian and human cells in culture, which cells produce Factor VIII.

S* In the example of this invention, provided below, we used affinity-purified human vWF to demonstrate 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 expressing factor VIII which would result in a cell line which either co-secretes factor VIII and vWf into che culture fluid to subseque-itly form the complex; or 25 assembles the factor VIII/vWf complex in either the endoplamic reticulum or the Golgi apparatus prior to exocytosis (see, for example, D. T. Bonthron et al.,

S.

'"Structure of prepro- von Willebrand factor and Its Expression in Heterologous Cells," Nature, 324, pp. 270-73 (1986)]. Most preferably, we produce recombinant factor VIII containing a deletion of a major part of the maturation polypeptide, in cells which have been cotransformed 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 activity which result from the methods of the present i -i 1 WO 88/05825 PCT/US88/00292 -9invention can be explained by either of the following two models. First, vWf pr.:ent 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 molecuJ, from extracellular proteolytic degradation. Alternatively, 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 purification steps include those used to purify natural and recombina.nt factor VIII [see, for example, L.-O.

Andersson et al., PNAS (USA), 83, pp. 2979-83 (1986)].

For example, the intermediate factor VIII-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 composition and methods for treatment of haemophilia A and in a variety of agents useful in treating uncontrolled 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., numan serum albumin or plasma preparations. See, e.g., Remington's Pharmaceutical Sciences W. Martin).

The resulting formulations will contain an amount of I i 1_

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II

ij 11

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WO 88/05825 PCT/US88/00292 modified factor VIII effective in the recipient to treat uncontrolled bleeding. Administration of these polypeptides, or pharmaceutically acceptable derivatives thereof, may be via any of the conventional 5 accepted modes of administration of factor VIII.

These include parenteral, subcutaneous, or intravenous 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.

WO 88/05825 PCT/US88/00292 -11- 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: 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 Sl, and subsequently digesting with KpnI which cleaves uniquely between the codons for Tyr 586 and Leu 587.

the synthetic oligonucleotide duplex fragment ATA ACT CGT ACT ACT CTT CAG TCA CTT TAT TGA GCA TGA TGA GAA GT GTC T CTA Gp Glu Ile Thr Arg Th- Thr Leu Gin Ser Asp 1649 1657 the 135 bp fragment obtained by digesting 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.

pUC18 digested with Kpnl and PstI.

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-fragnent ligation with Asp718 and PstI. The fragment encoding the RE fusion was used to replace the corresponding fragment in the expression plasmid for an earlier-created factor VI1t i -L Y WO 88/05825 PCT/US88/002E92 -12deletion, 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 Gln 744 and Asn 745. We removed the 5'AATT overhang with nuclease Sl, and then digested the plasmid at the unique Pvul site within the ampicillin resistance gene. We partially digested another aliquot with BamHl, 'which cleaves between the codons for Leu 1562 and Asp 1563. Wc 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 PstI, and then ligated the 624 bp fragment encoding the RE fusion to the resultant 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 inserting into RE a transcription unit directing the expression of agpt in animal cells. We isolated the transcription unit from plasmid pSVneo2911, a gift of Fred A, M. Asselbergso Plasmid pSVneo2911 was constru ted as follows: first, the SV40 HpaII-BamHI restriction fragment containing the eintire SV40 early rl' I I r"l wri WO 88/05825 PCT/US88/00292 -13region was inserted between Clal and BamHI into pBRd A. M. Asselbergs et al. "A Recombinant Chinese Hamster Ovary Cell Line Containing A 300-fold Amplified 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 restriction fragment encoding T antigen, was replaced in this intermediate plasmid (pSV81) with the large HindIII BamHI restriction fragment encoding agpt from pSV2neo 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 (promoter coding sequence polyadenylation sequence) for agpt, was blunt-ended with Kleaow enzyme and ligated into RE at the Klenow enzyme-treated Sall site.

The resultant plasmid, RE.neo, contained two SV40 replication origins, one 5' to the adenovirus-2 major late promoter and the other overlapping the SV40 early promoter which controls transcription ot 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 Southern and P. Berg, "Transformation of Mamrtalian 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 Saccharomvces 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 WO 88/05825 PCT/US88/00292 -14grow at 40 0 C W. Brockman and D. Nathans, PNAS (USA), 71, pp. 942-46 (1974)]. We co-transfected cells with plasmid RE.neo, which contains a SV40 origin of replication, a transcription unit for factor VIII, and a transcription unit for aminoglycoside phosphotransferase and a 10-fold molar excess of plasmid LTRtsA58, containing a transcription unit for a temperature sensitive SV40 T antigen allele. The mutant tsA58 virus is a temperaturesensitive mutant of SV40 which does not produce progeny at 39 0 C. The large T antigen protein specified by the tsA58 mutant is much more labile at the non-permissive temperature than wild type large T antigen protein Tegtmeyer et al., Journal of Virology, 16, pp. 168-78 (1975)].

We then tested transfectants which survived selection in the aminoglycoside antibiotic G418 (Geneticin G418 sulfate, Gibco Laboratories) for factor VIII expression. DNA sequences in these transfectants which are linked to a SV40 origin of replication are rapidly amplified by shifting the culture to the temperature permissive for T antigen expression (33 0 When the SV40 T antigen is expressed, it initiates multiple rounds ("onion ski,") of replication at an SV40 origin of replication, which results in the amplification of DNA sequences in the neighborhood 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.

jClone K [ATCC No. CRL 9206] was obtained by co-transfecting BSC40 with supercoiled RE.neo and supercoiled LTRtsA58 and selecting at 39.5 0 C for G418 resistance.

Clones Aat2.10 and Aat2.27 were obtained by co-transfecting BSC40 with RE.neo linearized at the unique Aat2 site and supercoiled LTRtsA58 and selecting at 39.5 0 C for G418 resistance.

-I i -r L -C L. i chain macromolecular precursor, which is later cleaved to yield the fragments which constitute "mature" factor VIII [see generally, W. J. Williams et al., WO 88/05825 PCT/US88/00292 We made our preferred cell lines by cotransfecting confluent cultures of BSC40' cells in 100 mm Petri dishes with 10 micrograms pLTRtsA58 and 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 0 the cells were replated at one-eighth the original density and cultured at 39.5 0 C in the presence of 700 pg/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 0 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 interfering with the chromogenic factor VIII assay).

We then put one plate into a 33 0 C incubator, and four days later, performed KabiVitrum's Coatesto Factor VIII assay, using conditioned medium for each line at both temperatures. We selected clones exhibiting the highest levels of activity at 33 0

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 0

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 pg/ml G418. At confluence, we replaced the culture fluid with 10 ml of fresh culture fluid and placed the T75 flask in a humidified, carbon dioxide incubator at 33 0 C. The synthesis of factor VIII was induced and factor VIII activity accumulated over a period of days in the culture fluid.

a f'actor, l Progress In Hematology, 13, pp. 279-309 i (1983), E. B. Brown, editor]. The vWf protein is missing in the bleeding disorder, von Willebrand WO 88/05825 PCT/US88/00292 -16- 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 pg/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 w'as affinity chromatography of the factor VIII/vWf complex on solid phase goat anti-human vWf [Andersson et al., PNAS, 83, pp. 2979-83 (1986)]. After washing the immunoabsorbent, the factor VIII was eluted with 0.6 M NaCI; 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 polyacrylamide 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 accumulated using culture fluid supplemented with 10 pg/ml 3 human vWf (the plasma concentration of human vWf is pg/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.

3- The dose-response relationship between human vWf concentration and factor VIII activity is depicted in Figure 2. In experiments designed to III WO 88/05825 PCT/US88/00292 -17measure the level of factor VIII accumulating in culture fluid in the presence of vWf, we observed that even 5 pg/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 containing no added human vWf, bovine vWf is copurified.

We determined this through amino acid sequence analysis of the major contaminants of affinity-purified factor VIII. Our observation suggests that bovine vWf is present in 10% fetal calf serum in a concentration sufficient to form a factor VIII/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.

.i l;l; |r I "The Chromatographic Separation of Factor VIII on Aminohexyl Sepharose," British Journal of Hematology, 43, pp. 669-74 (1979); M. Weinstein et al., "Analysis I c I ~l~r~ r- WO 88/05825 PCT/US88/00292 -18- 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 processes and compositions of this invention. Therefore, it will be appreciated that the scope of this invention is to be defined by the claims appended hereto rather than by the specific embodiments which have been presented hereinbefore by way of example.

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Claims (9)

1. A method of increasing the yield of factor VIII, as hereinbefore defined, comprising growing factor VIII secreting cells in the presence of vWf, said cells being transformed with a factor VIII coding sequence or being cell lines producing native factor VIII protein solely or predominantly.

2. The method according to claim 1, wherein the amount of vWf present comprises from 5 to 30 Ag of vWf per ml of culture fluid. e*

3. The method according to claii 1, wherein the amount of vWf present is 10 pg per ml of culture fluid.

4. The method according to claim 1, wherein the cells are unicellular mamm.lian host cells which have been transformed with a recombinant DNA molecule characterized by a DNA sequence coding on expression for factor VIII, as hereinbefore defined, said DNA sequence being operatively linked to an expression Scontrol sequence in said DNA molecule.

5. The method according to claim 4, wherein 9 the cells have been transformed with a recombinant DNA molecule which is additionally characterized 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. L ii :L j *i*rrr 20

7. The methods according to claim 1, wherein ae cells are a human cell line which secretes factor VIII, as hereinbefore defined.

8. A process for producing factor VIII, as hereinbefore defined, in high yield comprising growing a host which secretes factor VIII in a culture medium which contains vWf, collecting the tactot VIII/vWf complex and isolating factor VIII from the complex, said host being transformed with a factor VIII coding sequence, or being cell lines producing native factor VIII protein solely or predominantly.

9. The factor VIII/vWf complex produced according to the method of any one of claims 1-8. DATED this Seventh day of November, '1990. BiOGEN INC and MARK P PASEK by their Patent Attorneys CULLEN CO S @006 0000 S 0 00SS S 0 S @0 N'