JPH01501683A - Factor 8 high yield production method - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] 5. High yield production method of factor ■ Mitsutatsu The present invention relates to a high-yield production method for Factor (1). More specifically, the present invention provides vo n　Wi　1　Proliferate factor ■ producing cells in the presence of ebrand factor The present invention relates to a method for increasing the yield of Sake Factor ■.

V, J, Harder et al, '''5 standard N01eriC IattJre for FaCtOri and von Willebran d　Factor:A.

Recouendation By the International C onn1ttee on Thrombosis and Haeiostasi s, 'go hrombosis and river deaf m'm' D0.871-72 ( 1985). “Factor ■” ゛　 means coagulation precursor cofactor (procoagu lant cofactor) name, "von ko 111e brand factor" -” is the name of the adhesion factor of the bimolecular complex, and is hereinafter referred to as “Factor ■/V revision”. Ru.

Blood clotting factor: Factor ■ is an adhesion factor. It forms a non-covalent bond with VIJf'') and circulates in the plasma [L, W, Hoyer, Blood, 5B, DD, 1-13 (1981)].

Factor ■/Factor ■, a blood clot precursor component of VWf, was originally a single-chain polymer. Synthesized as a child precursor, but then cleaved to form the “mature” factor ■ resulting in fragments [general description is given by W. J. Williams et al. l, Hematology, pp, 1085-90. HcGraw -Hill, New York (1972)], the maturation factor Consists of two molecular chains linked via mu ions, i.e. = 740 amino acids Consisting of an amino terminal long chain and a carboxyl terminal short chain consisting of 684 amino acids has been done. The primary translation product of factor ■ is a single strand, in which the maturation factor -■ The long chain is separated from the short chain by a “mature polypeptide” of 908 amino acids. It is. Excision of this “mature polypeptide” is called Arg1B4B-Glu By proteolytic cleavage of the primary translation product at the peptide bond of 1649 Start. Once the initial break occurs, a series of proteolytic cleavages occur, producing a The long chain of R becomes shorter from the carboxyl terminus, resulting in 740 This is a completed long chain consisting of 684 amino acids, and a short chain consisting of 684 amino acids. Together with the chain, the maturation factor ■ is constructed [L, -0, Andersson et at, “l5olation andCharaCteriZatiO n of Hullan Factor ■: Molecular Forms in Commercial Factor VI Concentrate.

Cryoprecipitate, and Plasma, PNAS (U S^　83p.

2979-83 (1986)]. This complex is then released by thrombin. It is cleaved and activated at the rg 1689-Ser 1690 bond [D , Eaten et al, B10ChelliStr, 25゜p p, 505-12 (1986)].

von Wil 1ebrand factor same VI4f”) is a large polymeric blood A serous protein consisting of at least two subunits, which are linked by disulfide bonds. They are connected and become one.

vWf is a protein that plays an important role in the hemostatic process and is related to normal platelet aggregation and adhesion. are doing. In other words, when a blood vessel is injured, these four F's act as a mediator that attaches platelets to the basement membrane. In combination with the relatively large size of the polymer, platelet aggregation at the site of vascular injury is It exerts a collection function [T, S.

Zillllerllan et al,,ractor■/von Wi I1ebrandFactor, Progress in He1Ilat oloq, 13. Dp/279-309 (1983), E, B, B rown, editorl, bleeding disorder VOnWiIebran This VWf protein is missing in disease type I [A] [Z, H, Ruggeri & T, S, Ziuennan, “Variant von Willeb rand's Disease, j, Cl1n, InVeSt,, 65 ．． pp.

-1318-25 (1980)].

The mature V protein is synthesized through several steps. First, it consists of 2813 amino acids. Blood vessel endothelium as a prepro-VWf subunit with a molecular weight of 240,000 to 260,000 Synthesized in cells and mononuclear cells [E, A, Jaffe et al, 5y nthesis of Antihemophilic Factor Anti gen　By　Cu1tured　HumanEndothelial　Ce1 ls'', J, Cl1n, Invest, 52. pp.

2757-64 (1973); R, Nachman et al, 5 ynthesis of Factor ■ Antigen By Culture d Guinea Pig Hegakaryo-cytes, J, Cl1n , Invest, 60 pp, 914-21 (1977); C, L, Verweij et al,,”Full-lengthvan Will lebrand Factor (vWf) CDNA encodes aHi ghly Repetitive Protein Con5iderably Larger Than the Mature vWf 5ubunit,” , EHBOJ,, 5. Dp.

1939-47 (1986)]. After passing through charcoal-carbohydrate-added procerasin, A disulfide bond is formed between the dimers and the precursor is cleaved to form a mature dimer. to be accomplished. Special mechanisms called Weibel-Palade bodies located within endothelial cells A multimer consisting of up to 50 subunits gathers in the secretory vesicles of the brain and acts as a regulatory route. [L, A, 5porn et al.

“Inducible 5 creation of Large, Biolo gicallyPatent von Willebrand Factor Hultimers”, Ce1l.

46, l)p, 185-90 (1986)], release of VWf from endothelial cells. is induced by the action of thrombin, resulting in the Weibel-Palad The e-body ends up disappearing.

centre? The site(s) where the vector/v41 complex is formed and the mechanism of formation are still poorly understood. I don't know. One common view is that factor ■ is synthesized by hepatocytes. There is a theory that it is secreted later [)1. G, Zelechowska et a l.

°゛Old trastructura+ LOCaliZatiOn of Fac tor ■ Procoagulant Antigen in Human Li veruepatocytes", Na1Ure, 317. pp, 729 -30 (1985): L. Wion et al., “Distribu tion of Factor Vl mRNA and Antigen in Hu+nan Livsr and 0ther Ti5sues-Natur e, 317. pp, 726-28 (1985)]. According to this idea, minutes The secreted factor ■ is once taken up by adjacent sinusoidal endothelial cells [H, V , 5tel et al,, “Detection of Factor■/ Coagulant Antigen in Human Liver It will be spit out later. Another idea is that the complex is formed in plasma. There is a possibility that Once factor ■ is associated with VWf, Afterwards, at arginine 1689, the short chain of maturation factor Due to the cleavage action, factor ■ rises to the surface in a state where VWf is gone. After adhering to the platelet surface near the platelet, factor IXa and factor It circulates in the plasma with a half-life of 9-10 hours until it forms the original complex. Sewerin & L, -0゜Andersson, “Binding o” f Native and Thrombinal, ”Proteolyt ic Processing of Human Factor■.

Correlation of 5 specific Cleavages by 　ThrOIllbin.

FactorXa, and Activated Protein Cwit hActivation and Inactivation of Fact or■Coagulant Activity,”Biochemistr,” 25. pp, 505-12 (1986)].

Hemophilia A is a type of elastic hemorrhagic disease in which there is a deficiency in factor This is a disease caused by insufficient biological activity. In the treatment of hemophilia patients with acute bleeding In this case, Factor ① purified from normal serum using a conventional method is administered. g Various methods for clefting have been reported and are available in the literature [see below] Reference: Zimn+erman et al, United 5tates Patent4.361,509; 5 aundrey et at, Un Ited 5tates Patent4.578.218; E, G, D, T uddenham et al,'GoheProperties of Fa ctor■Coagulant ActivityPrepared by I mmunoadsorbent chromatography.

Journal of Laborator C11nical Medici ne, 93°pp, 40-53 (1979); D, E, G, Au5ten ,'goheChromatographic 5eparation of F actor■0nAIllinOheXyl 5epharose, "Br1 tissue Journal of Hematolo, 43. pp, 66 9-74 (1979); HJeinsteinet at, “AnalV SiS of Factor Wit coaguant Antigenin Normal, Thrombin treated, and Hea+op hilicPlasma”, PNAS (USA), 78.pp, 513 7-41 (1981); p.

J, Fay et al, “Purification and Char activation of A Highly Purified Hu manFactor Consisting of A Single Type e of Polypeptide Chain, “PNAS” (USA), 79.1) I), 7200-04 (1982); C, A, Fulcher & T, S, Zimmerman.

”Characterization of The Human Fact r■Procoagulant Protein With A Hetero logous Precipitatino Antibody”, PNAS (USA), 79. pp.

164B-52 (1982); F, Rotblat et al, Th romb.

Factor ■ is the key to the treatment of hemophilia, so the number of people who can produce it Much research has been carried out to date, e.g. by recombinant DNA technology [e.g. For example, refer to the following literature: GenetiC5In5titute, PCTAppli cation WO35101961; Genentech Europe nPatent Application 160,457; Chiron European Patent Application 150,735; J., J., Toole et al.

“Mo1ecular Cloning of a cDNA Encodin g Human Antihaeiophilic Factor” Nature e, 312. pp, 342-47 (1984); and W, 1. W ood et al, Nature, 312゜pp, 330-37 (1 984)]. However, recombinant factor ■ can be produced at a satisfactorily high yield. Establishing a high-yield production method for this is an ongoing issue. Plate The present invention attempts to solve the above problems by providing a high-yield production method for factor It is something that More specifically, the present invention provides factor Concerning a method for increasing the yield of factor ■ by multiplying production cells. . In this method, 5 to 30 μQ of V is present in the medium; or recombinant factor with a DNA' sequence encoding VWf. VWf is supplemented by transforming producer cells.

Rat 0! ! i amount of shellfish Results of several induction experiments with Loan K and culture without vWf This is a comparison of the results obtained by conducting the same experiment using a liquid.

Figure 2 shows that clone K was induced to produce human vWf at 0.5°10 and 20.30 μg/ ! Factor ■ was secreted into the culture medium containing various levels of dl and accumulated. The amount of the factor (■) is shown.

Figure 3 shows two other clones tested: Aat2.10 and Aat2.2. This figure shows the yield of factor ■ at 7.

Figure 4 depicts the plasmids RE and neo. ified)) Factor ■ gene is present, adenovirus-2 major phase promoter (adenovirus-2majcr 1ate promo tor).

Oimidan employee Oni Ao J In order to better understand the content of the present invention, a detailed description will be given below.

Terms used in the explanation and their detailed contents or factors ■... Molecule a i2B is a 5,000 polypeptide that, when matured and activated, plays a role in the blood coagulation cascade. At the same time, factor rXa of factor have the ability. Factor ■ in this application is also known as Factor ■:C. glycoproteins, factors ■Coagulation precursor active proteins, factors ■Coagulation active proteins Contains white matter [reviewed by J. Williams et al.

Her*atoloQV Do, 1056.1074.108110 On top of that In this application, we show that the main part of the maturation polypeptide of Factor ■ is missing. Polypeptides characterized by are also included in “Factor ■”. For example, maturing polyp ) What if the whole petit de is missing? Amino-terminal mature long chain and carbo It consists of a xyl-terminated mature short chain and both are carried together to form a single chain, or Proteins that are linked through calcium and other metal ions are also “factors”. ” include.

In addition, amino-terminal methionine such as f-Net-factor As long as the biological activity of the protein is essentially maintained, other amino acids may be omitted or added. Includes proteins characterized by additions and substitutions. Natural conflict between individuals Contains variant polypeptides. Furthermore, the degree of post-translational glycosylation may vary depending on the production host or site. Factors that may differ depending on the cellular environment of the tissue or the tissue ■Polypeptides All of these should be included in the category of “factor ■”.

von Willsbrand factor: the action that causes platelets to stick to blood vessel walls A polypeptide that functions as a carrier of Factor 2 in plasma.

The present invention relates to a method for mass-producing a high acid of Factor (1). More specifically, to proliferate Factor■ Production host or those who produce fine or natural factors■ with high yield It provides law. In the method of the present invention, a host that originally produces factor In the case of cells, 4F can be added to fresh culture medium, and due to the induced action of external factors, In host cells that produce factor ■, vlJf is added to the culture medium immediately before induction. can be added. Alternatively, vWf may be used as a factor in the host cell. They can also be produced simultaneously. In this case, the host cells are both V and factor It changes its shape with a DNA sequence that encodes it. During production of factor ■, culture solution 11 1! It is desirable that there be a V correction of 5 to 30 μQ per Il. Even more preferable Let this level be 10μQ.

In the examples of the present invention described later, the affinity-purified human VWf n vWf) was used, but recombinant VWf can also be used. For example, the VWf gene is 77 When introduced into a cell line that expresses vector ■, this cell line (1) contains 7? (2) whether the endoplasmic reticulum and vWf are secreted simultaneously to form a complex; Alternatively, after forming the factor ■/VWf complex in the GOIQi body, [For example: D, T, BOnthrOn et] al.

“5structure of orepro-von Willebrand factor and Its Expression in tfeterol ogous Ce1ls, Nature.

凰1)I), 270-73 (1986)]. Particularly preferably, vwr In cells co-transformed with the encoding DNA sequence, the mature polypeptide It is better to produce a recombinant factor (■) containing a deletion of a key part. The DN The A sequence is an expression control sequence (eXpression Control sequence). e) in the form of a recombinant DNA molecule functionally linked to

Although I don't want to be bound by theory, the factor ■ brought about by the method of the present invention We believe that high-yield production can be explained by one of two models.

One is that factor ■ is released from exocytotic Go1gi vesicles in mammalian host cells. Then, the V-synthesis present in the nutrient solution combines with this, and this causes extracellular proteolytic action. factor ■ molecules will be protected. Another thing is that VWf is a mammal hotel. It is taken up into a certain part of the endoplasmic reticulum or Go1gi body of the main cell and newly produced there. It is associated with octopus factor ■, and is probably a factor due to its lysoseem proteolytic action. By protecting ■, an environment is created in which it is easy for things to get out of the system.

Various conventional means can be used to purify Factor ① produced by the method of the present invention. I can do it. Among the most useful are natural and recombinant materials such as those described in the following references: Factor ■ There is a means of purification [1. -0. Andersson et al. .

PNAS (IJsA), 83. See ppi 2979-83 (1986) Light]. For example, the factor ■/vWf as an intermediate produced by the method of the present invention The complex can be dissociated by the method of AnderSSOn to obtain factor ■. can.

After purification, the factor (1) produced by the method of the present invention can be used as a therapeutic composition for hemophilia A. It can be used as a therapeutic agent or as a variety of drugs useful in the treatment of uncontrolled bleeding. I can do it.

The factor ■ produced by the method of the present invention can be prepared by a known method and used as a drug. Compositions useful as such can be prepared. Compositions of this type may also have pharmaceutical concerns. It is preferable to incorporate conventional carriers that do not cause problems, and other drugs and carriers. additives, adjuvants, excipients, etc., such as human serum albumin and certain blood preparations. can be done. For example, Remington's Pharn+aceutic al5sciences (E, W, Hartin). instructor The resulting preparation contains an activation factor ■ that is effective in treating unstoppable bleeding. It is normal for it to contain a certain amount. These polypeptide preparations or drugs The method of administration of these derivative formulations has been previously approved for Factor ■. Parenteral administration can be done by any of the methods listed above.

Both subcutaneous and intravenous injections are possible.

Compositions of the present invention for use in hemophilia A therapy may also be administered in a variety of forms. can be done. The preferred form depends on the intended administration method and the type of treatment. It depends on what you use it for. Dosage amount and rate of administration depend on a variety of factors. For example, whether to treat a patient with acute bleeding or not. It depends on whether it is done as a preventive measure. However, the amount of factor The level must be sufficient to stop blood and promote epithelial formation [Williams , Hematology pp, 1335-43].

Examples are provided below in order to better understand the content of the present invention. This example simply This is for illustrative purposes only and is not intended to limit the scope of the invention. stomach.

Taiougo In this example, we will describe the procedure for mass production of activation factor ① using the method of the present invention. . First, the main part or all of the mature polypeptide of the natural factor We created a cDNA sequence that encodes the activation factor molecule that has been deleted. . The restriction enzyme map of RE, neoli4 is shown in Figure 4, and it can be seen that RE The location of the deleted factor ■CDNA can be found.

RE, neo was created in several stages. First of all, RE [ARG 740- GLU 1649] [ATCC No, 53517] Deleted structure by following procedure 2 Made in stages.

In the first step, the four fragments were ligated to create an intermediate plasmid.

These four fragments are:

(1) 462 bp fragment...Full chain length factor ■Gene expression platform Separate the codons between Arg 740 and Ser 741 using HindIIl. Then, after removing 5'AGCT with nuclease S1, T obtained by causing specific cleavage between the yr 58 & and Leu 587 codons. Ta.

(2) Synthetic oligonucleotide double-stranded fragment...5' pGAA AT A ACT CGT ACT ACT CTT CAG TCACTT TAT TGA GCA TGA TGA GAA GTCAGT CTA Gp5゜ Glu Ile Thr Arg Thr Thr Leu Gln Ser Asp (3) 135bp fragment...First, complete with shi4-3^ Chain length factor ■ Decomposes gene expression plasmids.

As a result, the codon between Ser 1657 and ASp 1658 and Glu 1 411bl) fragment generated by decomposition between the codons of 794 and ASD 1795 was isolated. Next, this 411 bp fragment was degraded using Pst engineering, and A By cleaving between the codons of la 1702 and Va11703, a 135bp s'fra The 624 base pairs encoding the RE fusion are then extracted from the intermediate plasmid. The fragment was isolated. To do this, the The intermediate plasmid obtained was digested with ASp71B and pstI. RE fusion These 24 base pair fragments encode previously produced factor Corresponding fragments in expression plasmids for deletions (so-called QD deletions) replaced. In this QD deletion, the mature polypeptide (amino 11741 -1648), but the main part of the DNA sequence encoding it is missing. Approximately 90 amino acids in a peptide (four amino acids on the amino terminal side and a carboxylic acid) The 86 amino acids on the oxy-terminal side remain intact. To make a QD deletion form , Complete chain length factor ■ Gin a part of the gene expression plasmid with Kyogo R1 74 The codon between 4 and ASf'l 745 was partially resolved. 5' AATT suspension part This plasmid was removed with Crease S1 and then inserted into the ampicillin resistance gene. It was disassembled at different Pvu engineering sites. In addition, another part of the above expression plasmid Partially disassembled with Ban+l-1'l and Leu 1562 and ASI) 1563. Cleaved between codons. Insert the Ienow fragment into the 5°G^TC suspension part. Then, insert the above plasmid into the ampicillin resistance gene at Pvu 1. I took it apart. The two fragment mixtures thus formed are then combined and this They were ligated together using T4 DNA ligase. In the fusion thus generated, Gln A new Balt old site was formed between the codons 744 and ASp1563.

The expression plasmid for the QD deletion was completely degraded at the unique louse site. After dephosphorylating the 5° GTAC suspension with calf intestinal phosphatase, , this plasmid was partially degraded with PSt engineering, and then the RE fusion was encoded. The 624 bp fragment was linked to the fragment mixture generated in the above procedure. . The thus generated plasmid encoding the expression of the RE fusion is referred to as RE. .

Therefore, a map of RE and neo is shown in FIG. Expression of agpt in animal cells The RE, neo, was created by inserting a directing transcription unit into the RE. child The transcription unit was provided by Fred A. H. Asselbergs plus It was isolated from mido pS V neo 2911. This plasmid pSVneo 2911 was created as follows. First, S■40 early part (early v 5V40Hpa l-BamH ■ restriction fragment containing the entire [F, A, inserted between C1a ■ and Ban+)-II (7) of DBRd H, Asselbergs et al, “ARecoibinant C hinese Haister 0vary Ce1l Linecontai ning A 300-roldAJ)lified Tejraller Ofitle Hepatitis B Gentile Together with A Double Selection Marker Express sses High Levels of Viral Protein”, J , Mo1. Biol, 189. pp, 401-11 (1986) ].

Next, ■relatively small 1lind [l　-BamiHIIJ The limited fragment was transformed into pSV2 using a plasmid (pSV81) as an intermediate. Large Hindl [I-Bag] encoding agpt derived from rieo with the HIilJ limited fragment, replacing [P, 5outhern an d P, Berg, J, Mol.

A1. Genet, 1. DD, 327-41 (1982)], I made DSVneO2911. The complete transcription unit of agpt (promoter code) derived from pSvneo 2911, containing a polyadenylation sequence) The EcoRl-Ba1lHI restriction fragment was isolated using lenowlli. With a smooth end, RE tied to.

The thus obtained plasmid RE, neo contains two SV40 origins of replication. one at 5° to the adenovirus-2 major late promoter; The other is aminoglycoside 3”-phosphotransferase (0418/neo ) is an overshipped version of the SV40 early promoter that regulates gene transcription. there were. Thus, a positive selection marker was introduced into the RE plasmid and cultured in G418 medium. We created a stable cell line that proliferated [P, 5othern & P.

Berg, 'GoransforIIlati*n of Mammalian Ce1lst.

Antibiotic Re5istance with a Bacteri al Geneunder Control of the 3V40 Ear ly RegionPromofor”, J, Mo1. Appl, Ge netics, 1. pp, 327-41 (1982); A. Jime nez & J. Davies, “Expression of aTra nsposable Re5istance Element in Sacc harom ces-Cerevisiae, Nature 28mu DI), 869-71 (1980)].

Next, we obtained a cell line that synthesizes and secretes factor ① through induction. This thin The cell line was adapted from African green monkey B2O2 kidney cells to grow at 40°C. [1. H, Brockian & D.

Nathans, PNAS (USA), 71. PP, 942-46 (1974)], that is, B5C40 cells were simultaneously treated with the following two types of plasmids. Transformed. One is plasmid RE, neo, S■40? ! Manufacturing starting point, fa The transcription unit of the vector, the transcription unit of the aminoglycoside phosphotransferase Contains.

The other is a 10-fold molar excess of plasmid LTRtsASB, which is temperature-sensitive 5 Contains the transcription unit of the V40T antigen allele. Sudden displacement tSA 5B virus This is a temperature-sensitive SV40 mutant that does not produce progeny at 39°C. ts The T antigen protein specified by the A5B mutant differs from the wild type T antigen protein. [P, Tegtmeyer et al. al,, Journal of Virolo.

16, l) p, 16B-78 (1975)].

Next, aminoglycoside antibiotic G418 (, Gneticin ■, G418 mlll salt, Gibco LabOratOrieS) to survive by being culled in The expression of factor ■ in the transformed transformants was investigated.

The DNA sequences of these transformants, which are linked to the SV40 origin of replication, allow the medium to be Rapid amplification is achieved by shifting to the permissive temperature for antigen expression (33°C). 5V When the 40T antigen is expressed, it appears in a multi-layered, rounded shape ( “onion skin”) replication begins, resulting in amplification of DNA sequences near the origin. I will do it.

Aat 2.10°Aat 2.27 is suitable for the desired cell line or clone. , B5C40 Kataga transformant, which synthesizes and secretes recombinant factor ■ by inducing action. do. Clone K [ATCC No. CRL 9206] exceeds B5C40. Sen RE.

G418 resistance after simultaneous transformation of neo and supercoiled LTAtSA58 ・Also, clone Aat 2.10 was obtained by screening at 39.5℃. The same Aat 2.27 is a linear RE at the unique Aat-2 site, ne 041 and supercoiled LT RtsA SB were simultaneously transformed into B5C40. 8 resistances were screened at 39.5°C.

Preferred cell lines are prepared with 10 μg of E)L by standard calcium phosphate precipitation methods. TRtSA5B and 1.5μ9 RE.

Transfect a B5C40 cell culture in a congregate state in a Petri dish of diameter 1001111 with neo. I made it by converting it. Cultured for 48 hours in drug-free medium at nonpermissive temperature (39,5°C). Afterwards, these cells were replanted to the original density of 178 and 700μQ/1G41. 8 (Gibc.

Laboratories) at 39.5°C. Medium every 3 days I replaced it with a new one. Cloning ring is present after 15 days of transformation. Clones were isolated and transplanted into two 24-well culture dishes. These primary cell lines The cells were allowed to grow in the presence of 0418 at 39.5°C to reach a congregate state. Both sets of culture plates Both contain the antibiotic G418 but do not contain phenol red (the pH indicator is colored Complete medium (con+plete m edium) was used to replace the culture medium in the confluent state.

Next, one culture plate was placed in a 33°C culture device and removed after 4 days. Coat abiVitrui+ at two levels of temperature using culture supernatant for each cell line. st O factor ■ test was performed. The highest level at 33℃ for further investigation Clones showing activity were selected. In addition, at 39.5℃, the temperature cannot be ignored. There were no clones expressing factor ■ at levels of .

In the above clone group, 5% diacid was added in a wet state at 39.5°C in a T75 flask. The cultured fiII cells are grown in a culture device containing hydrogenated carbon and brought to confluence. created a factor ■. The medium used was DMEM-10% fetal bovine serum-4. mHl-Glutamine-201gHHE PES (pH 7,2)-720 Rarely happened with μQ/dG41B. Once the culture has reached a confluent state, transfer the culture medium to fresh culture medium. 10d and replaced with 5% carbon dioxide in a T75 flask kept at 33°C in a humid condition. The cells were then placed in a culture device. This induces the synthesis of factor The activity of factor (2) increased cumulatively over several days.

Just before moving the temperature level to the fresh medium used for cell culture, won Added ebrand factor. <-(Dvon Willebrand Fa The concentration range in the culture solution of the vector is 5 to 30μQ/wri! Met. I used By-products and It was mature human vWf (2050 amino acids) that had been obtained as follows. plasma faq The first step in the purification of the factor is the in-phase goat anti-human VWf factor/vWf. I Andersson by affinity chromatography method of complexes et al, PNAS.

83, Dp, 2979-83 (1986)]. After washing the immunoabsorbing material, remove the Factor ■ was eluted with 0.6M Naα. Naori Wf remains attached to the antibody. It was still. Subsequently, human VWf was eluted with 0.1 M glycine (pH 2,2), and the obtained The resulting solution was neutralized to pH 7.4 and then frozen. In this way, the affinity spirit As a result of analyzing the prepared materials on SDS polyacrylamide gel, it was found that mainly 22 It was found that it consisted of No. 0 K unit. 22011 block depending on the preparation material. Those containing the 7-minoterminal 130 K proteolytic fragment of the unit are also rare. Ta.

Next, KabiVitrium A B’s Coatest■ractor The activity of factor (■) was determined using method (2).

Looking at Figure 1, it can be seen that in the presence of vWf, the amount of factor ■ accumulated increases. Recognize. Activity level of factor ■ accumulated using culture medium not containing human VWf human VWf 10 μg/d (human VWf R degree in plasma is 10 μgad Human V The activity level of Factor ■ obtained when secreted into a culture medium containing Wf is approximately It turned out to be 3 times more expensive.

The dose-effect relationship between human vWf concentration and Factor 2 activity is shown in FIG. v As a result of a quantitative experiment of factor ■ that accumulates in a culture solution containing Wf, it was found that The activity of factor ■ obtained also in the presence of 5 μg/rIdl human VWf It was found that the amount increased by three times.

As can be seen in Figure 3, the activity level of factor ■ obtained by the presence of human VWf Bell increase is not a phenomenon specific only to clones. Clone Aat 2.10 ．． Similarly, an increase in the activity of factor (2) was observed in Aat 2.27.

Finally, when factor ■ is affinity-purified from the culture medium without adding human vWf, , it was also found that calf vWf was also purified at the same time. This is affinity purified This is a judgment based on the results of amino acid sequence analysis of the main contaminants of Factor ■. . Based on the above data, 10% fetal bovine serum contains factor ■/bvWf. There appears to be sufficient concentration of calf VWf to produce the complex. But that However, adding human VWf to the culture medium increases the amount of factor ■ produced. There is no difference. The reason for this is that only the subgroup consisting of multimers has the factor ■. The concentration of this type of multimer is 10% in fetal bovine serum, which makes it difficult to bind. This seems to be because it is lower than that of purified human VWf.

By the way, although several embodiments of the present invention have been shown so far, the basic structure of the present invention Other embodiments are shown in which modifications are made to increase the utility value of the method and configuration of the present invention. Needless to say, you can do that too. Therefore, the scope of the present invention is defined by the examples above. It should be understood that the scope of the following claims rather than the specific embodiments shown It is.

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Claims (9)

[Claims] 1. The basic structure is to proliferate Factor VIII-secreting cells in the presence of vwf. Factor VIII yield increase method. 2. The claimed invention, wherein the amount of vwf present is in the range of 5 to 30 μg per ml of culture fluid. The method described in Scope 1. 3. Claim 1, wherein the amount of vwf present is 10 μg per ml of culture fluid. Method described. 4. The cell is characterized by a DNA sequence encoding the expression of Factor VIII. a unicellular mammalian host cell transformed with a recombinant DNA molecule that The A sequence is operably linked to an expression control sequence in the DNA molecule. The method described in box 1. 5. The cells contain a recombinant DNA sequence that also features a DNA sequence encoding vwf. The DNA sequence is transformed with an NA molecule, and the expression of the DNA sequence is controlled in the DNA molecule. 5. The method of claim 4, wherein the method is associated with a node array. 6. The host is cultured BMT10, BSC1, BSC40, COS1, COS7, C selected from HO cells and other mammalian and human cells, The method according to claim 4. 7. Claim 1, wherein the cell is a human cell line that secretes Factor VIII. The method described in section. 8. Host cells that secrete factor VIII are grown in medium containing vwf. After collecting the factor vIII/vwf complex, extract the factor from this complex. - A method for high-yield production of Factor VIII, comprising isolating Factor VIII. 9. A file produced by any of the methods set forth in claims 1 to 8. Vactor VIII/vwf complex.