• 专利附录
  • 专利说明
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  • 引用文献
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  • 优先权
    • 专利摘要:
    In medium without protein to which the polyol copolymer has been added, the recombinant factor VIII is continuously continuously compared to mammalian cells by culturing the cells in the absence of protein derived from an animal such as albumin, preferably in the presence of trace metals such as copper. It can be manufactured in large quantities. In a very preferred embodiment, the medium contains polyglycol, copper sulfate, ferrous sulfate / EDTA complex known as Pluronic F-68, and salts of trace metals such as manganese, molybdenum, silicon, lithium and chromium. .
    公开号:KR19980081531A
    申请号:KR1019980013934
    申请日:1998-04-18
    公开日:1998-11-25
    发明作者:샴-유엔 찬；캐쓸린 해리스
    申请人:제임스에이.기블린；바이엘코포레이션；
    IPC主号:
    专利说明:

    Method for preparing recombinant factor VII in medium containing no protein
    The present invention relates generally to the preparation of recombinant factor VIII, and more particularly to the preparation of recombinant factor VIII in a medium free of serum or protein.
    Hemophilia A is an X-chain recessive hereditary disease that causes bleeding due to missing or defective Factor VIII molecules. Hemophilia patients are treated with factor VIII by controlling bleeding symptoms. Historically, factor VIII has been isolated from human plasma. However, treatment with plasma-derived factor VIII has been associated with transmission of several human viruses, such as hepatitis and human immunodeficiency virus.
    With the advent of recombinant DNA technology, the structure of human factor VIII and its genes has been revealed. Transcripts of genes derived from 26 exons are mRNA molecules of 9000 bases in length that encode large proteins of 2351 amino acids. Structural studies on factor VIII have shown that factor VIII is a glycoprotein containing a significant number of carbohydrate residues.
    CDNA encoding for factor VIII was cloned and stably expressed in young hamster kidney (BHK-21) and Chinese hamster ovary (CHO) cells. A method for commercially preparing recombinant factor VIII for the treatment of hemophilia A has been developed. Recombinant factor VIII is currently produced by genetically engineered mammalian cells, thus eliminating the need to rely on plasma and minimizing any possible risk for viral infection.
    Gene amplification was the method of choice for inducing high production cell lines of therapeutic protein. Amplification methods include linking a transcript encoding a protein of interest to an amplifiable marker, such as a dihydrofolate reductase. The transfection technique is then used to deliver the vector DNA to the host cell. Select cell populations with increased resistance to certain drugs, such as methotrexate. Stable cell clones are established by limiting dilution cloning. These cell clones are then adapted to serum-free production medium and the production of the desired protein is monitored.
    Human albumin was added as a stabilizer during the preparation and purification of labile proteins such as Factor VIII. Even if albumin is subjected to a viral inactivation step by bactericidal, it would be ideal if recombinant factor VIII could be prepared when human and animal serum proteins were completely absent. We have found that this is possible by using the novel cell culture medium. Details will be described later.
    In the absence of plasma proteins derived from humans or animals, a method of continuously producing a relatively large amount of recombinant factor VIII (rFVIII) from mammalian cells is a protein-free addition of a polyol polymer such as Pluronic F-68. Culturing the host cell of the mammal in a containing medium. Preferred media contain copper sulfate, ferrous sulfate / EDTA complex and salts of trace metals such as manganese, molybdenum, silicon, lithium and chromium.
    Recent advances in recombinant protein expression technology have made it possible to produce large amounts of protein in mammalian cells. Suitable host cells for the production of Factor VIII include cell lines such as young hamster kidney (BHK) cells, Chinese hamster ovary cells (CHO) and human fetal kidney (HEK) cells. Particularly preferred are young hamster kidney cells, especially those transfected with genes capable of engineering the expression of Factor VIII described by Wood et al. (1984), including derivatives such as clone variants and their progeny. This cell line was deposited with the American Type Culture Collection and assigned accession number ATCC CRL-8544.
    Desired host cell lines carrying the Factor VIII gene are typically suitable for growing in suspension culture in protein-free production medium with the addition of lipoproteins. The basal medium selected for culturing the host cell line is not critical to the present invention and may be any one or combination of those known in the art, suitable for culturing mammalian cells. Mediums such as Dulbecco's Modified Eagle medium, Ham's medium F-12, Eagle's Minimal Essential medium and RPMI-1640 medium are commercially available. Adding growth factors such as recombinant insulin is common in the art.
    Because of the susceptible nature of Factor VIII, the productivity of treated host cells decreases rapidly under protein-free conditions. Human serum albumin is commonly used as a serum-free culture aid in the preparation of recombinant proteins. Human serum albumin contains a number of carriers such as (1) carriers of fatty acids, cholesterol and fat-soluble vitamins, steroid hormones and growth factors, (2) protective agents against damage due to shear forces, (3) buffers against pH changes, and (4) osmotic pressure regulators. Has the function. Another important role of albumin is to protect prone proteins such as factor VIII from proteolysis, perhaps acting as a substrate for proteolytic enzymes.
    In addition, impurities present in the production of albumin may contribute to the stabilizing effect of albumin. Factors such as lipoproteins (Chan, 1996) are known to be able to replace human serum albumin for the production of recombinant factor VIII under serum-free conditions.
    The inventors' efforts to develop albumin free production media derived from human plasma have developed the present invention, ie, protein-free basal medium for the production of recombinant factor VIII. Medium consisting of modified Dulbecco's Minimal Essential medium and Ham's F-12 medium (50:50, weight ratio) supplemented with 10 μg / mol of recombinant insulin (Nucellin, Eli Lilly) and FeSO 4 · EDTA (50 μM) are preferred. Do. Except for the preparation of factor VIII, treated BHK cells grow well in the protein-free basal medium of the invention.
    Surprisingly, the addition of polyols such as Pluronic F-68 had no effect on growth, but improved the specific productivity of BHK cells for Factor VIII. As an unexpected finding, the addition of copper sulfate also improves the production of factor VIII further. In addition, the inclusion of trace metal salts such as manganese, molybdenum, silicon, lithium and chromium further increases factor VIII production. Subsequently, a continuous method was developed for producing factor VIII under protein-free conditions derived from human plasma. Further information regarding the use of pluronic polyols can be found in the literature of Papoutsakis (1991) and Schmolka (1977).
    Pluronic F-68 (manufactured by BASF), a polyglycol, is commonly used to prevent foam formation occurring in agitated cultures and to protect cells from shear stress and bubble damage in spawned cultures. . Pluronic F-68 is a nonionic block copolymer with an average molecular weight of 8400 consisting of poly (oxypropylene) (20 wt.%) Blocks at the center and poly (oxyethylene) blocks at both ends. The results of a thorough study of the role of Pluronic F-68 indicate that Pluronic F-68 acts as a surfactant to prevent cell damage by releasing bubbles formed in the bioreactor from the cells during stirring or sparging. appear. However, some researchers have found that Pluronic F-68 has a beneficial effect on growth under culture conditions with minimal shear forces (Mizrahi, 1975; Murhammer and Goochee, 1990). Co-purification of lipids with Pluronic F-68 during the purification of the product provides anecdotal evidence that the fluorogenic polymer can not only replace albumin as a surfactant, but also act as a carrier of lipids. In addition, Pluronic F-68 can prevent cells from dying against membrane damage before they can be repaired by direct insertion into the membrane. The role of Pluronic F-68 as a metal ion buffer is not entirely known.
    Although Floronic F-68 in the medium has been reported to increase volume productivity, the mechanism of action appears to maintain cell viability (Schneider, 1989; Qi, 1996). Certainly, it is the first time that Pluronic F-68 has shown to increase the specific production of certain protein products. Because of the similar viability and growth rate in our Pluronic F-68 containing and non-containing systems, maintenance of cell viability could be a mechanism of action of fluoric F-68 in our system. none. However, the effect of adding Pluronic F-68 is immediate and dramatic whatever the mechanism is.
    It can be expected that a series of other polyols will have a similar effect. Such other polyols include nonionic block copolymers of poly (oxyethylene) and poly (oxypropylene) having molecular weights ranging from about 1000 to about 16,000.
    The method of the present invention is also suitable for the use of perfusion and batch bioreactors in addition to conventional suspension culture techniques such as shake flasks, spinner flasks and roller bottle cultures. Following culturing of host cells, factor VIII can be recovered from the medium used by standard methods such as ultrafiltration or centrifugation. If necessary, the recovered Factor VIII can be purified by methods such as, for example, ion exchange or size exclusion chromatography, immuno-affinity or metal chelate chromatography.
    Human or animal protein-free medium used in the present invention is a cell culture medium containing no protein derived from a human or animal source. Proteins isolated from human or animal sources inherently carry the risk of causing viral contamination. Thus, the goal of human or animal protein-free medium is to eliminate or at least significantly reduce viral transmission.
    Example 1
    Young hamster kidney (BHK-21) cells transfected with genes capable of manipulating factor VIII expression were obtained from Genentech, Inc., in southern San Francisco, California, USA. Cell lines were prepared as detailed by Wood et al. (1984) and deposited in the American Type Culture Collection and received Accession Number ATCC CRL-8544. Clonal variants of this cell line were also obtained from Genentech and used in all examples.
    Ham's F-12 medium and Dulbecco's Minimal Essential medium (50:50, weight ratio), neuceline (recombinant insulin, 5-10 μg / ml), FeSO 4 EDTA (50 μM) and MgCl 2 (15 mM) Serum-free basal medium was used to culture BHK-21 cells containing genes encoding Factor VIII into suspension cultures in shake flasks. Cells were maintained and passed at 48 hour intervals. Cells were spun at 800 × g for 5 minutes, counted and reinoculated at a density of 1 × 10 6 cells per ml. Each flask contains 50-100 ml of fresh medium. The shake flask was placed on a rotator, incubated at 37 ° C. and maintained in suspension culture by gentle rotation at 90-110 rpm.
    The effect of polyol and copper sulfate (50 nM), such as Pluronic F-68 (0.1%) (represented by F-68 in the table below) on the production of Factor VIII, was tested in shake flasks. Factor VIII was quantified by chromogenic assay. The analyzer is commercially available as a test kit known as Coatest VIII: C / 4 and can be purchased from Baxter HealthCare Products. Cells were maintained following the procedure for 24 days. Factor VIII activity of each medium was measured in a Coatest VIII: C / 4 kit and is shown in Table 1 below.
    ConditionTiter (U / ml)Specific productivity (μU / cell / day)% Increase for basal medium Foundation badge0.15 ± 0.07 * 0.026 ± 0.0130 Basis Badge + F-68 (0.1%) ** 0.24 ± 0.040.052 ± 0.013200 Basal Medium + F-68 (0.1%) + Cu (50 nM) ** 0.42 ± 0.090.091 ± 0.013350* Mean ± standard deviation of 36 samples. Cells were monitored for Factor VIII preparation over a period of 24 days as described above. ** Titration results showed that 0.1% is the optimal amount of Pluronic F-68. Increasing concentration up to 0.3% had no effect on factor VIII preparation. Dose-response experiments showed that copper sulfate 50-800 nM was the optimal dose for the preparation of factor VIII.
    As shown in Table 1, the potency and specific productivity of BHK cells containing a gene encoding factor VIII, either by addition of Pluronic F-68 alone or preferably in combination with copper sulfate, under protein-free conditions Greatly improved.
    Example 2
    To further optimize the production conditions of factor VIII under protein-free conditions, trace metals were added to the protein-free production medium. The production of Factor VIII was then assessed by a continuous shake flask culture system for 16 days as described in Example 1. The data is shown in Table 2 below. In the absence of copper sulfate, trace metals had no effect on the productivity of Factor VIII. See Table 2.
    ConditionTiter (U / ml)Specific productivity (μU / cell / day)% Increase for basal medium + F-68 Basis Badge + F-680.46 ± 0.110.065 ± 0.0130 Basis Badge + F-68 + Cu0.53 ± 0.150.078 ± 0.026120 Basis Badge + F-68 + Cu + Metal * 0.73 ± 0.160.104 ± 0.026160* Metals include CuSO 4 · 5H 2 O (50 nM), MnSO 4 (3 nM), Na 2 SiO 3 · 9H 2 O (1.5 μM), [NH 4 ] 6 Mo 7 O 24 4H 2 O (3 nM ), CrK (SO 4 ) 2 .4H 2 O (1.5 nM) and LiCl (236 nM).
    Example 3
    The effect of trace metals and copper on the production production of factor VIII was further evaluated in a perfusion fermenter. Two 1.5 liter fermentors were inoculated with BHK clone variants at a density of 2 × 10 6 cells / ml using the basal medium described in Table 1. The fermentor was perfused at an amount of 0.5 liters / day. One fermentor was kept as a control and the other fermentor added the copper and trace metals listed in Table 2. Fermenters were maintained for 15 days at an average cell density of ˜2-3 × 10 6 cells / ml. As shown in Table 3, the addition of Pluronic F-68, copper and trace metals greatly increased the specific productivity of BHK cells with genes encoding factor VIII under protein-free conditions of continuous perfusion conditions. Improved. This production method can be easily applied to larger capacity fermenters (200 to 500 liters) equipped with cell holding devices such as settler.
    WorkSpecific productivity (μU / cell / day) Foundation badgeCu + metal One0.020.04 20.020.05 30.020.045 40.0180.05 50.020.05 60.0350.060 70.0250.055 80.020.04 90.0250.06 100.020.065 110.0250.070 120.0250.065 130.020.060 140.030.06 150.020.05
    The above examples are intended to illustrate the invention and should not be construed as limiting the invention, the invention is only limited by the claims.
    According to the present invention, novel cell culture media in which the serum proteins of human and animal, which are the most ideal conditions, are not present at all, can be used to continuously produce a relatively large amount of recombinant factor VIII. Thus, there is no need to rely on serum, reducing any risk for viral infection.
    references
    Bihoreau, N., et al., Eur. J. Biochem. 222: 41-48 (1994)
    Chan, S.Y., US Pat. No. 5,576,194 (1996)
    Eis-Hubinger, A.M., et al., Thromb. Haemost. 76: 1120 (1996)
    Mizrahi, A., J. Clin. Microbiol. 11-13 (1975)
    Murhammer, D. W., et al., Biotechnol. Prog. 6: 142-148 (1990)
    Papoutsakis, E.T., Trends in Biotechnology (Tibtech) 9: 316-324 (1991)
    Qi, Y-M., Et al., Cytotechnology 21: 95-109 (1996)
    Schmolka, I. R., J. Am. Oil Chemists' Soc. 54: 110-116
    Schneider, Y-J., J. Immunol. Meth. 116: 65-77 (1989)
    Wood, W., et al., Nature 312: 330-337 (1984)
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    Zhang, J., et al., Biotechnol. 33: 249-258 (1994)
    权利要求:
    Claims (15)
    [1" claim-type="Currently amended] Preparation of Recombinant Factor VIII from Mammalian Cells Comprising Recombinant Factor VIII Genes, comprising Cultivating Mammalian Host Cells Comprising Recombinant Factor VIII Genes in Human or Animal Protein-Free Medium Added Polyol Way.
    [2" claim-type="Currently amended] The method of claim 1 wherein the medium contains copper ions.
    [3" claim-type="Currently amended] The method of claim 1, wherein the polyol is Pluronic F-68 and is present in the medium at a concentration range of about 0.025 to about 0.2 weight percent.
    [4" claim-type="Currently amended] The method of claim 1, wherein the medium contains copper sulfate in an amount ranging from about 50 to about 800 nM.
    [5" claim-type="Currently amended] The method of claim 2, wherein the manganese ions are present in an amount ranging from about 1.5 to about 4.5 nM.
    [6" claim-type="Currently amended] The method of claim 2 wherein the molybdenum containing ions are present in an amount ranging from about 1.5 to about 4.5 nM.
    [7" claim-type="Currently amended] The method of claim 2, wherein the silicon containing ions are present in an amount ranging from about 75 to about 300 nM.
    [8" claim-type="Currently amended] The method of claim 2, wherein the chromium ions are present in an amount ranging from about 1.0 to about 4.0 nM.
    [9" claim-type="Currently amended] The method of claim 2 wherein the lithium ions are present in an amount ranging from about 120 to about 480 nM.
    [10" claim-type="Currently amended] The method of claim 1, wherein said mammalian host cell is selected from the group consisting of young hamster kidney cells, human fetal kidney cells, and Chinese hamster ovary cells.
    [11" claim-type="Currently amended] Recombinant factor VIII product prepared according to the method of claim 1, free of human or animal proteins.
    [12" claim-type="Currently amended] A human or animal protein-free cell culture medium for producing recombinant factor VIII, consisting of a basal medium containing a polyol.
    [13" claim-type="Currently amended] The medium according to claim 12, which contains copper ions.
    [14" claim-type="Currently amended] The medium according to claim 13, which contains at least one trace metal selected from the group consisting of manganese, molybdenum, silicon, chromium and lithium.
    [15" claim-type="Currently amended] The medium of claim 14 containing insulin.
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    同族专利:
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    EP0872487A2|1998-10-21|
    TR199800493A1|1998-11-23|
    NZ330183A|1999-08-30|
    DE69836164T2|2007-08-16|
    HU9800901A3|2001-08-28|
    HU224306B1|2005-07-28|
    CZ295049B6|2005-05-18|
    PL192072B1|2006-08-31|
    SK285684B6|2007-06-07|
    RU2222547C2|2004-01-27|
    UA66340C2|2004-05-17|
    CA2234215A1|1998-10-18|
    CA2234215C|2008-06-17|
    TW490469B|2002-06-11|
    US6171825B1|2001-01-09|
    BR9801092A|2000-01-11|
    AU748731B2|2002-06-13|
    ES2273380T3|2007-05-01|
    KR100496111B1|2005-09-09|
    JP4257685B2|2009-04-22|
    PL325862A1|1998-10-26|
    ZA9803234B|1998-10-22|
    DK0872487T3|2007-02-05|
    IL124123A|2006-07-05|
    HU9800901D0|1998-07-28|
    CZ115698A3|1998-11-11|
    EP0872487B1|2006-10-18|
    MY115583A|2003-07-31|
    US5804420A|1998-09-08|
    EP0872487A3|1999-10-27|
    CN1127518C|2003-11-12|
    ID20193A|1998-10-22|
    DE69836164D1|2006-11-30|
    SK49798A3|1998-12-02|
    BR9801092B1|2013-04-02|
    HK1018797A1|2004-07-23|
    AU6198298A|1998-10-22|
    JPH10295397A|1998-11-10|
    HU9800901A2|1999-05-28|
    CN1210866A|1999-03-17|
    PT872487E|2007-01-31|
    AT342921T|2006-11-15|
    CO4790112A1|1999-05-31|
    AR012451A1|2000-10-18|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    法律状态:
    1997-04-18|Priority to US08/844,714
    1997-04-18|Priority to US08/844,714
    1997-04-18|Priority to US8/844,714
    1998-04-18|Application filed by 제임스에이.기블린, 바이엘코포레이션
    1998-11-25|Publication of KR19980081531A
    2005-09-09|Application granted
    2005-09-09|Publication of KR100496111B1
    优先权:
    申请号 | 申请日 | 专利标题
    US08/844,714|US5804420A|1997-04-18|1997-04-18|Preparation of recombinant Factor VIII in a protein free medium|
    US08/844,714|1997-04-18|
    US8/844,714|1997-04-18|
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