MXPA98003051A - Preparation of recombinant factor viii in a protei free medium - Google Patents

Abstract

The present invention relates to the production of recombinant Factor VIII in relatively large amounts, on a continuous basis, from mammalian cells in the absence of any animal derived protein, such as albumin, by culturing the cells in a medium protein-free supplemented with polyol copolymers, preferably in the presence of trace metals such as copper. In highly preferred embodiments, the medium includes a polyglycol known as Pluronic F-68, copper sulfate, ferrous sulfate / AEDT complex and trace metal salts such as manganese, molybdenum, silicon, lithium and

Description

PREPARATION OF RECOMBINANT FACTOR VIII IN A PROTEIN FREE MEDIUM FIELD OF THE INVENTION The present invention relates generally to the manufacture of recombinant Factor VIII and specifically to the manufacture of recombinant Factor VIII in a serum or protein free medium. BACKGROUND OF THE INVENTION Hemophilia A is a recessive genetic disorder linked to the X chromosome that is due to a defective or deficient Factor VIII molecule, causing an orographic tendency. To control bleeding episodes, hemophiliacs are treated with Factor VIII. Historically, Factor VIII has been isolated from human blood plasma. However, therapy with plasma-derived Factor VIII has been associated with the transmission of several human viral diseases, such as hepatitis and the human immunodeficiency virus. With the advent of recombinant DNA technology, the structure of Factor VIII and its gene have been elucidated. The transcription product of the gene, which is derived from 26 exons, is a messenger RNA molecule of ~ 9000 bases in length, which codes for a large protein of 2351 amino acids. Studies REF: 27977 The structural effects of Factor VIII indicate that it is a glycoprotein that contains a significant number of carbohydrate residues. The cDNA encoding Factor VIII has been cloned and stably expressed in lactating hamster kidney cells (BHK-21) and in Chinese hamster ovary (CHO) cells. Commercial processes have been developed to produce recombinant Factor VIII for the treatment of hemophilia A. Recombinant Factor VIII is currently manufactured by genetic engineering processes in mammalian cells, thus obviating the use of plasma and minimizing any possible risk of transmission viral. Amplification of the gene has been the method to be selected to derive high production cell lines for therapeutic proteins. The amplification strategy involves ligating a transcriptional unit that codes for the desired protein to an amplifiable marker, such as dihydrofolate reductase. Then, transection techniques are applied to transfer the DNA vector to the recipient cells. Cell populations are selected for their increased resistance to the selected drug, such as methotrexate. The establishment of a clone of stable cells is achieved by limiting the dilution of the cloning. These clones Cells are subsequently adapted to a serum-free production medium and their production of the desired protein is recorded. For labile proteins such as Factor VIII, human albumin has been added as a stabilizer during the preparation and purification procedures. Although albumin is subjected to a stage of viral inactivation by pasteurization, it would be ideal if Factor VIII could be manufactured in complete absence of blood proteins from humans and animals. Now it has been found that this is possible using a new cell culture medium. The details are described below. BRIEF DESCRIPTION OF THE INVENTION The method for the continuous production of relatively large amounts of recombinant Factor VIII (rFVIII) from mammalian cells, in the absence of any plasma protein derived from animals or humans, comprises the culture of mammalian host cells. in a protein free medium supplemented with a polyol polymer, such as Pluronic F-68. The preferred medium includes copper sulfate, a complex of ferrous sulfate / AEDT (ethylenediaminetetraacetic acid) and salts of trace metals such as manganese, molybdenum, silicon, lithium and chromium.

DETAILED DESCRIPTION OF THE INVENTION Recent advances in recombinant protein expression technology have made possible the production of proteins in large quantities in mammalian cells. Suitable host cells for the production of Factor VIII include cell lines such as lactating hamster kidney cells (BHK)., Chinese hamster ovary cells (CHO) and human embryonic kidney cells (HEK). Particularly preferred are lactating hamster kidney cells, specifically those transfected with a gene capable of directing Factor VIII expression, as described in Wood et al. (1984) (including derivatives such as clonal variants and progeny thereof). Such cell line has been deposited in the North American Type Culture Collection (ATCC) and has been assigned the access number ATCC CRL-8544. The desired host cell line carrying the Factor VIII gene is typically adapted to grow as a suspension culture in a protein-free production medium which is supplemented with lipoprotein. The basal medium selected to cultivate the host cell line is not determinant for the present invention and can be any or a combination of those known in the art which are suitable for culturing mammalian cells. Commercially available media such as Eagle's medium modified by Dulbecco, Ham's medium D-12, Eagle's minimal essential medium and medium ROMI-1640, and the like. The addition of growth factors such as recombinant insulin is conventional in the art. Due to the labile nature of Factor VIII, the productivity of genetically engineered host cells is severely reduced under protein-free conditions. Human serum albumin is commonly used as a serum-free culture supplement for the production of recombinant proteins. Human serum albumin serves many functions, including: (1) as a vehicle for fatty acids, cholesterol and lipophilic vitamins, steroid hormones and growth factors; (2) as a protective agent against damage due to cutting forces; (3) as a regulator for pH changes; and (4) as a regulator for changes in osmotic pressure. Another critical function of albumin is probably to protect labile proteins such as Factor VIII from proteolysis, serving as a substrate for proteases. The impurities present in the preparations of Albumin may also contribute to the stabilizing effect thereof. Factors such as lipoprotein (Chan, 1996) have been identified as a replacement for human serum albumin, for the production of recombinant Factor VIII under serum-free conditions. The attempt to develop an albumin-free production medium derived from human plasma led to the invention described herein, a protein-free basal medium for the production of Factor VIII. The preferred medium consists of Dulbecco's minimal essential medium and Ham's F-12 medium (50:50, by weight) supplemented with recombinant insulin (Nucellin, Eli Lilly) at μg / ml and FeS04-AEDT (50 μM). With the exception of Factor VIII production, BHK cells genetically engineered grow well in this protein-free basal medium. Surprisingly, the addition of a polyol, such as Pluronic F-68, had no effect on growth but enhanced the specific productivity of BHK cells for Factor VIII. Incidentally, the addition of copper sulfate further increases the production of Factor VIII. Likewise, the inclusion of a trace metal panel, such as manganese, molybdenum, silicon, lithium and chromium, causes - - additional increases in the production of Factor VIII. Then a continuous process for the production of Factor VIII was developed, under conditions free of proteins derived from human plasma. Additional information can be found regarding the use of Pluronic polyols in Papoutsakis 81991) and Schmolka (1977). Pluronic F-68, a polyglycol (BASF, Wyandot) is commonly used to prevent the presence of foam in shaking cultures and to protect cells from shearing stress and damage caused by bubbles in bubbling cultures. Pluronic F-68 is a nonionic block copolymer with an average molecular weight of 8400, consisting of a central block of poly (oxypropylene) (20% by weight) and poly (oxyethylene) blocks at both ends). Extensive research on the function of Pluronic F-68 indicates that it acts as a surfactant and prevents damage to cells by allowing cells to move away from the bubbles formed in bioreactors during agitation or bubbling. However, several researchers have observed beneficial effects of Pluronic F-68 on growth under culture conditions in which agitation is minimal (Mizrahi, 1975, Murhammer and Goochee, 1990). The copurification of lipids with Pluronic F-68 during the purification of the product, provides anecdotal evidence that the Pluronic polymer can replace albumin not only as a surfactant, if not that also acts as a vehicle for lipids. Pluronic F-68 can also prevent cells with membrane damage from dying before they can make repairs, possibly by direct intercalation in the membrane. The function of Pluronic F-68 acting as a regulator of metal ions is completely unknown. Although there are reports that Pluronic F-68 in culture media can increase volumetric productivity, the mechanism of action seems to be the maintenance of cell viability (Schneider, 1989; Qi, 1996). As far as is known, this is the first time that Pluronic F-68 has been considered to increase the specific production of a particular protein product. Since the viability and growth rates are comparable in the present system with and without Pluronic F-68, maintenance of cell viability can not be the mechanism of action of Pluronic F-68 in the system herein. However, the effect of the addition of Pluronic F-68 is immediate and drastic, whatever its mechanism. It is anticipated that a range of other polyols It could have similar effects. Such other polyols include nonionic block copolymers of poly (oxyethylene) and poly (oxypropylene) having molecular weights ranging from about 1000 to about 16,000. In addition to conventional suspension culture techniques such as shake flasks, spin flasks and rotating bottles, the method of the present invention is also applicable for use with perfusion and in batches of bioreactors. After culturing the host cells, Factor VIII can be recovered from the medium used by normal methodologies, such as ultrafiltration or centrifugation. If desired, the recovered Factor VIII can be purified, for example, by ion exchange or by size exclusion chromatography, immunoaffinity or metal chelate chromatography, and the like. As used herein, the term "protein-free medium of animal or human origin" is a cell culture medium that is free of any protein that has been derived from a human or animal. Proteins that are isolated from animals or humans inherently have the risk of introducing viral contamination. The goal of a protein-free medium of human or animal origin, then, - lo ¬ is to eliminate or at least greatly reduce the risk of viral transmission. Example 1: Lactating hamster kidney cells (BHK-21) transfected with a gene capable of directing Factor VIII expression were obtained from Genentech, Inc., South San Francisco, California, E.U.A. The cell line was prepared in the manner described in Wood et al. (1984) and was deposited in the North American Type Culture Collection and given accession number ATCC CRL-8544. A clonal variant of this cell line was also obtained from Genentech, Inc., and used in all the Examples. BHK-21 cells containing the gene encoding Factor VIII were grown in suspension in flasks using a serum-free basal medium containing the following: Ham F-12 medium and Dulbecco minimum essential medium (50:50). , by weight), Nucellin (recombinant insulin, 5-10 μg / ml), FeS04-AEDT (50 μM), and MgCl2 (15 mM). Cells were maintained and passaged at 48 hour intervals. The cells were centrifuged at 800 x g for 5 minutes, counted and reseeded at a density of 1 x 10 ml cells. Each flask contains 50 to 100 ml of fresh medium. The shake flasks were placed on a rotator, incubated at 37 ° C and kept in the form of suspension culture shaking gently at a speed of 90 and 110 r.p.m. The effect of a polyol such as Pluronic F-68 (0.1 ° a), hereinafter shown as F-68, and copper sulfate (50 nM) on the production of Factor VIII, was studied in the flasks. Factor VIII was quantified by a chromogenic assay. The test is commercially available in the form of a test kit known as Coatest VIII: C / 4 and is available from Baxter HealthCare Products. The cells were maintained by this procedure for 24 days. The activity of Factor VIII in each medium, determined by the Coatest VIII: C / 4 team, is shown in Table 1. TABLE 1 Average value of 36 samples ± standard deviation.

The production of Factor VIII of the cells was evaluated in a period of 24 days in the manner described above. The titration experiments showed that a 0.1% concentration is the optimal dose of Pluronic F-68. Increasing the concentration to 0.3% did not have an impact on the production of Factor VIII. Dose-response experiments revealed that an amount of 50 to 800 nM copper sulfate is optimal for the production of Factor VIII. As shown in Table 1, the addition of Pluronic F-68 alone or, preferably, in combination with copper sulfate, significantly increased the titer and specific productivity of BHK cells containing the gene encoding Factor VIII, under protein-free conditions. Example 2: To further optimize the production of Factor VIII under protein free conditions, trace metals were added to the protein-free production medium. Then, the production of Factor VIII was evaluated by the continuous shake flask culture system, in the manner described in Example 1 for 16 days. The data are shown in Table 2. In the absence of copper sulfate, the trace metals had no effect on the productivity of Factor VIII. See Table 2.

TABLE 2 The metals include CuS0 • 5H20 (50 nM), MnS04 (3 nM) Na2SiO-9H20 (1.5 μM), [NH4] 6M07O24 '4H20 (3 nM), CrK (S04) 2'4H20 (1.5 nM), and LiCl (236 nM). Example 3: The effect of trace metals and copper on the production of Factor VIII was further evaluated in a percussion fermentor. Two 1.5-liter trowelers were inoculated with the clonal variant of BHK cells at a density of 2 x 10 cells / ml using the basal medium described in Table 1. The fermenter was perfused at a rate of 0.5 liters / day. One fermentor was kept as control and the other was supplemented with copper and trace metals as those described in Table 2. The fermentors were maintained for 15 days with an average cell density of ~ 2-3 x 10 cells / ml. As shown in Table 3, the addition of Pluronic F- 68, copper and trace metals significantly increased the specific productivity of the BHK cells carrying the gene encoding Factor VIII, under protein-free conditions and continuous perfusion conditions. This production method can be easily adapted to larger fermenters (200 to 500 liters) equipped with cellular retention devices, such as settling tanks. TABLE 3 The above Examples are provided as a means of illustrating the present invention and are not intended to limit it, which will be defined by the claims. References: Bihoreau, N., et al. , Eur. J. Biochem. 222: 41-48 (1994) Chan, S.Y., U.S. 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 ai., Biotechnol. Prog. 6: 142-148 1990) Papoutsakis, E.T., Trends in Biotechnoloogy (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) Xu, D., et al. , China J. Biotech. 11: 101-107 (1995) Zhang, J., et al. , Biotechnol. 33: 249-258 (1994) It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention. Having described the invention as an antecedent, what is contained in the following is claimed as property. *****

Claims (15)

1. CLAIMS 1. A method for the production of recombinant Factor VIII from mammalian cells carrying the gene for it, characterized in that it comprises culturing the host cells of mammalian origin, in a medium free of proteins of animal or human origin, supplemented with polyols .
2. 2. The method according to claim 1, characterized in that the medium includes copper ions.
3. The method according to claim 1, characterized in that the polyol is Pluronic F-68 and is present in the medium at a concentration ranging from about 0.025 to about 0.2% by weight.
4. 4. The method according to claim 1, characterized in that the medium includes copper sulfate in an amount ranging from about 50 to about 800 nM.
5. 5. The method according to claim 2, characterized in that the manganese ions are present in an amount ranging from about 1.5 to about 4.5 nM.
6. 6. The method according to claim 2, characterized in that the ions that contain molybdenum are present in an amount ranging from about 1.5 to about 4.5 nM.
7. The method according to claim 2, characterized in that the silicon-containing ions are present in an amount ranging from about 75 to about 300 nM.
8. The method according to claim 2, characterized in that the chromium ions are present in an amount ranging from about 1.0 to about 4.0 nM.
9. 9. The method according to claim 2, characterized in that the silicon ions are present in an amount ranging from about 120 to about 480 nM.
10. The method according to claim 1, characterized in that the mammalian host cells are selected from the group consisting of lactating hamster kidney cells, human embryonic kidney cells and Chinese hamster ovary cells.
11. 11. A recombinant Factor VIII product prepared according to the method of claim 1, characterized in that the product is free of proteins of animal or human origin.
12. 12. A cell culture medium free of proteins of animal or human origin, for production of Recombinant Factor VIII, characterized in that it comprises a basal medium including a polyol.
13. 13. The medium according to claim 12, characterized in that it includes copper ions.
14. 14. The medium according to claim 13, characterized in that it includes at least one trace metal that is selected from the group consisting of manganese, molybdenum, silicon, chromium and lithium.
15. 15. The medium according to claim 14, characterized in that it includes insulin.