Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1998-09-04
2001-01-09
Carlson, Karen Cochrane (Department: 1653)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S383000, C435S404000, C435S406000, C530S383000
Reexamination Certificate
active
06171825
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field
This disclosure is concerned generally with the manufacture of recombinant Factor VIII and specifically with the manufacture of recombinant Factor VIII in a serum or protein free medium.
2. Prior Art
Hemophilia A is an X-linked recessive genetic disorder that is due to a defective or deficient Factor VIII molecule, resulting in a hemorrhagic 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 transmission of several human viruses, such as hepatitis and human immunodeficiency viruses.
With the advent of recombinant DNA technology, the structure of human Factor VIII and its gene has been elucidated. The transcription product of the gene, which is derived from 26 exons, is a messenger RNA molecule of ~9000 bases in length, coding for a large protein of 2351 amino acids. Structural studies of Factor VIII indicate that it is a glycoprotein containing a significant number of carbohydrate residues.
The cDNA coding for Factor VIII has been cloned and stably expressed in baby hamster kidney (BHK-21) and Chinese hamster ovary (CHO) cells. Commercial processes have been developed to produce recombinant Factor VIII for treatment of hemophilia A.
Recombinant Factor VIII is currently manufactured by genetically engineered mammalian cells, thus obviating the reliance on plasma and minimizing any possible risk of virus transmission.
Gene amplification has been the method of choice to derive high production cell lines for therapeutic proteins. The amplification strategy involves the linking of a transcriptional unit encoding the desired protein to an amplifiable marker such as dihydrofolate reductase. Transfection techniques are then applied to transfer the vector DNA to recipient cells. Cell populations are selected for increased resistance to the drug of choice such as methotrexate. The establishment of a stable cell clone is accomplished by limiting dilution cloning. These cell clones are then adapted to a serum-free production medium and monitored for production of the desired protein.
For labile proteins such as Factor VIII, human albumin has been added as a stabilizer during the preparation and purification procedures. Although the albumin is subjected to a viral inactivation step by pasteurization, it would be ideal if recombinant Factor VIII could be manufactured in the complete absence of human and animal blood proteins. I have now found this is possible by using novel cell culture media. Details are described below.
SUMMARY OF INVENTION
The method for the continuous production of relatively large quantities of recombinant Factor VIII (rFVIII) from mammalian cells in the absence of any human or animal-derived plasma proteins comprises culturing the mammalian host cells in a protein-free medium supplemented with a polyol polymer such as Pluronic F-68 and copper ions. The preferred medium includes copper sulfate, a ferrous sulfate/EDTA complex, and the salts of trace metals such as manganese, molybdenum, silicon, lithium, and chromium. Alternatively we have also found that addition of copper ions alone (without polyol polymers) in the protein free medium may be used to enhance productivity of rFVIII in recombinant mammalian host cells, as described below under Additional Studies.
DETAILED DESCRIPTION OF THE INVENTION
Recent advances in recombinant protein expression technology have made possible the production of protein in large quantities in mammalian cells. Host cells suitable for Factor VIII production include cell lines such as baby hamster kidney (BHK) cells, Chinese hamster ovary (CHO) cells, and human embryonic kidney (HEK) cells. Particularly preferred are baby hamster kidney cells, specifically those transfected with a gene capable of directing the expression of Factor VIII as described in Wood et al. (1984) (including derivatives such as clonal variants and progeny thereof). Such a cell line has been deposited with the American Type Culture Collection and has been assigned the accession number ATCC CRL-8544.
The desired host cell line carrying the Factor VIII gene is typically adapted to grow as suspension cultures in a protein-free production medium which is supplemented with lipoprotein. The basal medium chosen for culturing the host cell line is not critical to the present invention and may be any one of, or combination of those known to the art which are suitable for culturing mammalian cells. Media such as Dulbecco's Modified Eagle Medium, Ham's Medium F-12, Eagle's Minimal Essential Medium, and RPMI-1640 Medium, and the like, are commercially available. 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 the 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 carrier for fatty acids, cholesterol and lipophilic vitamins, steroid hormones and growth factors; (2) as a protective agent against damages due to shear forces; (3) as a buffer for pH changes; and (4) as an osmotic pressure regulator. Another critical role of albumin is perhaps to protect labile proteins such as Factor VIII from proteolysis by serving as a substrate for proteases.
The impurities present in albumin preparations may also contribute to the stabilizing effect of albumin. 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.
Our attempt to develop a production medium free of human plasma-derived albumin led to the inventions of this disclosure, basal protein-free media for recombinant Factor VIII production. The preferred medium consists of modified Dulbecco's Minimum Essential Medium and Ham's F-12 Medium (50:50, by weight) supplemented with recombinant insulin (Nucellin, Eli Lilly) at 10 &mgr;g/ml, and FeSO
4
•EDTA (50 &mgr;M). With the exception of Factor VIII production, engineered BHK cells 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 the BHK cells for Factor VIII. Serendipitously, the addition of copper sulfate further enhances the production of Factor VIII. Also the inclusion of a panel of trace metals such as manganese, molybdenum, silicon, lithium, and chromium lead to further increases in Factor VIII production. A continuous process was then developed for Factor VIII production under human plasma-derived protein-free conditions. Further information regarding the use of Pluronic polyols can be found in Papoutsakis (1991) and Schmolka (1977).
Pluronic F-68, a polyglycol, (BASF, Wyandot) is commonly used to prevent foaming that occurs in stirred cultures, and to protect cells from shear stress and bubble damage in sparged cultures. Pluronic F-68 is a nonionic block copolymer with an average molecular weight of 8400, consisting of a center block of poly(oxypropylene) (20% by weight) and blocks of poly(oxyethylene) at both ends. Extensive research on the role of Pluronic F-68 indicates that Pluronic F-68 acts as a surfactant and prevents damage to cells by allowing the drainage of cells away from bubbles formed in the bioreactors during stirring or sparging. However, several investigators have noticed beneficial effects of Pluronic F-68 on growth under culture conditions in which shear is minimal (Mizrahi, 1975; Murhammer and Goochee, 1990). Co-purification of lipids with Pluronic F-68 during product purification provides anecdotal evidence that the Pluronic polymer may substitute for albumin not only as a surfactant, but may also act as a carrier for lipids. Pluronic F-68 may also prevent membrane damage from killing cells b
Chan Sham-Yuen
Harris Kathleen
Bayer Corporation
Carlson Karen Cochrane
Giblin James A.
Schnizer Holly
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