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 .about.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.