|
One alternative to antihaemophilic factor VIII isolated from plasma, is the Recombinant Factor
VIII (rFactor VIII) manufactured by biotechnological methods.
The factor VIII protein need no longer be isolated from numerous donations of blood
or plasma, the heterogeneous initial material, but can be produced from the cells of
an individual and precisely characterized marnmalian cell line. This new technology
requires not only the design of an optimized and standardized manufacturing process but also a control system with more than 600 tests in order to guarantee maximum safety for the user. In 1984 the human factor VIII gene was first characterized (Gitschier, 1984).
A DNA-fragment (cDNA) containing the nucleic acid sequences necessary for
coding the factor VIII-protein was inserted in an expression vector.
By  applying biochemical methods the vector (pAIVIL 3p.8c1) which carries
the factor VIII gene was integrated into the chromosomes of the cells of a
hamster kidney cell line (BHK). This cell line has all prerequisites to
manufacture complex glycosilated proteins like human factor VIII and to
secrete it into the culture medium. The produced factor VIII (rFactor VIII)
has to be physiologically active. Cells that produce large amounts
of the factor VIII protein were adapted to industrial production scale.
The factor VIII producing R3-cell line which was derived this way
was
precisely characterized according to the inserted gene and
its safety (see  table 1). Afterwards small aliquots of the cells were frozen in liquid nitrogen and stored
as identical sources of cells for every Recombinant Factor VIII production campaign.
The vector contains the regulatory sequences of a promoter and a terminator that
enable the transcription of genetic information by the host cell. The dehydrofolate
reductase gene (DHFR) serves the selection of transfected cells.
|
