Hemophilia A is an inherited disorder caused by deficiency or abnormality of FVIII, which is required for the efficient clotting of blood. Human FVIII is synthesized as a 2351-amino acid single chain precursor protein containing homologous domains. The 256-kD single chain protein is proteolytically processed intracellularly to a metal ion-linked heterodimer of a 90- to 200-kD heavy chain and an 80-kD light chain. In vivo, FVIII circulates as an inactive cofactor that requires further proteolytic cleavage to exert its coagulation activity. The 186-kb gene encoding human FVIII and the 7.2-kb cDNA sequences are known. rFVIII has been expressed from both the intact cDNA and from mutated cDNA's lacking B-domain. Highly purified rFVIII from cell-culture systems was shown to have a good safety profile and was introduced as replacement therapy in 1993. Similarly to the plasma derived product, about 20% of treated patients with rFVIII develop inhibitory antibodies that is the most common and serious complication of the replacement therapy. rFVIII expression in mammalian cells containing amplified copies of FVIII cDNA is 2-3 orders of magnitude lower than the expression of other recombinant proteins. Only about one unit or 200ng/ml per 10 6 cells per day is produced by this method. This could be due to the large size and complex protein modifications of FVIII including proteolytic processing, N- and O-linked glycosylation, and tyrosine-sulfation, which are required for function. Studies have also suggested that FVIII mRNA is unstable and does not accumulate efficiently inside transfected CHO cells. Sequences in the coding regions of the FVIII gene that have inhibitory effect upon expression by blocking transcriptional elongation or by silencing transcription have been reported. A major portion of rFVIII is inefficiently transported from the endoplasmic reticulum to Golgi, leading to inefficient secretion from cells, which is somewhat improved by the coexpression of vWF. Additionally, the protein is very susceptible to proteolytic degradation and requires vWF for protection from proteases. All these factors make rFVIII molecule particularly sensitive to the variation in the production process that can result in the increased heterogeneity of the product. The goal of the proposed project is to simulate common manufacturing deviations which occur during fermentation and reveal its impact on structure and function of rFVIII. Initially, we will express rFVIII in CHO cells using laboratory scale bioreactor, BIOFLO 110 that controls precisely fermentation process. Changing temperature, media composition and number of passages will modulate conditions of cell culture. Studies of post-translational modifications and potency of the expressed rFVIII will allow us to evaluate the significance of implemented changes. The effect on CHO cell physiology will be evaluated by the studies of mRNA expression of selected processing enzymes. The FVIII """"""""knock-out"""""""" mouse, that models replacement therapy for Hemophilia A will be used to verify in vivo relevance of the structural heterogeneity of rFVIII protein.
