Impact of the cell culture condition on the expression o

Investigators

Abstract

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 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 7.2 kb cDNA sequences are known. rFVIII has been manufactured from both intact cDNA and from mutated cDNA's lacking B domain. Highly purified rFVIII from cell-culture systems was introduced as replacement therapy in 1993 and was shown to have a good safety record. However, similarly to the plasma derived product, about 20% of treated patients with rFVIII develop inhibitory antibodies, which is the most common and serious complication of the replacement therapy. Expression of the rFVIII in the mamalian cells is 2 to 3 orders of magnitude lower than the expression of other recombinant protein. This could be due to the large size of protein and complex modifications of FVIII molecule including proteolytic processing, N- and O-linked glycosylation, and tyrosine-sulfation. Studies has also suggested that rFVIII RNA is unstable and does not efficiently accumulates in the cells. All these factors make the rFVIII protein particularly sensitive to the variation in the production process that can results in the unpredictable heterogeneity of the product. The goal of our project is to simulate manufacturing deviations during the scaled down fermentation process and revealed its impact on the cell culture metabolism and structure of rFVIII. The B-domain deleted recombinant FVIII will be expressed in 293 human cells that have been adapted to serum free medium and suspension growth. Cell culture will be performed in a small-scale bioreactor, BIOFLO 110 that allows us to control parameters of cell culture. In the series of pilot experiments we have changed temperature and medium composition (concentration of serum) to modulate cell culture condition. We are in the process of profiling the mRNA expression of the 293 cells using the microarray technique. This allows to generate "snap-shots" of the changes in cDNA chip pattern in the activation or silencing of mRNA transcripts and to identify predictive genes that can be use as biomarkers. These biomarkers reveal the metabolic status of the cells following culture insult, including information pertinent to the intracellular synthesis of recombinant proteins. This allows us to correlate the character of the deviation in the cell culture process with metabolic changes occurring within cell substrate and activity of the recombinant protein. The DNA based microarray method in conjuction with routinely used in-process controls will improve the control of cell culture process and add a new dimension to the characterization of the quality of recombinant plasma proteins.