SUPER-CHO - A CELL-LINE CAPABLE OF AUTOCRINE GROWTH UNDER FULLY DEFINED PROTEIN-FREE CONDITIONS

Chinese Hamster Ovary (CHO) cells are widely used for the large scale production of recombinant biopharmaceuticals. Growth of the CHO-K1 cel l line has been demonstrated in serum-free medium containing insulin, transferrin and selenium. In an attempt to get autocrine growth in pro tein-free medium, DNA coding for insulin and transferrin production wa s transfected into CHO-K1 cells. Transferrin was expressed well, with clones secreting approximately 1000 ng/10(6)cells/24h. Insulin was poo rly expressed, with rates peaking at 5 ng/10(6)cells/24h. Characterisa tion of the secreted insulin indicated that the CHO cells were incompl etely processing the insulin molecule. Site-directed mutagenesis was u sed to introduce a furin (prohormone converting enzyme) recognition se quence into the insulin molecule, allowing the production of active in sulin. However, the levels were still too low to support autocrine gro wth. Further investigations revealed insulin degrading activity (presu mably due to the presence of insulin degrading enzymes) in the cytopla sm of CHO cells. To overcome these problems insulin-like growth factor I (instead of insulin) was transfected into the cells. IGF-1 was comp letely processed and expressed at rates greater than 500 ng/10(6)cells /24h. In this paper we report autonomous growth of the transfected CHO -K1 cell line expressing transferrin and IGF-1 in protein-free medium without the addition of exogenous growth factors. Growth rates and fin al cell densities of these cells were identical to that of the parent cell line CHO-K1 growing in insulin, transferrin, and selenium supplem ented serum-free media.