Design of an efficient medium for insect cell growth and recombinant protein production

We report the development of a new serum-free medium based on the use of fa ctorial experiments. At first, a variety of hydrolysates were screened usin g a fractional factorial approach with High-Five cells. From this experimen t yeastolate ultrafiltrate was found to have, by far, the most important ef fect on cell growth. Furthermore, Primatone RL* was found to remarkably pro long the stationary phase of Sf-9 and High-Five cell cultures. The optimal concentrations for yeastolate and Primatone were determined to be 0.6 and 0 .5%, respectively, on the basis of a complete factorial experiment. This ne w medium, called YPR. supported good growth of both Sf-9 and High-Five cell s in batch cultures, with maximal densities of 5.4 and 6.1 x 10(6), cells/m l, respectively. In addition, both cell lines achieved good growth in biore actor batch culture and had a prolonged stationary phase of 3-4 d in YPR me dium compared to Insect-XPRESS medium. The ability of the new medium to sup port recombinant protein expression was also tested by infecting Sf-9 or Hi gh-Five cells at high density (2 x 10(6) cells/ml) with a baculovirus expre ssing secreted placental alkaline phosphatase (SEAP). The maximum total SEA P concentration after 7 d was about 43 IU/ml (58 mg/L) and 28 IU/ml (39 mg/ L) for High-Five and Sf-9 cells, respectively.