A novel autoregulated proliferation-controlled production process using recombinant CHO cells

Controlled proliferation bioprocesses have shown great enhancement of heter ologous protein production. This novel technology has been implemented here using a multicistronic expression unit encoding the product gene and a cyt ostatic cell-cycle-arresting gene (p27) under control of a single tetracycl ine-repressible (tet(off)) promoter. The strict genetic linkage of both gen es allows the dissection of the production process into a nonproductive gro wth phase (dicistronic expression unit repressed) followed by a proliferati on-inhibited production phase (dicistronic expression unit induced) when th e cells have reached an optimal cell density. Based on rapid degradation of the external repressible agents tetracycline (tet) and doxycycline (dox) i n the cell culture medium, we developed a self-regulated process for transi tion from the growth phase to the production phase in a fashion that is dep endent only on the starting cell population and the initial concentration o f the tetracyclines. With this process, no change in medium is required to accomplish the transition from growth to production phase. The two-phase bi oprocess achieved here by tet switch-controlled proliferation is reliable a nd allows a growth-arrested production phase of at least 7 days, during whi ch cells remain in a well-defined, highly viable physiological state and sh ow enhanced heterologous protein production. This Tet(SWITCH) process is re adily adaptable to a variety of industrial processes designed for productio n of difficult-to-express protein pharmaceuticals. (C) 1999 John Wiley & So ns, Inc.