Temperature control of growth and productivity in mutant Chinese-hamster ovary cells synthesizing a recombinant protein

The use of a temperature switch to control the growth and productivity of temperature-sensitive (ts) mutants was investigated to extend the productive life span of recombinant Chinese hamster ovary (CHO) cells in batch culture. Bromodeoxyuridine was used at 39-degrees-C to select mutagenized CHO-K1 cells, which resulted in the isolation of 31 temperature-sensitive mutants that were growth inhibited at 39-degrees-C. Two of these mutants were successfully transfected with the gene for tissue inhibitor of metalloproteinases (TIMP) using glutamine synthetase amplification, and a permanent recombinant cell line established (5G1-B1) that maintains the ts phenotype. Continuous exposure to the nonpermissive temperature (npt) of 39-degrees-C led to a rapid decline in cell viability. However, a temperature regime using alternating incubations at 34-degrees-C and 39-degrees-C arrested the 5G1-B1 cells while retaining a high cell viability for up to 170 h in culture. The specific production rate of the growth-arrested cells was 3-4 times that of control cultures maintained at a constant 34-degrees-C over the crucial 72-130-h period of culture, which resulted in a 35% increase in the maximum product yield. Glucose uptake and lactate production both decreased in arrested cells. Flow cytometric analysis indicated that 5G1-B1 cells arrested in the G1 or G0 phase of the cell cycle, and no major structural damage was caused to these cells by the alternating temperature regime. These results demonstrate that growth-arrested ts CHO cells have increased productivity compared to growing cultures and maintain viability for longer periods. The system offers the prospect of enhancing the productivity of recombinant mammalian cells grown in simple batch fermentors. (C) 1993 John Wiley & Sons, Inc.