No abstract

Medium optimization is an important strategy that can lead to several fold increase in the production of proteins in cell culture. However, the usual methods of medium optimization are complex and time consuming. Urokinase is a widely employed thrombolytic drug for the treatment of stroke. We describe here medium optimization for maximizing urokinase production by HT-1080 cells using supplementation of specific amino acids. The new specifically designed method resulted in 240 % increase in urokinase productivity.

in Wiley InterScience (www.interscience.wiley.com).Urokinase was produced in a hollow fiber reactor using HT-1080 human fibrosarcoma cells. External modulation comprised replenishing of the medium in the extracapillary space, reducing the serum concentration in the extracapillary space from 10% to 2% and increasing flow rate of the circulating medium in the intracapillary space from 20 to 80 mL/min, each according to a specific protocol. More than sixfold increase was observed in the cumulative urokinase production for two and three medium replenishing modulations of the extracapillary space. After 15 days of continuous operation, the highest cumulative urokinase obtained was 1.63 Â 10 6 PU/mL. SDS-PAGE and zymogram study established that the urokinase obtained was in the high molecular weight range of 54 kDa. The effect of external modulation on cumulative urokinase production was visualized as trajectories with respect to the ratio of lactic acid production rate (LPR) to the glucose uptake rate (GUR). The collective external modulation data showed two separate physiological regions in the cumulative urokinase vs. LPR/GUR plane. The HT-1080 cells exhibited two distinct morphologies in these regions that may be related to acidosis and metastasis. These regions also correspond to low and high urokinase productivity.

The effect of culture temperature on urokinase production by HT-1080 cell line was studied in batch culture and hollow fiber reactor. Small-scale t-flask experiments revealed that urokinase production could be enhanced and media utilization could be reduced by lowering the culture temperature in production phase. Urokinase production was scaled up using a hollow fiber perfusion reactor system. Temperature of culture was maintained at the physiological 37 °C during growth phase that extended up to 12 days in hollow fiber bioreactor. Subsequently, in the production phase, culture temperature was lowered to 34 °C. Decrease in culture temperature resulted in a significant increase in urokinase production. Proteolytic degradation and inhibition was also minimized. The medium utilization rate was decreased at lower temperature, and hence, a higher economy of production could be obtained.