Chinese hamster ovary (CHO) cells, producing human recombinant tissue plasminogen activator (tPA), were grown with mannose (5, 20 and 40 mM) instead of glucose at 31, 33 and 37 degrees C. The highest tPA concentration (1.5 mg l(-1) at 144 h of cultivation) and tPA specific production rate (47 ng 10(-6) cell h(-1)) were obtained at 31 degrees C and 40 mM mannose. Regardless of the temperature or mannose concentration used, an inverse relationship between the specific growth rate and tPA specific production rate was observed, suggesting that tPA production rate would be directly controlled by the growth rate.

The tPA production by CHO cells cultures conducted at different culture temperatures and mannose concentrations was studied. The aim of this work was to evaluate the effect of low culture temperatures and mannose concentrations (instead glucose) on cell growth and tPA production, establishing an possible relationship between the growth rate and the tPA synthesis. CHO TF 70R cell line (Pharmacia & Upjohn) was grown in BIOPRO1 (Lonza, Belgium) medium. Batch cultures were performed in spinner flasks (150 mL) with 50 mL of culture medium at different temperatures (31, 33 and 37 • C) and mannose concentrations (5, 20, and 40 mM). Number of viable cells, cell viability, and mannose concentration were determined according to described previously. tPA concentration was measured by a commercial ELISA kit. Specific cell growth rate (µ) and tPA specific production rate (q tPA ) were calculated using the integral of viable cells (IVC) and accumulate variation of the concentrations. The results showed similar maximum cell concentration at 33 and 37 • C when comparing cultures conducted at 20 and 40 mM mannose. However, the cell concentration was around of 2 times lower at 31 • C, demonstrating that cell growth is affected by lower temperatures. It is interesting to point out that cell viability before 96 h was higher than 90% for all cultures. Only a slight change in the maximum tPA concentration was obtained when temperature was reduced from 37 to 33 • C and an important increase (1.5-2.1 times) was produced at 31 • C. As expected, a decrease in the cultivation temperature determined a lower µ and by lowering the culture temperature from 37 to 33 or 31 • C (under all the mannose concentration tested) an increase in the q tPA was observed. In light of this observation, a possible link between µ and q tPA was determined. Thus, regardless of the culture conditions, a monotonic increase in the q tPA (from 20 to 47 ng 10 −6 cell h −1 ) was obtained when the µ decreases from 0.028 to 0.008 h −1 , indicating that µ influences the tPA biosynthesis rate whether if this is caused by mannose concentration or by culture temperature. Overall, the findings showed that a higher mannose concentration (40 mM) and lower culture temperature (31 • C) determined a significant increase in the tPA production. An inverse relationship between µ and q tPA is obtained if culture temperature or mannose concentration were varied, suggesting that tPA biosynthesis rate would be directly controlled by the growth rate.