A recombinant DNA Chinese hamster ovary (CHO) cell line which produces tissue-type plasminogen activator (t-PA) was cultivated continuously in suspension with a constant dilution rate of 0.5 day-1. The cultivation consisted of four phases with four different ammonium chloride concentrations (0, 2.5, 5, and 7.5 mM) in the feed medium, causing a reactor ammonium concentration of up to 8 mM. Cell growth was not inhibited by these high ammonium concentrations, as cell densities of around 2.3 x 10(6) cells mL-1 were established. In contrast, the production of t-PA was reduced under high ammonium concentration. The decrease in specific t-PA production could be due to either a negative ammonium influence on productivity or a limitation of medium components, e.g., amino acids. Cell metabolism was changed under high ammonium concentrations, seen most clearly by a decrease in specific ammonium production by a factor of 8 and an increase in specific alanine production of 30%.

A recombinant DNA Chinese hamster ovary (CHO) cell line that produces tissue-type plasminogen activator (tPA) was cultivated continuously in suspension with a constant dilution rate of 0.5 day with three different asparagine concentrations in the feed (0.05, 2.55 and 7.55 mM). The up-shift in asparagine concentration caused an up-shift in asparagine consumption [15.7 and 31.4 nmol (10(6) cells)-1 h-1] and intracellular concentration (2.19 and 18.7 mM). The up-shift was accompanied by an increased production of ammonium, glycine and alanine, and a metabolic shift whereby the cells began to produce aspartate and glutamate, which were consumed before the shift. The tPA production was reduced in the up-shift culture. This might be explained by ammonium inhibition, but alternatively by a surprising down-shift in the intracellular concentration of many amino acids, a down-shift that was not observed in the extracellular concentrations or consumption rates. For efficient physiological engineering of mammalian cells it is necessary to include both extracellular and intracellular measurements and to consider the transport into and out of the cells.

A high density hybridoma perfusion culture was established by separating and recycling cells from the product stream to the reactor using a simple external sedimentation-based separator-an inclined modified Erlenmeyer flask. After 3 weeks, when the optimal perfusion rate of 1.0 day-1 had been reached, viable cell density stabilized at around 10 x 10(6) cells ml-1, a level five times that obtained by simple batch culture. The efficiency of the separator was enhanced by cell flocculation. Specific antibody productivity, which was initially 0.4 micrograms 1 x 10(6) cells-1 h-1, decreased to half that value while cell density was increasing, but recovered to the initial level when the culture finally stabilized at a high cell density. During the final phase, when viable cell density and specific antibody production were high, there was a marked shift in metabolism. Consumption of the two most important substrates for energy generation, glucose and glutamine, caused their broth concentrations to decrease to 1.5 mM and 1 mM, respectively, from input medium concentrations of 25 mM and 10 mM, respectively. At the same time there was an increase in the specific production of glycine and aspartate, their broth concentrations reaching 1.5 mM and 0.02 mM, respectively. We suggest that this shift in metabolism results in enhanced production of ATP from glutamine. The specific glucose consumption and lactate production also indicate that there is a shift to more energy efficient metabolism. The mechanism whereby this leads to enhanced specific antibody production remains to be elucidated. Nevertheless, the combination of high cell density and enhanced productivity obtained with the present perfusion culture resulted in a high monoclonal antibody production-100 mg 1-1 d-1.

No abstract

When a transfected CHO cell, that produces tissue-type Plasminogen Activator, t-PA, was transferred from a medium based on 5% Fetal Calf Serum, FCS, to a medium based on 0.8% casein peptone with variable glutamine and asparagine content, it was observed, that the growth of the cells changed from anchorage dependant to suspension culture giving more reproducible cultivations. In the FCS culture t-PA was unstable, observed as a decline in t-PA concentration after 250 h. This decline in t-PA concentration was not observed in the serum free culture, although there was a decline in productivity after 200 h. This change in production profile may be attributed to either no proteolytic attack from serum or by scavenging of proteolytic activities produced by the cells from the peptone peptides. Increasing amounts of glutamine/asparagine gave higher production of t-PA in synchrony with an increasing production of ammonia/ammonium ions. Ammonia inhibition does not seem to be a key factor for this cell line as seen with many others.