Kinetic study of hybridoma cell growth in continuous culture: II. Behavior of producers and comparison to nonproducers.

A hybridoma cell line, AFP-27-P, was cultivated in continuous culture under glucose-limited conditions. The viable cell concentration, dead-cell concentration, and cell volume all varied with the dilution rate. A model previously developed for a nonproducing clone of the same cell line, AFP-27-NP, was extended to describe the behavior of the cells. The relationship between the specific growth rate and glucose concentration is described by a function similar to the Monod model. A threshold glucose concentration and a minimum specific growth rate are incorporated; the model is meaningful only at glucose concentration and a minimum specific growth rate are incorporated; the model is meaningful only at glucose concentrations and specific growth rates above these levels. The relationship between the death rate and the glucose concentration is described by an inverted Monod-type function. Furthermore, the yield coefficient based on glucose is constant in the lower range of specific growth rates and changes to a new constant value in the upper range of specific growth rates. No maintenance term for glucose consumption is used; in the plot of specific glucose consumption rate vs. specific growth rate, the line intercepts the specific growth rate at a value close to the minimum growth rate. The productivity of antibody as a function of the specific growth rate is described by a mixed type model with a noon-growth-associated term and a negative-growth-associated term. The values for the model parameters were determined from regression analysis of the steady state data.

Comparison of growth kinetics of producing and nonproducing hybridoma cells in batch culture.

Several clones of nonproducing cells were isolated from a continuous culture of hybridoma cells, which were originally producing antibody. Their behavior was compared to that of the producing cells in batch culture. The growth kinetics of five out of six clones exhibited higher specific growth rate, higher yield of cell mass on glutamine, and lower yields of lactate and ammonium. The implications of the comparisons for growth of hybridoma cultures are discussed.

Kinetic study of hybridoma cell growth in continuous culture. I. A model for non-producing cells.

The kinetic behavior of a nonproducing hybridoma clone AFP-27-NP was investigated in continuous culture under glucose-limited conditions. A total of more than 21, 000 h of cultures were operated at dilution rates ranging from 0.01 to 0.06 h(-1). The viable cell concentrations, dead cell concentrations, and cell volumes all varied with the dilution rate. A steady-state model was developed based on the biomass concentration and the glucose concentration. The specific growth rate as a function of glucose concentration is described by a model similar to the Monod model with a threshold glucose concentration and a minimum specific growth rate incorporated; the model is meaningful only at glucose concentrations and specific growth rates above these levels. A death rate is included in the model which is described by an inverted Monod-type function of glucose concentration. The yield coefficient based on glucose is constant in the lower range of specific growth rates and changes to a new constant value in the upper region of specific growth rates. No maintenance term for glucose consumption was needed; in the plot of specific glucose consumption rate vs. specific growth rate, the line intercepted the specific growth rate axis at a value close to the minimum growth rate. The values for the model parameters were determined from regression analysis of the steady-state data. The model predictions and experimental results fit very well.

Cell volume measurement as an estimation of mammalian cell biomass.

Measurements of volume distributions and dry weight are made on hybridoma cells in culture. The volume of viable hybridoma cells is significantly larger than that of nonviable cells. During exponential growth, the volume of the viable hybridoma cells is found to be significantly larger than that during other stages of batch culture. Proportionality is found between the volume of the cells and their dry weight, indicating that the volume data can be used in conjunction with cell concentration data as a practical technique for indirect measurement of the biomass concentration present in a culture. Comparison of dry weight concentrations in continuous culture to predictions from the volume data shows very good agreement.

The loss of antibody productivity in continuous culture of hybridoma cells.

Two hybridoma lines, HB8178 and AFP-27, were grown in continuous culture. The concentrations of viable cells as well as those of various nutrients and metabolites reached steady-state values. The concentrations of either total IgG or antigen-specific antibody, however, failed to reach steady-state values but rather continuously decreased over the course of the cultures. The fraction of antibody-producing cells in the total cellular population also continuously decreased in the AFP-27 cultures. Comparison of the specific antibody productivity based on either the entire population or the antibody-producing fraction of the population over time suggests that the decrease in productivity was at least partly due to the occurrence of a nonproducing subpopulation of cells.

Effect of glucose on the cultivation of mammalian cells.

Most of the glucose consumed by mammalian cells cultivated in vitro is converted to lactate. The glucose consumption rate appears to be affected by glucose concentration. In a batch cultivation of cells the glucose concentration can be manipulated at a low level by programmed feeding of glucose. In such a culture the specific consumption rate of glucose and the fraction of glucose converted to lactate can be reduced. This reduced conversion rate of glucose to lactate appears to coincide with an increased oxygen uptake rate. A possible consequence of such programmed feeding of glucose is the increased oxidation of glutamine and the concurrent increased production of ammonium. For the cultivation of hybridoma cells high concentrations of ammonium and lactate can be growth inhibitory. It is suggested that the identification of the optimum cultivation conditions is necessary if such a programmed feeding is to be used to increase cell concentration and medium utilization efficiency.