The effects of elevated lactic acid concentration on the cell cycle kinetics of hybridoma cell growth and antibody production in batch culture were studied using conventional methods based on population-average data analysis and using flow cytometry based on single-cell data analysis. When 33 mM lactic acid was initially present, the true specific growth rate was reduced by 37% and the cell specific antibody production rate increased by a factor of 2.6 relative to a control culture with no additional lactic acid. DNA content distributions measured during balanced exponential growth were not affected by lactic acid concentration indicating lactic acid has a uniform effect on cell growth throughout the cell cycle. There was little or no effect on single-cell distributions of intracellular antibody content measured for the total population and for each cell cycle phase. The net rate of total antibody synthesis was found to be independent of specific growth rate. This implies that the balance of the total amount of antibody synthesized is shifted from cellular accumulation towards secretion when specific growth rate decreases. Our data predict that a maximum specific secretion rate of 2.7 pg per cell per h could be achieved if the specific growth rate was reduced to zero. The rates of secretion in the G1 and S phases increased with decreasing specific growth rate, while the rate of secretion in the G2 + M phase remained relatively constant. Under the assumptions that (a) at the fastest growth rate, secretion in the G1 phase is negligible and (b) the rate of synthesis increases exponentially as cells proceed from the S phase to the G2 + M phase, our data predict that for the slowest growth rate, the rate of secretion in G2 + M is approx. 3-times that in the G1 phase and 5-times that in the S phase.
Bibliographical noteFunding Information:
The authors wish to acknowledge the Graduate School of the University of Minnesota and the National Science Foundation for partial support of this work (grant BCS-9100385). SJK is the recipient of a National Science Foundation Graduate Fellowship. Miss Shirley Hwang assisted with the glucose, lactic acid, and total protein assays. The authors are grateful to Dr. G. Dunny of the Institute for Advanced Studies in Biological Process Technology at the University of Minnesota for the use of the microtiter plate reader.
- Cell cycle kinetics
- Flow cytometry
- Hybridoma cell culture
- Lactic acid
- Monoclonal antibody production