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Cell Culture Engineering

Animal cells are used largely in the production of proteins such as antibodies and other pharmaceutical products. With increasing demand for these products, there is likewise a parallel effort to find ways in which to inrease the culture efficiency and productivity. These efforts take form in many ways. Among these are attempts to develop better reactor control algorithms and the formulation of mathematical models that will sufficiently describe the cell culture process. These are hoped to give a better understanding of the nature and limitations of animal cell culture and will, in the process, enhance production.

Mammalian cells, under typical conditions, convert most of the glucose consumed into lactic acid, that has adverse effects on cell growth. The accumulation of lactate limits the maximum cell concentration achieved in cultures. Fed-batch cultures have been used to cultivate hybridoma cells where glucose was controlled and maintained at low levels. Dynamic nutrient feeding based on on-line measurement of oxygen uptake rate (OUR) was employed to control the feeding rate by estimating the metabolic demand of the cells. This strategy led to a significant reduction in lactic acid production.

The altered metabolic state of hybridoma cells could also be maintained in continuous cultures to give a high steady state cell concentration of 5x106 cells/ml. This concentration was achieved by altering the cellular metabolism in a fed-batch prior to switching to continuous mode. The strategy was used to achieve various distinct steady states with the molar ratio of lactate produced to glucose consumed (L/G) ranging from 0.03 to 0.5. In comparison, a control initiated from a batch culture had a L/G of 1.4 and yielded a cell concentration of only 1.5e6 cells/ml.

The different steady states were analyzed to demonstrate the differences in glucose and amino acid metabolism under different metabolic states. The specific uptake rates of glucose and amino acids were found to reduce by an order of magnitude with the reduction in L/G. For some amino acids, the specific rates were lower even at higher extracellular concentrations, indicating a possible down-regulation of the amino acid transporters. A few amino acid transporter activities were studied using radiolabeled substrates to analyze their characteristics under differing metabolism. Intracellular fluxes were also calculated using Metabolic Flux Analysis and were found to vary over the different metabolic states. Specifically, the fluxes around pyruvate were seen to redistribute indicating potential enzymatic regulation. To probe the energy metabolism, the activity of mitochondria was observed using a vital stain Rhodamine 123. A distinct difference was observed between the mitochondrial activity and distribution in cells of different metabolism. The specific rate of antibody production was found to be the same for all the metabolic states studied. These results demonstrate the potential of controlling cell metabolism, to reduce the metabolite production, in order to achieve high cell and product concentrations in mammalian cell cultures. The altered metabolic state could be maintained in continuous cultures, while increasing the feed nutrient concentration, to achieve 1.0e7 cells/ml.

Subsections:
Laser Probe for On-line Estimation of Cell Growth

Selected Recent Publications :

Zhou, W., Europa, L.F. and Hu, W. -S. (1998) "Controlling mammalian cell metabolism in bioreactors" Korean J. Microbiol. Biotechnol., 8, 1, 8-13

Current Graduate Students

Rashmi Korke B. Chem. Engg. University of Bombay 1997 korke@cems.umn.edu	Jongchan Lee B.S. Chemical Technology Seoul National University 1995 M.S. Chemical Technology Seoul National University 1997 jlee@cems.umn.edu	Mugdha Deshpande B. Chem. Engg. University of Mumbai 1999 mugdha@cems.umn.edu