High viable cell concentration fed‐batch cultures of hybridoma cells through on‐line nutrient feeding

Abstract

A hybridoma cell line was cultivated in fed-batch cultures using a low-protein, serum-free medium. On-line oxygen uptake rate (OUR) measurement was used to adjust the nutrient feeding rate based on glucose consumption, which was estimated on-line using the stoichiometric relations between glucose and oxygen consumption. Through on-line control of the nutrient feeding rate, not only sufficient were supplied for cell growth and antibody production, but also the concentrations of glucose and other important nutrients such as amino acids were maintained at low levels during the cell growth phase. During the cultivation, cell metabolism changed from high lactate production and low oxygen consumption to low lactate production and high oxygen consumption. As a result the accumulation of lactate was reduced and the growth phase was extended. In comparison with the batch cultures, in which cells reached a concentration of approximately 2 x 10(6) cells/mL, a very high concentration of 1.36 x 10(7) cells/mL with a high cell viability (>90%) was achieved in the fed-batch culture. By considering the consumption of glucose and amino acids, as well as the production of cell mass, metabolites, and antibodies, a well-closed material balance was established. Our results demonstrate the value of coupling on-line OUR measurement and the stoichiometric relations for dynamic nutrient feeding in high cell concentration fed batch cultures.

Expression of anti‐apoptosis genes alters lactate metabolism of Chinese Hamster Ovary cells in culture

“…Numerous strategies have been devised to address the accumulation of excessive lactate build-up including (1) maintaining low medium glucose concentrations (Kurokawa et al, 1994;Xie and Wang, 1993;Zhang et al, 2004;Zhou et al, 1995), (2) feeding alternative sugars, including fructose (Martinelle et al, 1998, Altamirano et al, 2004Wlaschin and Hu, 2007), (3) partially knocking out lactate dehydrogenase (LDH) expression by homologous recombination or siRNA technology (Chen et al, 2001;Kim and Lee, 2007a); (4) over-expression of pyruvate carboxylase (Kim and Lee, 2007b); (5) use of dichloracetate (DCA), a pyruvate dehydrogenase (PDH) activator (via PDH kinase inhibition) (Stacpoole et al, 2003) and (6) oxamic acid, an LDH competitive inhibitor (Mothersill and Seymour, 1986). While the above strategies have been partially successful in reducing the lactate concentration, an alternative approach to consider is stimulating mitochondrial respiration in order to enhance culture performance.…”

“…4). For this reason, a number of researchers have developed process methodologies such as restricting glucose feed in order to minimize lactate accumulation in mammalian cell cultures (Kurokawa et al, 1994;Xie and Wang, 1993;Zhang et al, 2004;Zhou et al, 1995).…”

“…Previous researchers have detected lactate consumption in hybridoma, NS0 myeloma and CHO cultures (Burky et al, 2008;deZengotita et al, 2000;Pascoe et al, 2007;Zhou et al, 1995Zhou et al, , 1997. However, these cells typically exhibit lactate production in the exponential phase and transition to consumption in the stationary phases to suggest that lactate can represent both an intermediate by-product and a carbohydrate fuel source (Brooks, 1985;Burky et al, 2008).…”

The availability of glucose to CHO cells affects the intracellular lipid-linked oligosaccharide distribution, site occupancy and the N-glycosylation profile of a monoclonal antibody

“…Feeding strategies have been reported in the literature to reduce lactate production. Limited glucose supply is deliberately induced to moderately high cell density in order to prolong the productive stationary phase (Cruz et al, 1999;Zhou et al, 1995). However, this is complicated by the fact that nutrient deprivation has an adverse effect on cell growth, productivity and may even sabotage product quality.…”

Enhanced glycoprotein production in HEK-293 cells expressing pyruvate carboxylase

“…At the process level, the apparent deregulation of glucose and glutamine uptake rates when these nutrients are present in excess has led to the implementation of controlled feeding strategies to maintain low but non-limiting levels of the main nutrient concentrations (Europa et al, 2000;Gambhir et al, 1999;Kurokawa et al, 1994;Lee et al, 2003;Haggstrom, 1992, 1994;Omasa et al, 1992aOmasa et al, , 1992bSiegwart et al, 1999;Zhou et al, 1995). Such strategies have proven effective at reducing the overall yield of lactate on glucose, but require on-line monitoring of nutrient concentrations and automatic feed rate adjustment during the culture.…”