Metabolic flux analysis of CHO cell metabolism in the late non‐growth phase

Abstract

Chinese hamster ovary (CHO) cell cultures are commonly used for production of recombinant human therapeutic proteins. Often the goal of such a process is to separate the growth phase of the cells, from the non-growth phase where ideally the cells are diverting resources to produce the protein of interest. Characterizing the way that the cells use nutrients in terms of metabolic fluxes as a function of culture conditions can provide a deeper understanding of the cell biology offering guidance for process improvements. To evaluate the fluxes, metabolic flux analysis of the CHO cell culture in the non-growth phase was performed by a combination of steady-state isotopomer balancing and stoichiometric modeling. Analysis of the glycolytic pathway and pentose phosphate pathway (PPP) indicated that almost all of the consumed glucose is diverted towards PPP with a high NADPH production; with even recycle from PPP to G6P in some cases. Almost all of the pyruvate produced from glycolysis entered the TCA cycle with little or no lactate production. Comparison of the non-growth phase against previously reported fluxes from growth phase cultures indicated marked differences in the fluxes, in terms of the split between glycolysis and PPP, and also around the pyruvate node. Possible reasons for the high NADPH production are also discussed. Evaluation of the fluxes indicated that the medium strength, carbon dioxide level, and temperature with dissolved oxygen have statistically significant impacts on different nodes of the flux network.

Responses of CHO-DHFR cells to ratio of asparagine to glutamine in feed media: cell growth, antibody production, metabolic waste, glutamate, and energy metabolism

“…The respiratory quotient of cells was constant in metabolic flux analysis, and the value was one (Sengupta et al 2011;Ferreira et al 2005;Duarte et al 2014;Ahn and Antoniewicz 2012). So, the specific rate of CO 2 production was equal to that of O 2 consumption.…”

“…Glutamine and asparagine are the two most consumed amino acids in mammalian cell culture. To achieve high-specific growth rate, cell density, and productivity, the culture should be fed with high levels of glutamine or asparagine (Wlaschin and Hu 2006;Jain and Kumar 2008;Lu et al 2013;Sengupta et al 2011). Specifically, glutamine and asparagine are mutually related to each other not only in transportation but also in metabolism (Wipf et al 2002;Huang et al 2007;Kobayashi et al 2015;Xu et al 2014).…”

Scale-down of CHO cell fed-batch cultures

“…These studies support that the increased NADH generation flux during the SI phase is mainly generated from a higher flux through the TCA cycle. However, Sengupta et al 72 and Templeton et al 73 showed by using isotopic labeling that the flux through both the oxidative PPP as well as through the TCA cycle increased during the non-growth phase. In addition, Ahn and Antoniewicz 60 reported a significant increase in oxidative PPP together with a constant TCA flux from the growth phase to the stationary phase.…”

“…At the moment, fed-batch is the most common industrial process for CHO cell cultures, due to its ease of operation and flexibility. Three different culture phases can normally be discerned in a fed-batch culture: an exponential growth phase, a stationary phase, and a death phase 60,71,72 . The transitions between these phases are caused by the changes in culture conditions (e.g.…”

“…Martínez et al 61 showed that a CHO cell line grown in a batch culture had a six times higher energy efficiency when consuming lactate as compared to the situation where lactate was produced. Using isotopic labeling in combination with metabolic flux analysis, Sengupta et al 72 and Templeton et al 73 both reported higher metabolic fluxes through the pentose phosphate pathway (PPP) and TCA cycle during the stationary phase as compared to the growth phase of CHO cell cultures. Furthermore, Templeton et al 73 showed that the high PPP and TCA activities are associated with peak mAb production.…”