Elevated pCO₂ affects the lactate metabolic shift in CHO cell culture processes

Abstract

The shift from lactate production to consumption in CHO cell metabolism is a key event during cell culture cultivations and is connected to increased culture longevity and final product titers. However, the mechanisms controlling this metabolic shift are not yet fully understood. Variations in lactate metabolism have been mainly reported to be induced by process pH and availability of substrates like glucose and glutamine. The aim of this study was to investigate the effects of elevated pCO2 concentrations on the lactate metabolic shift phenomena in CHO cell culture processes. In this publication, we show that at elevated pCO2 in batch and fed‐batch cultures, the lactate metabolic shift was absent in comparison to control cultures at lower pCO2 values. Furthermore, through metabolic flux analysis we found a link between the lactate metabolic shift and the ratio of NADH producing and regenerating intracellular pathways. This ratio was mainly affected by a reduced oxidative capacity of cultures at elevated pCO2. The presented results are especially interesting for large‐scale and perfusion processes where increased pCO2 concentrations are likely to occur. Our results suggest, that so far unexplained metabolic changes may be connected to increased pCO2 accumulation in larger scale fermentations. Finally, we propose several mechanisms through which increased pCO2 might affect the cell metabolism and briefly discuss methods to enable the lactate metabolic shift during cell cultivations.

Perfusion cultures require optimum respiratory ATP supply to maximize cell‐specific and volumetric productivities

“…As NADH is oxidized to NAD + either via the formation of lactate or via oxidative phosphorylation, the relative use of the two reactions plays a crucial role setting the cellular carbon efficiency and the ATP generation. The finding is also reflected by the redox variable R (Brunner et al, 2018;Nolan & Lee, 2011) revealing a negative correlation between R and q mAb for each steady state ( Figure 6). The more NADH is produced in glycolysis and not transported into mitochondria for oxidative phosphorylation, the fewer antibodies are produced.…”

“…The latter is based on the variation of perfusion and bleed rates, which is one way to decouple substrate availability and cell density (Clincke, Mölleryd, Zhang, et al, ; Takuma et al, ). Possible improvements in metabolic efficiency including the switch from lactate production to consumption have only been previously analyzed in distinct fed‐batch phases using tools such as flux analysis (Brunner, Doppler, Klein, Herwig, & Fricke, ; Ivarsson, Noh, Morbidelli, & Soos, ; Mulukutla, Gramer, & Hu, ) and measurements of intracellular metabolite pools (Luo et al, ). Although beneficial effects of glucose limitation on the cellular phenotype have been described for perfusion processes, the influence on the underlying metabolism and the connection between adenosine triphosphate (ATP) supply and productivity has not yet been unravelled.…”

“…The latter is based on the variation of perfusion and bleed rates, which is one way to decouple substrate availability and cell density (Clincke, Mölleryd, Zhang, et al, 2013;Takuma et al, 2007). Possible improvements in metabolic efficiency including the switch from lactate production to consumption have only been previously analyzed in distinct fed-batch phases using tools such as flux analysis (Brunner, Doppler, Klein, Herwig, & Fricke, 2018;Ivarsson, Noh, Morbidelli, & Soos, 2015;Mulukutla, Gramer, & Hu, 2012) and measurements of intracellular metabolite pools (Luo et al, 2012).…”

Metabolic flux balance analysis during lactate and glucose concomitant consumption in HEK293 cell cultures

“…line measurement of metabolic cell activity, also supports the idea of a more active metabolism during cell growth in Phase P2_B when pH was not controlled. In other words, a concomitant consumption of glucose and lactate was detected in the second phase of non-pH-controlled cultures, whereas a sequential consumption of glucose and afterward lactate was observed in pH-controlled cultures.3.2 | Respiratory activity of isolated mitochondria in lactate containing buffer points out an alternative metabolic pathway of lactate transport into mitochondria Several authors have reported the consumption of lactate by mammalian cells(Altamirano et al, 2006, Brunner et al, 2017, Martínez et al, 2013, Mulukutla et al, 2012. This fact has extensively been described in the literature(Gagnon et al, 2011;Kuwae, Ohda, Tamashima, Miki, & Kobayashi, 2005;Zagari, Jordan, Stettler, Broly, & Wurm, 2013).…”

“…Also, Osman, Birch, and Varley (2001) demonstrated that lactate consumption in NS0 cells can be induced by reducing pH below 7.5, but in such conditions, growth rate and glucose uptake rate decreased. Recently, Brunner, Doppler, Klein, Herwig, and Fricke (2017) have shown lactate consumption in CHO cultures is a consequence of high CO 2 levels. However, our group has observed a different metabolic behavior regarding lactate production and consumption in HEK293 cultures (Liste-Calleja, Lecina, Lopez-Repullo, et al, 2015).…”