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

Abstract

Background: Optimization of chemically defined medium has been a critical way to produce monoclonal antibody. Usually, amount of glutamine was added into the feed medium, but a half of asparagine was added. Our study found that asparagine was important in the antibody production phase. Increasing the ratio of asparagine to glutamine in feed medium for enhancement of antibody production in CHO-DHFR cell culture would be an efficient way. Results:We optimized the total amount and the ratio of the two vital amino acids in feed medium to increase antibody production. In this work, we have demonstrated that feeding medium of high ratio between asparagine and glutamine (FB-H) can enhance the cell density after reaching the stationary phase. Moreover, FB-H was shown to improve cell maintenance, and increased the antibody production. The metabolic flux analysis proved that ratio of asparagine to glutamine had little influence on glycolysis. Furthermore, the TCA cycle of FB-H was enhanced by 20 % compared to that of low ratio of asparagine to glutamine (FB-L). And the energy metabolism of FB-H was 22.6 % higher than that of FB-L. For the later, lactate can be less produced in FB-H. Conclusions:We should improve the ratio between asparagine and glutamine in feed medium properly under the premise of no influence on cell growth to achieve high mAb producing goal.

Oxygen Uptake Rate Soft-Sensing via Dynamic kLa Computation: Cell Volume and Metabolic Transition Prediction in Mammalian Bioprocesses

“…However, all cells tended to alter their metabolic activity to a different state at a certain point, regardless of whether they were cultured in a dynamic or static process. In all fed-batch cultivations, we observed a clear effect of Gln consumption on the excretion of ammonia, Ala and Glu, as expected due to their direct connection to Gln metabolism (Doverskog et al, 1997; Zhang et al, 2016). A clear link was also evident between Asp and Glu (see Figure 7D).…”

“…The variations in the amino acid concentrations of glutamine (Gln), glutamate (Glu), asparagine (Asn), aspartate (Asp), and alanine (Ala) are of particular importance in mammalian cell culture (Zhang et al, 2016). Though Glu and Gln exhibited a similar trend over the course of the fed-batch for all experimental runs, Ala, Asn, and Asp progressed differently (see Appendix Figure 2).…”

“…In a recent study, the ratio of asparagine and glutamine was found to be important, to some extent, in terms of process performance (Zhang et al, 2016). However, glutamine was depleted and asparagine concentrations were sufficiently high throughout the process (Figure 7C) and did not exhibit significant dependence.…”

“…Stirred tank reactors (STRs) are currently the most widely used bioreactor type to cultivate aerobic organisms in suspension culture or on carriers. In aerobic upstream bioprocesses, the oxygen uptake rate (OUR) is crucial for cellular activity and a good indicator of changes in the metabolic state of the culture (Deshpande and Heinzle, 2004; Wahrheit et al, 2015), which can be induced by changing substrate availabilities (Toye et al, 2010; Niklas et al, 2011; Young, 2013; Zhang et al, 2016). Thus, in the context of implementing Quality by Design and Process Analytical Technology (QbD/PAT) in bioprocesses, the OUR could be an informative process indicator (Sommeregger et al, 2017).…”

Model Transferability and Reduced Experimental Burden in Cell Culture Process Development Facilitated by Hybrid Modeling and Intensified Design of Experiments

“…Unsupervised learning ( Figure 3B ), correlation to the target variables ( Figure 3C ), and whether available previous in-depth knowledge proved to be highly valuable for the initial selection of potential inputs, which displayed importance and were available for all investigated scales. This workflow enables the reduction from the initial 11 inputs down to 6, which were identified to be relevant in all performed DoEs: the two CPPs, the aspartate-to-glutamate ratio, glutamine, asparagine, and alanine, which are also confirmed by literature to be of high impact for CHO cultivations ( Carrillo-Cocom et al, 2015 ; Fan et al, 2015 ; Zhang et al, 2016 ; Ghaffari et al, 2020 ). Due to this procedure, the final hybrid model could be applied for both scales and is more generic and its performance more robust when presented with new data.…”