Development of a fed-batch culture process for enhanced production of recombinant human antithrombin by Chinese hamster ovary cells

Kuwae, Shinobu; Ohda, Toyoo; Tamashima, Hiroshi; Miki, Hajime; Kobayashi, Kaoru

doi:10.1263/jbb.100.502

Cited by 42 publications

(28 citation statements)

References 33 publications

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“…It can be noticed that the pH decrease has been already shown to decrease glucose uptake and to promote the cell oxidative metabolism (Yoon et al 2005;Kuwae et al 2005;Burky et al 2007). The pH decrease occurring after 45 h in Erlenmeyer flask culture could have induced this metabolism shift.…”

Section: Glycolysismentioning

confidence: 99%

Combination of yeast hydrolysates to improve CHO cell growth and IgG production

et al. 2012

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Many studies underlined the great benefits of hydrolysates used as additives in animal free media on cell culture performances. However, to precisely define hydrolysate supplementation strategies, a deeper understanding of their effect on cell growth and protein production is required. In the present study, the effect of addition of one yeast extract (YE) and two yeast peptones (named YP.A and YP.B) in a chemically defined medium was first assessed on cell culture performances. Interestingly, specific effects were found depending on the degree of degradation of yeast hydrolysates. The YE at 1 g L -1 increased the maximal cell density by 70 %, while a mixture of YE (1 g L -1 ) and YP.A (4 g L -1 ) increased IgG production by 180 %. These conditions were then evaluated on the CHO cell kinetics all over cultures. Hydrolysates extended the cell growth phase in Erlenmeyer flask and increased the maximal growth rate in bioreactor up to 20 %. Cell growth stimulation induced by hydrolysates addition was linked with energetic metabolism improvement suggesting that they promote oxidative pathway. Furthermore, hydrolysates provided an additional source of substrate that supported cell growth despite glutamine limitation.

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Section: Glycolysismentioning

confidence: 99%

Combination of yeast hydrolysates to improve CHO cell growth and IgG production

et al. 2012

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“…Therefore, glucose and Gln should be maintained at low concentrations but carefully monitored to ensure that they are not consumed below limitation levels. Kuwae and Ohda (2005) also reported a similar phenomenon, that is, cell metabolism shifted from net lactate production to lactate consumption in late cultivation phases.…”

Section: Discussionmentioning

confidence: 62%

“…When there is sufficient Gln in the medium, Gln metabolism proceeds mainly through the a-ketoglutarate and alanine pathway. When Gln is restricted, Gln metabolism begins to switch to generate a-ketoglutaric acid and the ammonia pathway, since the ammonia pathway produces more ATP, thereby alleviating the decline in energy substrates (Kuwae and Ohda 2005;Paredes et al 1998). The co-consumption of lactate and Ala after 250 h of cultivation suggests that the decrease in residual glucose concentration caused a limited availability of pyruvate from the glycolytic pathway and then led to the simultaneous consumption of lactate and Ala. Lactate and Ala can be metabolized via pyruvate as a source of energy to yield ATP.…”

Section: Discussionmentioning

confidence: 99%

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Bioprocess development for the production of mouse-human chimeric anti-epidermal growth factor receptor vIII antibody C12 by suspension culture of recombinant Chinese hamster ovary cells

Deng

Wang

et al. 2011

Cytotechnology

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The mouse-human chimeric anti-epidermal growth factor receptor vIII (EGFRvIII) antibody C12 is a promising candidate for the diagnosis of hepatocellular carcinoma (HCC). In this study, 3 processes were successfully developed to produce C12 by cultivation of recombinant Chinese hamster ovary (CHO-DG44) cells in serum-free medium. The effect of inoculum density was evaluated in batch cultures of shaker flasks to obtain the optimal inoculum density of 5 9 10 5 cells/mL. Then, the basic metabolic characteristics of CHO-C12 cells were studied in stirred bioreactor batch cultures. The results showed that the limiting concentrations of glucose and glutamine were 6 and 1 mM, respectively. The culture process consumed significant amounts of aspartate, glutamate, asparagine, serine, isoleucine, leucine, and lysine. Aspartate, glutamate, asparagine, and serine were particularly exhausted in the early growth stage, thus limiting cell growth and antibody synthesis. Based on these findings, fedbatch and perfusion processes in the bioreactor were successfully developed with a balanced amino acid feed strategy. Fed-batch and especially perfusion culture effectively maintained high cell viability to prolong the culture process. Furthermore, perfusion cultures maximized the efficiency of nutrient utilization; the mean yield coefficient of antibody to consumed glucose was 44.72 mg/g and the mean yield coefficient of glutamine to antibody was 721.40 mg/g. Finally, in small-scale bioreactor culture, the highest total amount of C12 antibody (1,854 mg) was realized in perfusion cultures. Therefore, perfusion culture appears to be the optimal process for small-scale production of C12 antibody by rCHO-C12 cells.

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“…Here we used glutamine limitation. The production processes should be run in the fed-batch mode, not only in order to increase productivity and to prevent the formation of inhibiting overflow metabolites (Butler 2005;Kuwae et al 2005;Xie and Wang 2006;Ansorge et al 2010;Gnoth et al 2007) but also to increase process reproducibility.…”

Section: Introductionmentioning

confidence: 99%

Increasing batch-to-batch reproducibility of CHO cultures by robust open-loop control

et al. 2010

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In order to guarantee the quality of recombinant therapeutic proteins produced in mammalian cell systems, the straightforward approach in industry is to run the processes as reproducible as possible. It is first shown that considerable distortions in the currently operated processes appear when the initial cell density deviates from its nominal value. Small deviations in the initial cell mass may lead to severe deviations from the desired biomass trajectory. Next, it is shown how to design a fed-batch production process in such a way that it is robust with respect to variations in the viable cell density. A simple open loop strategy is proposed for that purpose. Here we show for the first time at animal cell cultures (CHO cells) that by means of an appropriate glutamine feed rate profile F(t), which keeps the specific growth rate of the cells on a predefined value below its maximal value while maintaining the viabilities on a high level, the diverging viable cell count profiles change over into a robust converging set of profiles. The CHO cells used to validate the procedure could be focused to any specific growth rates below l max .

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Development of a fed-batch culture process for enhanced production of recombinant human antithrombin by Chinese hamster ovary cells

Cited by 42 publications

References 33 publications

Combination of yeast hydrolysates to improve CHO cell growth and IgG production

Combination of yeast hydrolysates to improve CHO cell growth and IgG production

Bioprocess development for the production of mouse-human chimeric anti-epidermal growth factor receptor vIII antibody C12 by suspension culture of recombinant Chinese hamster ovary cells

Increasing batch-to-batch reproducibility of CHO cultures by robust open-loop control

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