Shake flasks are widely used to culture microorganisms, but they do not allow for pH control without additional infrastructure. In the presence of a carbon source like glucose, culture pH typically decreases due to overflow metabolism and can limit the growth of microorganisms in shake flasks. In this study, we demonstrate the use of magnesium hydroxide-loaded pH managing hydrogels (m-pHmH) for in situ base release to counter the decrease in culture pH in shake flasks using Escherichia coli as a model organism, in both complex and mineral salts medium. Base release from m-pHmH is shown to increase with decreasing pH (22-fold increase in release rate from pH 8 to 5), thus providing feedback from culture pH. The addition of m-pHmH resulted in better pH maintenance and higher biomass yields of E. coli K12 in media containing glucose as a carbon source. The use of m-pHmH with additional buffer resulted in pH being maintained above 6.9 while pH decreases below 5 without m-pHmH. We demonstrate one application of such in situ pH management to increase the volumetric plasmid yield from E. coli in shake flask culture. In situ glucose release through a hydrogel to mimic fed-batch culture along with the addition of m-pHmH resulted in a 395 % increase in volumetric plasmid yield to 38 μg/ml in shake flask culture.
Inorganic phosphate (P ) is an essential ion involved in diverse cellular processes including metabolism. Changes in cellular metabolism upon long term adaptation to P limitation have been reported in E. coli. Given the essential role of P , adaptation to P limitation may also result in metabolic changes in animal cells. In this study, we have adapted CHO cells producing recombinant IgG to limiting P conditions for 75 days. Not surprisingly, adapted cells showed better survival under P limitation. Here, we report the finding that such cells also showed better growth characteristics compared to control in batch culture replete with P (higher peak density and integral viable cell density), accompanied by a lower specific oxygen uptake rate and cytochrome oxidase activity towards the end of exponential phase. Surprisingly, the adapted cells grew to a lower peak density under glucose limitation. This suggests long term P limitation may lead to selection for an altered metabolism with higher dependence on glucose availability for biomass assimilation compared to control. Steady state U- C glucose labeling experiments suggest that adapted cells have a higher pyruvate carboxylase flux. Consistent with this observation, supplementation with aspartate abolished the peak density difference whereas supplementation with serine did not abolish the difference. This supports the hypothesis that cell growth in the adapted culture might be higher due to a higher pyruvate carboxylase flux. Decreased fitness under carbon limitation and mutations in the sucABCD operon has been previously reported in E. coli upon long term adaptation to P limitation, suggestive of a similarity in cellular response among such diverse species. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:749-758, 2017.
Passaging and expansion of animal cells in lean maintenance medium could result in periods of limitation of some nutrients. Over time, such stresses could possibly result in selection of cells with metabolic changes and contribute to heterogeneity. Here, we investigate whether selection of Chinese Hamster Ovary (CHO) cells under glutamine limitation results in changes in growth under glutamine‐replete conditions. In glutamine‐limiting medium, compared to control cells passaged in glutamine‐rich medium, the selected cells showed higher glutamine synthetase (GS) activity and attained a higher peak viable cell density (PVCD). Surprisingly, in glutamine‐replete conditions, selected cells still showed a higher GS activity but a lower PVCD. We show that in glutamine‐replete medium, PVCD of selected cells was restored on (a) inhibition of GS activity with methionine sulfoximine, (b) supplementation of aspartate—without affecting GS activity, and (c) supplementation of serine, which is reported to inhibit GS in vitro. Consistent with the reported effect of serine, inhibition of GS activity was observed upon serine supplementation along with reduced growth of cells under glutamine‐limiting conditions. The latter observation is important for the design of glutamine‐free culture medium and feed used for GS‐CHO and GS‐NS0. In summary, we show that CHO cells selected under glutamine limitation have superfluous GS activity in glutamine‐replete medium, which negatively affects their PVCD. This may be due to its effect on availability of aspartate which was the limiting nutrient for the growth of selected cells in glutamine‐replete conditions.
BACKGROUND Shake flasks are widely used for evaluating mammalian cells in suspension. Lack of pH control can contribute to differences in culture performance between them and bioreactors. This study evaluates whether a previously reported in situ base releasing hydrogel (pHmH) to counter pH decrease can enable shake flask cultures to better mimic bioreactor cultures. RESULTS Compared with bioreactor culture, fed‐batch cultures of a recombinant Chinese hamster ovary (CHO) cell‐line in shake flasks without pHmH showed a decrease in pH to 6.6, accompanied by 40, 60 and 22% lower peak cell density, lactate accumulation, and immunoglobulin G (IgG) titer, respectively. Use of pHmH allowed shake flasks to maintain pH above 6.8 and reduced this difference to 20, 30, and 15%, respectively, thus enabling culture performance in shake flasks to better mimic the bioreactor. IgG glycosylation profiles were similar in identically fed cultures across all three platforms. Application of pHmH hydrogel during clone screening was evaluated by comparing correlation between titers for five recombinant CHO clones in bioreactors and shake flasks with and without pHmH; a higher correlation was found in shake flasks with pHmH than without. CONCLUSION In situ base release through hydrogel can allow identically fed fed‐batch cultures in shake flasks to better mimic cell growth, lactate accumulation and IgG titers in bioreactors, without additional infrastructure. © 2018 Society of Chemical Industry
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