Abstract
Purpose
The accumulation of carbon dioxide during large-scale culture of animal cells brings adverse effects, appropriate aeration strategies alleviate CO2 accumulation while improper reactor operation may lead to the presence of low CO2 partial pressure (pCO2) condition as occurs in many industrial cases. Thus, this study aims to reveal the in-depth influence of low pCO2 on Chinese Hamster Ovary (CHO) cells for providing a reference for design space determination of CO2 control with regard to the Quality by Design (QbD) guidelines.
Methods and results
The headspace air over purging caused the ultra-low pCO2 (ULC) where the monoclonal antibody production as well as the aerobic metabolic activity were reduced. Intracellular metabolomics analysis indicated a less efficient aerobic glucose metabolic state under ULC conditions. Based on the increase of intracellular pH and lactate dehydrogenase activity, the shortage of intracellular pyruvate could be the cause of the deficient aerobic metabolism, which could be partially mitigated by pyruvate addition under ULC conditions. Finally, a semi-empirical mathematical model was used to better understand, predict and control the occurrence of extreme pCO2 conditions during the cultures of CHO cells.
Conclusion
Low pCO2 steers CHO cells into a defective metabolic state. A predictive relation among pCO2, lactate, and pH control was applied to get new insights into CHO cell culture for better and more robust metabolic behavior and process performance and the determination of QbD design space for CO2 control.
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Abbreviations
- CHO:
-
Chinese hamster ovary
- CO2 :
-
Carbon dioxide
- pCO2 :
-
CO2 partial pressure
- vvm:
-
Air volume/culture volume/min
- TCA:
-
Tricarboxylic acid
- pHi:
-
Intracellular pH
- LDH:
-
Lactate dehydrogenase
- LDHi:
-
Intracellular LDH
- LC–MS:
-
Liquid chromatography–mass spectrometry
- qPCR:
-
Real-time quantitative PCR
- OPLS-DA:
-
Orthogonal partial least squares discriminant analysis
- qO2 :
-
Specific oxygen uptake rate
- PFK:
-
Phosphofructokinase
- mAb:
-
Monoclonal antibody
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Supplementary information
Table S1. Description of the UHPLC gradient method used in this study.
Table S2. Significantly differentiated metabolites with ULC group (0.4 vvm) versus control group (0.05 vvm).
Fig. S1 Effect of TCA cycle intermediates addition on the growth and mAb production of CHO cells during the fed-batch cultures under 1% (ULC) and 5% (Ctrl) pCO2 conditions
Fig. S2 Gaps of intracellular pH during the fed-batch cultures between ULC (0.4 vvm) and control (0.05 vvm) groups (a), and intracellular LDH activities with different buffer pH under 32°C (b).
Fig. S3 Effect of amiloride and lansoprazole on CHO cell proliferation.
Fig. S4 Regulation of the central carbon metabolism under normal pCO2 condition (a) and ultra-low pCO2 condition (b) (Key enzymes are labelled).
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Zhao, L., Wang, C., Wang, J. et al. Low CO2 partial pressure steers CHO cells into a defective metabolic state. Biotechnol Lett 45, 1103–1115 (2023). https://doi.org/10.1007/s10529-023-03404-9
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DOI: https://doi.org/10.1007/s10529-023-03404-9