engleski

An omics approach to rational feed Enhancing growth in CHO cultures with NMR metabolomics and 2D-DIGE proteomics

Expression of recombinant proteins exerts stress on cell culture systems, affecting the expression ofendogenous proteins, and contributing to the depletion of nutrients and accumulation of waste metabo-lites. In this work, 2D-DIGE proteomics was employed to analyze differential expression of proteinsfollowing stable transfection of a Chinese Hamster Ovary (CHO) cell line to constitutively express a heavy-chain monoclonal antibody. Thirty-four proteins of significant differential expression were identifiedand cross-referenced with cellular functions and metabolic pathways to identify points of cell stress.Subsequently, 1D-1H NMR metabolomics experiments analyzed cultures to observe nutrient deple-tion and waste metabolite accumulations to further examine these cell stresses and pathways. Fromamong fifty metabolites tracked in time-course, eight were observed to be completely depleted from theproduction media, including: glucose, glutamine, proline, serine, cystine, asparagine, choline, and hypox-anthine, while twenty-three excreted metabolites were also observed to accumulate. The differentiallyexpressed proteins, as well as the nutrient depletion and accumulation of these metabolites correspondedwith upregulated pathways and cell systems related to anaplerotic TCA-replenishment, NADH/NADPHreplenishment, tetrahydrofolate cycle C1 cofactor conversions, limitations to lipid synthesis, and redoxmodulation. A nutrient cocktail was assembled to improve the growth medium and alleviate these cellstresses to achieve a ∼75% improvement to peak cell densities.

CHO cells ; 2D-DIGE ; Proteomics ; NMR ; Metabolomics

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