| Summary: | Valuable recombinant therapeutic proteins are routinely produced from Chinese hamster ovary (CHO) cells in fed-batch cultivations. An improved understanding of the physiological factors that affect cell proliferation, survival, productivity and product quality in fed-batch could contribute to facilitate general access to these products. This work describes the investigation of autophagy and glutamine metabolism in CHO cells for the purpose of increasing fed-batch process performance. The close link between glutamine deprivation and autophagy was found to greatly affect process performance, with an increase of the cellular lysosomal compartment correlated with decreased cell-specific productivity. The increased autophagic activity upon glutamine withdrawal was confirmed by the formation of GFP-LC3 fluorescent puncta and by an LC3 autophagic flux assay. The use of 3-methyl adenine (3-MA) to inhibit autophagy increased the yield of recombinant tissue plasminogen activator (t-PA) by 2.8-fold, without compromising the glycosylation capacity of the cells given that the t-PA fucosylation, galactosylation and sialylation all increased. A more comprehensive study of glutamine metabolism and autophagy performed, including by investigating 2 additional CHO cell lines expressing different antibody proteins. The mitochondrial and lysosomal changes in response to glutamine deprivation varied substantially between cell lines, illustrating how the susceptibility to autophagy can be cell-line dependent. Integrating the combined effect of enhanced proliferation (achieved through modulation of glutamine metabolism) and inhibition of autophagy (by treatment with 3-MA), a maximum 4.6-fold increase of t-PA production was obtained in fed-batch culture. Finally, autophagy and glutamine metabolism were explored in cancer cell lines, and produced original findings on the potential for Raman spectroscopy to analyze live cell physiological responses to conditions that trigger autophagy. Overall, this study illustrates the potential for a fruitful interaction between basic scientific research and applied biotechnology. The investigation of response mechanisms to cellular stress provided opportunities to both improve industrial processing and open new perspectives for basic biological research.
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