Mitochondria are the powerhouse of a cell that produces cellular energy, especially adenosine triphosphate (ATP), to fuel cell biosynthesis and protein production. In this study, we aim at inducing mitochondrial biogenesis in Chinese hamster ovary (CHO) cells to increase cellular ATP levels as well as recombinant protein productivity. Focusing on the culture process engineering, we tested two chemical additives, resveratrol and nicotinamide adenine dinucleotide (NAD), in CHO cell cultures to reinforce mitochondrial biogenesis. Resveratrol and NAD treatment resulted in an increase in specific productivity (q p ) by 10-30% and 40-50% compared to the control, respectively. In addition, NAD exhibited an increase in viable cell densities, culture longevity and specific lactate uptake rates. Moreover, along with an increase in the ATP level, mitochondrial DNA (mtDNA) copy number and mRNA expression of the mitochondrial biogenesis-related genes were also up-regulated compared to the control. In conclusion, although there might be varied cell line-specific impacts on cell growth, inducing mitochondrial biogenesis using chemical additives would be an effective strategy for improving recombinant proteins productivity in CHO cells culture.
Aerobic glycolysis and its by‐product lactate accumulation are usually associated with adverse culture phenotypes such as poor cell viability and productivity. Due to the lack of knowledge on underlying mechanisms and accompanying biological processes, the regulation of aerobic glycolysis has been an ongoing challenge in culture process development for therapeutic protein productivity. Nicotinamide adenine dinucleotide (NAD+), a coenzyme and co‐substrate in energy metabolism, promotes the conversion of inefficient glycolysis into an efficient oxidative phosphorylation (OXPHOS) pathway. However, the effect of NAD+ on Chinese hamster ovary (CHO) cells for biopharmaceutical production has not been reported yet. In this work, we aimed to elucidate the influence of NAD+ on cell culture performance by examining metabolic shifts and mAb productivity. The supplementation of NAD+ increased the intracellular concentration of NAD+ and promoted SIRT3 expression. Antibody titer and the specific productivity in the growth phase were improved by up to 1.82‐ and 1.88‐fold, respectively, with marginal restrictions on cell growth. NAD+ significantly reduced the accumulation of reactive oxygen species (ROS) and the lactate yield from glucose, determined by lactate accumulation versus glucose consumption (YLAC/GLC). In contrast, OXPHOS capacity and amino acid consumption rate increased substantially. Collectively, these results suggest that NAD+ contributes to improving therapeutic protein productivity in bioprocessing via inducing an energy metabolic shift.
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