Chinese hamster ovary (CHO) cells are preferred hosts for the production of recombinant biopharmaceuticals. Efforts to optimize these bioprocesses have largely relied on empirical experience and our knowledge of cellular behavior in culture is incomplete. More recently, comprehensive investigations of metabolic network operation have started to be used to uncover traits associated with optimal growth and recombinant protein production. In this work, we used (1) H-nuclear magnetic resonance ((1) H-NMR) to analyze the supernatants of glutamine-synthetase (GS)-CHO cell clones expressing variable amounts of an IgG4 under control and butyrate-treated conditions. Exometabolomic data revealed accumulation of several metabolic by-products, indicating inefficiencies at different metabolic nodes. These data were contextualized in a detailed network and the cellular fluxomes estimated through metabolic flux analysis. This approach allowed comparing metabolic activity across different clones, growth phases and culture conditions, in particular the efficiency pertaining to carbon lost to glycerol and lactate accumulation and the characteristic nitrogen metabolism involving high asparagine and serine uptake rates. Importantly, this study shows that early butyrate treatment has a marked effect on sustaining high nutrient consumption along culture time, being more pronounced during the stationary phase when extra energy generation and biosynthetic activity is fueled to increase IgG formation. Collectively, the information generated contributes to deepening our understanding of CHO cells metabolism in culture, facilitating future design of improved bioprocesses.

Chinese hamster ovary (CHO) cells are the predominant host for production of therapeutic glycoproteins. In particular, the glutamine-synthetase (GS) expression system has been widely used in the biopharmaceutical industry for efficient selection of high-yielding clones. However, much remains unclear on how metabolic wiring affects culture performance. For instance, asparagine and serine have been observed to be the largest nitrogen sources taken up by GS-CHO cells, but their roles in biosynthesis and energy generation are poorly understood. In this work, a comprehensive profiling of extracellular metabolites coupled with an analysis of intracellular label distributions after 1-(13) C-pyruvate supplementation were used to trace metabolic rearrangements in different scenarios of asparagine and serine availability. The absence of asparagine in the medium caused growth arrest, and was associated with a dramatic increase in pyruvate uptake, a higher ratio of pyruvate carboxylation to dehydrogenation and an inability for de novo asparagine synthesis. The release of ammonia and amino acids such as aspartate, glutamate, and alanine were deeply impacted. This confirms asparagine to be essential for these GS-CHO cells as the main source of intracellular nitrogen as well as having an important anaplerotic role in TCA cycle activity. In turn, serine unavailability also negatively affected culture growth while triggering its de novo synthesis, confirmed by label incorporation coming from pyruvate, and reduced glycine and formate secretion congruent with its role as a precursor in the metabolism of one-carbon units. Overall, these results unfold important insights into GS-CHO cells metabolism that lay a clearer basis for fine-tuning bioprocess optimization.

To investigate the mechanisms by which the hypothalamic peptide GHRH influences cell division, we analyzed its effects on the proliferation of two different cell lines: CHO-4, an ovary-derived cell line, and GH3, a pituitary-derived cell line. We found that GHRH induces the proliferation of pituitary-derived cells but inhibits the proliferation of ovary-derived cells. We further characterized this dual effect of GHRH to find that the cytoplasmic signals induced by this hormone are similar in both cell lines. Moreover, in CHO-4 cells GHRH stimulates two well-known positive cell cycle regulators, c-myc and cyclin D1, but is unable to induce the degradation of the negative cell cycle regulator p27(Kip1). Significantly, when the Pit-1/GHF-1 gene is exogenously expressed in CHO-4 cells, the negative effect of GHRH on the proliferation of these cells is attenuated. Furthermore, when the levels of Pit-1 are downregulated by siRNA in GH3-GHRHR cells, the positive effects of GHRH on the proliferation of these cells are diminished. These findings add to our understanding of the molecules involved in the regulation of cell proliferation by GHRH, as we demonstrate for the first time that Pit-1 is not only required to drive the expression of the GHRH receptor, as previously described, but is also needed for the downstream effects that occur after its activation to modulate cell proliferation. These data suggest that the regulation of cell proliferation in response to a specific growth factor depends in certain cell populations on the presence of a tissue-specific transcription factor.