Adjustments to CHO cell physiology were recently observed during implementation of a Raman spectroscopy-based glucose and lactate control strategy. To further understand how these cells, under monoclonal antibody (mAb) production conditions, utilized the extra lactic acid fed, we performed a comprehensive semi-quantitative and time-dependent analysis of the exometabolome. This study focused on the CHO cell's metabolic shift from the fifth day of culture. We compared relative levels of extracellular metabolites in the absence or presence of a 2 g/L lactic acid setpoint while glucose was kept at 4 g/L. Our hypothesis is that extra lactic acid would supply more pyruvate, favoring oxidative phosphorylation. We subsequentially uncovered several carnitine derivatives as biomarkers of the simultaneous activation of TCA anaplerotic pathways as well as a carbon-buffering pathway. CHO cells exhibited a balance between intermediates from (i) amino acid catabolism, (ii) fatty acid β-oxidation, and (iii) pyruvate from glycolysis and lactic acid; and the secretion of their intermediate carnitine derivatives. In addition, 3-hydroxy-methyl-glutaric acid (HMG) and mevalonate syntheses were found as biomarkers of alternative acyl group removal. Together, under a limited capacity to assimilate the surplus of acyl-CoA groups as well as an ability to maintain the acyl-CoA: free CoA ratio for proper and continuous functioning of the TCA cycle, CHO cells activate the carnitine-buffering system, HMG, and mevalonate pathways.

中文翻译：

---

外代谢组分析揭示了与葡萄糖和乳酸共同喂养的 CHO 细胞的分批补料培养物中肉碱缓冲途径的激活

最近在实施基于拉曼光谱的葡萄糖和乳酸控制策略期间观察到对 CHO 细胞生理的调整。为了进一步了解这些细胞在单克隆抗体 (mAb) 生产条件下如何利用补充的乳酸，我们对外代谢组进行了全面的半定量和时间依赖性分析。这项研究的重点是 CHO 细胞从培养第五天开始的代谢转变。我们比较了在不存在或存在 2 g/L 乳酸设定点而葡萄糖保持在 4 g/L 的情况下细胞外代谢物的相对水平。我们的假设是额外的乳酸会提供更多的丙酮酸，有利于氧化磷酸化。我们随后发现了几种肉碱衍生物作为同时激活 TCA 回补途径和碳缓冲途径的生物标志物。CHO细胞在中间体之间表现出平衡（i ) 氨基酸分解代谢，( ii ) 脂肪酸β-氧化，和 ( iii ) 来自糖酵解和乳酸的丙酮酸；以及它们的中间肉碱衍生物的分泌。此外，发现 3-羟基-甲基-戊二酸 (HMG) 和甲羟戊酸合成是替代酰基去除的生物标志物。总之，在同化过量酰基辅酶A基团的能力有限以及维持酰基辅酶A：游离辅酶A比率以确保TCA循环正常和连续运行的能力下，CHO细胞激活肉碱缓冲系统HMG和甲羟戊酸途径。