Department of Biological Sciences and Bioengineering, Inha University, Incheon, Republic of Korea.
Division of Biological Science and Technology, Yonsei University, Wonju, Gangwon-do, Republic of Korea.
Biotechnol J. 2024 Aug;19(8):e2400311. doi: 10.1002/biot.202400311.
In the previous study, the culture medium was treated with nicotinamide adenine dinucleotide (NAD) under the hypothesis that NAD regeneration is a major factor causing excessive lactate accumulation in Chinese hamster ovary (CHO) cells. The NAD treatment improved metabolism by not only reducing the Warburg effect but also enhancing oxidative phosphorylation, leading to enhanced antibody production. Building on this, four NAD precursors - nicotinamide mononucleotide (NMN), nicotinic acid (NA), nicotinamide riboside (NR), and nicotinamide (NAM) - were tested to elevate intracellular NAD+ levels more economically. First, the ability of CHO cells to utilize both the salvage and Preiss-Handler pathways for NAD biosynthesis was verified, and then the effect of NAD precursors on CHO cell cultures was evaluated. These precursors increased intracellular NAD levels by up to 70.6% compared to the non-treated group. Culture analysis confirmed that all the precursors induced metabolic changes and that NMN, NA, and NR improved productivity akin to NAD treatment, with comparable integral viable cell density. Despite the positive effects such as the increase in the specific productivity and changes in cellular glucose metabolism, none of the precursors surpassed direct NAD treatment in antibody titer, presumably due to the reduction in nucleoside availability, as evidenced by the decrease in ATP levels in the NAD precursor-treated groups. These results underscore the complexity of cellular metabolism as well as the necessity for further investigation to optimize NAD precursor treatment strategies, potentially with the supplementation of nucleoside precursors. Our findings suggest a feasible approach for improving CHO cell culture performances by using NAD precursors as medium and feed components for the biopharmaceutical production.
在之前的研究中，培养基中添加了烟酰胺腺嘌呤二核苷酸（NAD），假设 NAD 的再生是导致中国仓鼠卵巢（CHO）细胞中乳酸积累过多的主要因素。NAD 处理不仅通过减少瓦伯格效应，而且通过增强氧化磷酸化来改善代谢，从而提高抗体产量。在此基础上，测试了四种 NAD 前体 - 烟酰胺单核苷酸（NMN）、烟酸（NA）、烟酰胺核苷（NR）和烟酰胺（NAM） - 以更经济地提高细胞内 NAD+水平。首先，验证了 CHO 细胞利用补救和普雷希特-哈勒尔途径合成 NAD 的能力，然后评估了 NAD 前体对 CHO 细胞培养的影响。与未处理组相比，这些前体将细胞内 NAD 水平提高了高达 70.6%。培养分析证实，所有前体都诱导了代谢变化，并且 NMN、NA 和 NR 提高了与 NAD 处理相当的生产力，具有可比的整体活细胞密度。尽管前体增加了特异性生产率和细胞葡萄糖代谢的变化等积极影响，但与 NAD 处理相比，没有一种前体在抗体滴度上超过直接 NAD 处理，这可能是由于核苷可用性的降低，正如 NAD 前体处理组中 ATP 水平的降低所证明的那样。这些结果强调了细胞代谢的复杂性，以及需要进一步研究来优化 NAD 前体处理策略的必要性，可能需要补充核苷前体。我们的研究结果表明，使用 NAD 前体作为生物制药生产中的培养基和饲料成分，是一种可行的提高 CHO 细胞培养性能的方法。
