Mild hypothermia upregulates myc and xbp1s expression and improves anti-TNFα production in CHO cells

Chinese hamster ovary (CHO) cells are the most frequently used host for commercial production of therapeutic proteins. However, their low protein productivity in culture is the main hurdle to overcome. Mild hypothermia has been established as an effective strategy to enhance protein specific productivity, although the causes of such improvement still remain unclear. The self-regulation of global transcriptional regulatory factors, such as Myc and XBP1s, seems to be involved in increased the recombinant protein production at low temperature. This study evaluated the impact of low temperature in CHO cell cultures on myc and xbp1s expression and their effects on culture performance and cell metabolism. Two anti-TNFα producing CHO cell lines were selected considering two distinct phenotypes: i.e. maximum cell growth, (CN1) and maximum specific anti-TNFα production (CN2), and cultured at 37, 33 and 31°C in a batch system. Low temperature led to an increase in the cell viability, the expression of the recombinant anti-TNFα and the production of anti-TNFα both in CN1 and CN2. The higher production of anti-TNFα in CN2 was mainly associated with the large expression of anti-TNFα. Under mild hypothermia myc and xbp1s expression levels were directly correlated to the maximal viable cell density and the specific anti-TNFα productivity, respectively. Moreover, cells showed a simultaneous metabolic shift from production to consumption of lactate and from consumption to production of glutamine, which were exacerbated by reducing culture temperature and coincided with the increased anti-TNFα production. Our current results provide new insights of the regulation of myc and xbp1s in CHO cells at low temperature, and suggest that the presence and magnitude of the metabolic shift might be a relevant metabolic marker of productive cell line.中国仓鼠卵巢细胞（Chinese hamster ovary, CHO）是商业生产治疗性蛋白最常用的宿主细胞。然而，其培养过程中较低的蛋白比生产率是亟待突破的主要障碍。轻度低温已被证实是提升蛋白比生产率的有效策略，但其产量提升的具体调控机制仍未明确。诸如Myc与XBP1s等全局转录调控因子的自我调控，似乎参与了低温环境下重组蛋白产量的提升过程。本研究评估了CHO细胞批式培养体系中，低温对myc与xbp1s表达的影响，以及二者对细胞培养表现与细胞代谢的调控作用。研究选取了两株分泌抗TNFα的CHO细胞株，二者具有截然不同的表型：分别为最高细胞生长型（CN1）与最高抗TNFα比生产型（CN2），并采用批式培养体系，在37、33及31℃三个温度下进行培养。无论在CN1还是CN2细胞株中，低温培养均可提升细胞存活率、重组抗TNFα的表达量以及抗TNFα的总产量。CN2株中更高的抗TNFα总产量，主要与其抗TNFα的高表达水平相关。在轻度低温条件下，myc与xbp1s的表达水平分别与最大活细胞密度及抗TNFα比生产率呈直接正相关。此外，细胞同时发生了代谢转变：从乳酸产生转向乳酸消耗，从谷氨酰胺消耗转向谷氨酰胺产生；且培养温度越低，这种代谢转变越显著，同时该转变与抗TNFα产量的提升相契合。本研究结果为低温培养下CHO细胞中myc与xbp1s的调控机制提供了新的学术视角，并提示代谢转变的存在与程度，可作为高产细胞株的潜在代谢标志物。