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首页> 外文期刊>Biotechnology Progress >A Single Nutrient Feed Supports Both Chemically Defined NSO and CHO Fed-Batch Processes: Improved Productivity and Lactate Metabolism
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A Single Nutrient Feed Supports Both Chemically Defined NSO and CHO Fed-Batch Processes: Improved Productivity and Lactate Metabolism

机译：单一营养饲料可同时支持化学成分确定的NSO和CHO补料分批过程：提高的生产率和乳酸代谢

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A chemically defined nutrient feed (CDF) coupled with basal medium preloading was developed to replace a hydrolysate-containing feed (HCF) for a fed-batch NSO process. The CDF not only enabled a completely chemically defined process but also increased recombinant monoclonal antibody titer by 115%. Subsequent tests of CDF in a CHO process indicated that it could also replace the hydrolysate-containing nutrient feed in this expression system as well as providing an 80% increase in product titer. In both CDF NSO and CHO processes, the peak lactate concentrations were lower and, more interestingly, lactate metabolism shifted markedly from net production to net consumption when cells transitioned from exponential to stationary growth phase. Subsequent investigations of the lactate metabolic shift in the CHO CDF process were carried out to identify the cause(s) of the metabolic shift. These investigations revealed several metabolic features of the CHO cell line that we studied. First, glucose consumption and lactate consumption are strictly complementary to each other. The combined cell specific glucose and lactate consumption rate was a constant across exponential and stationary growth phases. Second, Lactate dehydrogenase (LDH) activity fluctuated during the fed-batch process. LDH activity was at the lowest when lactate concentration started to decrease. Third, a steep cross plasma membrane glucose gradient exists. Intracellular glucose concentration was more than two orders of magnitude lower than that in the medium. Fourth, a large quantity of citrate was diverted out of mitochondria to the medium, suggesting a partially truncated tricarboxylic acid (TCA) cycle in CHO cells. Finally, other intermediates in or linked to the glycolytic pathway and the TCA cycle, which include alanine, citrate, isocitrate, and succinate, demonstrated a metabolic shift similar to that of lactate. Interestingly, all these metabolites are either in or linked to the pathway downstream of pyruvate, but upstream of fumarate in glucose metabolism. Although the specific mechanisms for the metabolic shift of lactate and other metabolites remain to be elucidated, the increased understanding of the metabolism of CHO cultures could lead to future improvements in medium and process development.
机译：开发了化学成分确定的营养饲料（CDF）和基础培养基预加料，以代替分批加料NSO工艺的含水解物的饲料（HCF）。 CDF不仅实现了完全化学上确定的过程，而且使重组单克隆抗体的效价提高了115％。随后在CHO过程中对CDF进行的测试表明，该CDF也可以代替此表达系统中的含水解物的营养物质进料，并且可使产品效价提高80％。在CDF NSO和CHO过程中，乳酸的峰值浓度都较低，而且更有趣的是，当细胞从指数生长期过渡到静止生长期时，乳酸代谢显着从净生产转向净消耗。随后对CHO CDF过程中的乳酸代谢变化进行了调查，以确定代谢变化的原因。这些研究揭示了我们研究的CHO细胞系的几种代谢特征。首先，葡萄糖消耗和乳酸消耗是严格互补的。组合的细胞特异性葡萄糖和乳酸消耗速率在指数生长期和静止生长期是恒定的。其次，补料分批过程中乳酸脱氢酶（LDH）的活性发生波动。当乳酸浓度开始降低时，LDH活性最低。第三，存在陡峭的跨质膜葡萄糖梯度。细胞内葡萄糖浓度比培养基中的葡萄糖浓度低两个数量级以上。第四，大量柠檬酸盐从线粒体转移到培养基中，表明CHO细胞中部分截短的三羧酸（TCA）循环。最后，糖酵解途径和TCA循环中或与之相关的其他中间体（包括丙氨酸，柠檬酸盐，异柠檬酸盐和琥珀酸盐）表现出与乳酸相似的代谢变化。有趣的是，所有这些代谢产物都在丙酮酸下游，或在葡萄糖代谢的富马酸上游的途径中或与之相关。尽管乳酸和其他代谢物的代谢转移的具体机制仍有待阐明，但对CHO培养物代谢的了解的增加可能会导致培养基和工艺开发的进一步改善。

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