Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, Brisbane, Queensland, Australia.
Charles Perkins Centre, The University of Sydney, Sydney, New South Wales, Australia.
Biotechnol Bioeng. 2018 Sep;115(9):2315-2327. doi: 10.1002/bit.26724. Epub 2018 May 22.
Lactate is one of the key waste metabolites of mammalian cell culture. High lactate levels are caused by high aerobic glycolysis, also known as the Warburg effect, and are usually associated with adverse culture performance. Therefore, reducing lactate accumulation has been an ongoing challenge in the cell culture development to improve growth, productivity, and process robustness. The pyruvate dehydrogenase complex (PDC) plays a crucial role for the fate of pyruvate, as it converts pyruvate to acetyl coenzyme A (acetyl-CoA). The PDC activity can be indirectly increased by inhibiting the PDC inhibitor, pyruvate dehydrogenase kinase, using dichloroacetate (DCA), resulting in less pyruvate being available for lactate formation. Here, Chinese hamster ovary cells were cultivated either with 5 mM DCA or without DCA in various batch and fed-batch bioreactor processes. In all cultures, DCA increased peak viable cell density (VCD), culture length and final antibody titer. The strongest effect was observed in a fed batch with media and glucose feeding in which peak VCD was increased by more than 50%, culture length was extended by more than 3 days, and the final antibody titer increased by more than twofold. In cultures with DCA, lactate production and glucose consumption during exponential growth were on average reduced by approximately 40% and 35%, respectively. Metabolic flux analysis showed reduced glycolytic fluxes, whereas fluxes in the tricarboxylic acid (TCA) cycle were not affected, suggesting that cultures with DCA use glucose more efficiently. In a proteomics analysis, only few proteins were identified as being differentially expressed, indicating that DCA acts on a posttranslational level. Antibody quality in terms of aggregation, charge variant, and glycosylation pattern was unaffected. Subsequent bioreactor experiments with sodium lactate and sodium chloride feeding indicated that lower osmolality, rather than lower lactate concentration itself, improved culture performance in DCA cultures. In conclusion, the addition of DCA to the cell culture improved culture performance and increased antibody titers without any disadvantages for cell-specific productivity or antibody quality.
乳酸是哺乳动物细胞培养的关键废物代谢物之一。高乳酸水平是由高需氧糖酵解引起的，也称为沃伯格效应，通常与不良的培养性能有关。因此，减少乳酸积累一直是细胞培养开发中的一个持续挑战，目的是提高生长、生产力和过程稳健性。丙酮酸脱氢酶复合物（PDC）对丙酮酸的命运起着至关重要的作用，因为它将丙酮酸转化为乙酰辅酶 A（乙酰-CoA）。PDC 活性可以通过使用二氯乙酸（DCA）抑制丙酮酸脱氢酶激酶来间接增加，从而减少可用于形成乳酸的丙酮酸量。在这里，中国仓鼠卵巢细胞在各种分批和补料分批生物反应器工艺中要么用 5mM DCA 培养，要么不用 DCA 培养。在所有培养物中，DCA 均增加了峰值活细胞密度（VCD）、培养时间和最终抗体滴度。在补料分批培养中观察到的效果最强，其中补料分批培养中培养基和葡萄糖的补料使峰值 VCD 增加了 50%以上，培养时间延长了 3 天以上，最终抗体滴度增加了两倍以上。在含有 DCA 的培养物中，指数生长期的乳酸生成和葡萄糖消耗平均减少了约 40%和 35%。代谢通量分析显示糖酵解通量降低，而三羧酸（TCA）循环通量不受影响，表明 DCA 培养物更有效地利用葡萄糖。在蛋白质组学分析中，仅鉴定出少数差异表达的蛋白质，表明 DCA 作用于翻译后水平。抗体质量在聚集、电荷变体和糖基化模式方面不受影响。随后用乳酸钠和氯化钠补料的生物反应器实验表明，较低的渗透压而不是较低的乳酸浓度本身，改善了 DCA 培养物中的培养性能。总之，向细胞培养物中添加 DCA 可提高培养性能并增加抗体滴度，而不会对细胞特异性生产力或抗体质量产生任何不利影响。
