Goudar Chetan, Biener Richard, Zhang Chun, Michaels James, Piret James, Konstantinov Konstantin
Research & Development, Process Sciences, Bayer HealthCare, Biological Products Division, 800 Dwight Way, Berkeley, CA 94710, USA.
Adv Biochem Eng Biotechnol. 2006;101:99-118. doi: 10.1007/10_020.
Cellular physiology and metabolism were monitored using a quasi real-time combination of on-line and off-line data to estimate metabolic fluxes in an established bioreaction network. The utility of this approach towards optimizing bioreactor operation was demonstrated for CHO cells cultivated in 15 L perfusion reactors at 20 x 10(6) cells/mL. Medium composition and dilution rates were changed to obtain several steady states with varying glucose and glutamine concentrations. When cells were restored to initial culture medium and perfusion rate conditions after being exposed to lower glucose and glutamine concentrations, the pyruvate flux into the TCA cycle was increased 30% while the pyruvate flux through lactate was decreased 30%, suggesting steady-state multiplicity. By appropriately altering cellular metabolism, perfusion bioreactors can operate at lower perfusion rates without significant accumulation of inhibitory metabolites such as lactate. Changes in glucose, lactate and glutamine uptake/production rates had significant effects on the calculation of other fluxes in the network. Sensitivity analysis of these key metabolic fluxes highlighted the need for accurate and reliable real-time sensors. Overall, rapid observation of metabolic fluxes can be a valuable tool for bioprocess development, monitoring and control. The framework presented in this study offers a convenient means for quasi real-time estimation of metabolic fluxes and represents a step towards realizing the potential of metabolic flux analysis for accelerated bioprocess optimization.
利用在线和离线数据的准实时组合监测细胞生理学和代谢，以估计既定生物反应网络中的代谢通量。对于在15 L灌注反应器中以20×10⁶个细胞/mL培养的CHO细胞，证明了这种方法对优化生物反应器操作的实用性。改变培养基组成和稀释率以获得几种具有不同葡萄糖和谷氨酰胺浓度的稳态。当细胞在暴露于较低葡萄糖和谷氨酰胺浓度后恢复到初始培养基和灌注速率条件时，进入三羧酸循环的丙酮酸通量增加30%，而通过乳酸的丙酮酸通量降低30%，表明存在稳态多重性。通过适当地改变细胞代谢，灌注生物反应器可以在较低的灌注速率下运行，而不会显著积累诸如乳酸等抑制性代谢物。葡萄糖、乳酸和谷氨酰胺摄取/产生速率的变化对网络中其他通量的计算有显著影响。对这些关键代谢通量的敏感性分析突出了对准确可靠的实时传感器的需求。总体而言，代谢通量的快速观察可以成为生物过程开发、监测和控制的宝贵工具。本研究中提出的框架为准实时估计代谢通量提供了一种便捷方法，并代表了朝着实现代谢通量分析在加速生物过程优化方面的潜力迈出的一步。
