Feng Qiang, Mi Li, Li Ling, Wang Xian-Hui, Chen Zhi-Nan
Cell Engineering Research Centre, Fourth Military Medical University, Xi'an 710033, China.
Sheng Wu Gong Cheng Xue Bao. 2003 Sep;19(5):593-7.
On-line analysis and control are critical for the optimization of product yields in animal cell culture. The close monitor of viable cell number helps to gain a better insight into the metabolism and to refine culture strategy. In this study, we use the oxygen uptake rate (OUR) to estimate the number of viable cell and the OUR-based feed-back control strategy for nutrients feeding to improve the efficiency of cell culture. A hybridoma cell line (HAb18) was cultured in fed-batch and perfusion model using serum free medium in 5L CelliGen Plus bioreactor (NBS Co., American) and 5L Biostat B bioreactor (Braun Co., Germany). The system and the method for online monitoring OUR in bioreactors, based on the dynamic measurement of dissolved oxygen (DO), were developed. The method of on-line cell concentration estimation was established based on the relationship between the growth of the hybridoma and the uptake rate of oxygen. This method was then used to determine OUR and the concentrations of cell, antibody, glucose, lactate, glutamine and ammonia in the bioreactors at given times. The relationship between OUR and nutrients metabolism was studied and OUR-based feed-back control strategy, which used the state deltaOUR = 0 as the regulation point, was established and used to control the rates of nutrients or medium feeding rate in the perfusion culture. The results showed that there was close relationship between OUR, concentration of live cells, productivity of antibody and consumption of glutamine. The sudden decrease in OUR may be caused by glutamine depletion, and with different delay times, the viable cell concentration and antibody productivity also decreased. The further analysis revealed the linear relationship between OUR and the density of live cells in the exponential growth phase as qOUR = (0.103 +/- 0.028) x 10(-12) mol/cell/h. These findings can be applied to the on-line detection of live cell density. Our study also indicated that by adjusting the perfusion rate with OUR-based feed-back control strategy, it is feasible to continuously increase in viable cell density and antibody concentration in the perfusion culture.
在线分析与控制对于优化动物细胞培养中的产品产量至关重要。密切监测活细胞数量有助于更好地了解细胞代谢并优化培养策略。在本研究中，我们使用氧气摄取率（OUR）来估算活细胞数量，并采用基于OUR的反馈控制策略来控制营养物质的添加，以提高细胞培养效率。使用无血清培养基在5L CelliGen Plus生物反应器（美国NBS公司）和5L Biostat B生物反应器（德国Braun公司）中，以分批补料和灌注模式培养杂交瘤细胞系（HAb18）。基于溶解氧（DO）的动态测量，开发了生物反应器中在线监测OUR的系统和方法。基于杂交瘤细胞生长与氧气摄取率之间的关系，建立了在线细胞浓度估算方法。然后使用该方法在给定时间测定生物反应器中OUR以及细胞、抗体、葡萄糖、乳酸、谷氨酰胺和氨的浓度。研究了OUR与营养物质代谢之间的关系，并建立了以状态deltaOUR = 0为调节点的基于OUR的反馈控制策略，用于控制灌注培养中营养物质或培养基的进料速率。结果表明，OUR、活细胞浓度、抗体产量和谷氨酰胺消耗之间存在密切关系。OUR的突然下降可能是由于谷氨酰胺耗尽引起的，且延迟时间不同，活细胞浓度和抗体产量也会下降。进一步分析表明，在指数生长期，OUR与活细胞密度之间存在线性关系，即qOUR = (0.103 ± 0.028) x 10(-12) mol/细胞/h。这些发现可应用于活细胞密度的在线检测。我们的研究还表明，通过基于OUR的反馈控制策略调整灌注速率，在灌注培养中持续提高活细胞密度和抗体浓度是可行的。
