Konstantinov K B, Tsai Y, Moles D, Matanguihan R
Bayer Corporation, Berkeley, California 94710, USA.
Biotechnol Prog. 1996 Jan-Feb;12(1):100-9. doi: 10.1021/bp950044p.
The strategies for control of the feed rate in high-density perfusion cultures of animal cells are limited to several simple schemes. While in an industrial environment simplicity is seen as a major advantage, the need for more elaborate closed-loop control methods that can improve process stability in long-term continuous cultures is also well understood. What has prevented the application of the advanced control strategies known from theory is the lack of reliable real-time information that can be used to close the feedback loop. Among the variables that are appropriate for direct feedback control of the perfusion rate, high priority should be given to the glucose concentration. Unlike some other environmental variables, such as dissolved oxygen and pH, it provides unambiguous information which facilitates the selection of the right feed rate. The present paper describes the application of a closed loop control scheme, known as a "glucose-stat", to the long-term cultivation of Chinese hamster ovary cells in a high-density (35-40 million cells/mL) perfusion process. The monitoring and control system worked successfully for more than 2.5 months without any signs of performance degradation. In targeting industrial application, issues such as reliability, sterility, and accuracy, are given high priority. The implementation of the glucose monitoring system, which is the main part of the control complex, is addressed in details. The performance of the perfusion culture was evaluated at four different glucose set points, providing essential information about process optimization. It became evident that the perfusion culture was operated in the so-called "high-gain" zone (where the system is highly sensitive to the dilution rate), which justifies the application of a feedback control. The on-line glucose concentration was also used by an embedded expert system which drove the process through the batch and the perfusion phase, achieving total computer control of the feed rate. In summary, the proposed glucose monitoring and control technique proved to be a reliable biotechnology tool which can be applied with confidence at an industrial scale to either microbial or mammalian cell cultures.
在动物细胞高密度灌注培养中，控制进料速率的策略仅限于几种简单方案。在工业环境中，简单性被视为主要优势，但人们也清楚地认识到需要更精细的闭环控制方法，以提高长期连续培养过程的稳定性。理论上已知的先进控制策略之所以未能得到应用，是因为缺乏可用于闭合反馈回路的可靠实时信息。在适合直接反馈控制灌注速率的变量中，葡萄糖浓度应被高度优先考虑。与一些其他环境变量（如溶解氧和pH值）不同，它提供明确的信息，便于选择合适的进料速率。本文描述了一种称为“葡萄糖恒化器”的闭环控制方案在高密度（3500 - 4000万个细胞/毫升）灌注过程中对中国仓鼠卵巢细胞进行长期培养的应用。监测和控制系统成功运行了两个半月以上，没有任何性能下降的迹象。在针对工业应用时，可靠性、无菌性和准确性等问题被高度重视。详细阐述了作为控制复合体主要部分的葡萄糖监测系统的实施情况。在四个不同的葡萄糖设定点对灌注培养的性能进行了评估，提供了有关过程优化的重要信息。很明显，灌注培养是在所谓的“高增益”区域（系统对稀释率高度敏感）运行的，这证明了反馈控制的应用是合理的。嵌入式专家系统也使用在线葡萄糖浓度，该系统在分批培养和灌注阶段驱动过程，实现了进料速率的完全计算机控制。总之，所提出的葡萄糖监测和控制技术被证明是一种可靠的生物技术工具，可以放心地应用于工业规模的微生物或哺乳动物细胞培养。
