Miller W M, Blanch H W, Wilke C R
Biotechnol Bioeng. 2000 Mar 20;67(6):853-71.
Hybridomas are finding increased use for the production of a wide variety of monoclonal antibodies. Understanding the roles of physiological and environmental factors on the growth and metabolism of mammalian cells is a prerequisite for the development of rational scale-up procedures. An SP2/0-derived mouse hybridoma has been employed in the present work as a model system for hybridoma suspension culture. In preliminary shake flask studies to determine the effect of glucose and glutaminE, it was found that the specific growth rate, the glucose and glutamine metabolic quotients, and the cumulative specific antibody production rate were independent of glucose concentration over the range commonly employed in cell cultures. Only the specific rate of glutamine uptake was found to depend on glutamine concentration. The cells were grown in continuous culture at constant pH and oxygen concentration at a variety of dilution rates. Specific substrate consumption rates and product formation rates were determined from the steady state concentrations. The specific glucose uptake rate deviated from the maintenance energy model(1) at low specific growth rates, probably due to changes in the metabolic pathways of the cells. Antibody production was not growth-associated; and higher specific antibody production rates were obtained at lower specific growth rates. The effect of pH on the metabolic quotients was also determined. An optimum in viable cell concentration was obtained between pH 7.1 and 7.4. The viable cell number and viability decreased dramatically at pH 6.8. At pH 7.7 the viable cell concentration initially decreased, but then recovered to values typical of pH 7.1-7.4. Higher specific nutrient consumption rates were found at the extreme pH values; however, glucose consumption was inhibited at low pH. The pH history also influenced the behavior at a given pH. Higher antibody metabolic quotients were obtained at the extreme pH values. Together with the effect of specific growth rate, this suggests higher antibody production under environmental or nutritional stress.
杂交瘤在生产各种各样的单克隆抗体方面的应用越来越广泛。了解生理和环境因素对哺乳动物细胞生长和代谢的作用是开发合理放大培养程序的先决条件。在本研究中，采用了一种源自SP2/0的小鼠杂交瘤作为杂交瘤悬浮培养的模型系统。在初步的摇瓶研究中，为了确定葡萄糖和谷氨酰胺的影响，发现特定生长速率、葡萄糖和谷氨酰胺代谢商以及累积特定抗体产生速率在细胞培养常用的浓度范围内与葡萄糖浓度无关。仅发现谷氨酰胺摄取的特定速率取决于谷氨酰胺浓度。细胞在恒定pH值和氧气浓度下以不同的稀释率进行连续培养。从稳态浓度确定特定底物消耗速率和产物形成速率。在低特定生长速率下，特定葡萄糖摄取速率偏离维持能量模型(1)，这可能是由于细胞代谢途径的变化。抗体产生与生长无关；在较低的特定生长速率下获得了更高的特定抗体产生速率。还确定了pH值对代谢商的影响。在pH 7.1至7.4之间获得了活细胞浓度的最佳值。在pH 6.8时，活细胞数量和活力急剧下降。在pH 7.7时，活细胞浓度最初下降，但随后恢复到pH 7.1 - 7.4的典型值。在极端pH值下发现了更高的特定营养物消耗速率；然而，在低pH值下葡萄糖消耗受到抑制。pH值历史也影响给定pH值下的行为。在极端pH值下获得了更高的抗体代谢商。连同特定生长速率的影响，这表明在环境或营养应激下抗体产量更高。
