多孔微载体无血清培养rCHO细胞生产u-PA

在30L搅拌式反应器中无血清培养分泌尿激酶型纤溶酶原激活剂(u-PA)的DNA重组CHO细胞，定期部分更换Cytopore多孔微载体，使生长在多孔微载体中的细胞不断更新繁殖，解决大规模细胞培养中的细胞凋亡问题。在91d连接换液培养过程中，细胞密度可维持在(1.3～2.6)×107／mL，活细胞比率维持在90％以上。在7.5L搅拌罐中培养细胞，利用外部周期性压力振荡刺激并结合载体更新技术，可减轻密度效应对细胞生长和表达的影响，在一定程度上提高细胞在高密度培养条件下的表达水平。在67d连续换液培养中，细胞最高密度为2.64×107／mL，活细胞比率维持在95％以上。与稳压操作相比，利用周期变压刺激技术可提高产量10％～20％，且可降低葡萄糖厌氧代谢生成乳酸的转化率，利用4步纯化工艺，从含u-PA约135g的2100L上清中获得约80gu-PA(单链比例约为90％)。

Production of u-PA with rCHO Cell Culture on Porous Microcarriers in Serum-free Growth Medium

A novel technique was developed to deal with apoptosis in large-scale animal cell culture. By means of replacing part of Cytopore porous microcarriers at regular intervals, a rCHO cell line, which produces urokinase-type plasminogen activitor(u-PA), was cultivated continuously with serum-free medium in a 30 L stirred tank for 91 days. The cell density was maintained at (1.3-2.6) x 10(7)/mL, and > 90% of cells was viable. In order to reduce the effect of cell density on cell growth and expression, a cyclic pressure oscillation was exerted on a 7.5 L reactor headspace to enhance cell expression at high cell density to a certain extent. During the 67 days of medium-replacement culture, the maximal cell density reached 2.64 x 10(7)/mL, and cell viability was always kept above 95% when combined with microcarrier-replacement. Compare to control culture, culture with cyclic pressure oscillation could enhance cell expression level and reduce the ratio of glucose metabolized anaerobically to produce lactate. With four-step purification process, about 80 g u-PA(approximately 90% scu-PA) was recovered from approximately 2100 liters supernatant which contained approximately 135 g u-PA.