Forschungszentrum Jülich GmbH, Institute of Biotechnology, D-52425, Jülich, Germany.
Cytotechnology. 1997 May;24(1):19-30. doi: 10.1023/A:1007913128209.
The monoclonal-antibody production of an immobilized hybridoma cell line cultivated in a fluidized-bed reactor was monitored on-line for nearly 900 h. The monoclonal antibody concentration was determined by an immuno affinity-chromatography method (ABICAP). Antibodies directed against the product, e.g. IgG, were immobilized on a micro-porous gel and packed in small columns. After all IgG present in the sample was bound to the immobilized antibodies, unbound proteins were removed by rinsing the column. Elution of the bound antibodies followed and the antibodies were determined by fluorescence. The analytical procedure was automated with a robotic device to enable on-line measurements. The correlation between the on-line determined data and antibody concentrations measured by HPLC was linear.A sampling system was constructed, which was based on a pneumatically actuated in-line membrane valve integrated into the circulation loop of the reactor. Separation of the cells from the sample stream was achieved by a depth filter made of glass-fibre, situated outside the reactor. Rapid obstruction of the filter by cells or cell debris and contamination of the sample system was avoided by intermittent rinsing of the sample system with a chemical solution. The intermittent rinsing of the filter, which had a surface of 4.8 cm(2), resulted in an operational capacity of up to 40 samples (1.0 l total sample volume). Both the sampling system and the analytical device functioned without failure during this long-term culture.The culture temperature was varied between 34 and 40 °C. Raising the temperature from 34 up to 37 °C resulted in a simultaneous increase of growth and specific antibody production rate. Specific metabolic rates of glucose, lactate, glutamine and ammonium stayed constant in this temperature range. A further enhancement of temperature up to 40 °C had a negative effect on the growth rate, whereas the specific monoclonal antibody production rate showed a small increase. The other specific metabolic rates also increased in the temperature range between 38 to 40 °C.
在流化床反应器中培养的固定化杂交瘤细胞系的单克隆抗体生产进行了近 900 小时的在线监测。单克隆抗体浓度通过免疫亲和层析法（ABICAP）确定。针对产物的抗体，例如 IgG，固定在微孔凝胶上并填充在小柱中。在样品中存在的所有 IgG 与固定化抗体结合后，通过冲洗柱子去除未结合的蛋白质。然后洗脱结合的抗体，并用荧光法测定抗体。分析过程通过机器人设备自动化，以实现在线测量。在线测定的数据与通过 HPLC 测定的抗体浓度之间存在线性相关性。构建了一种采样系统，该系统基于集成在反应器循环回路中的气动致动在线膜阀。通过位于反应器外部的玻璃纤维制成的深度过滤器从样品流中分离细胞。通过用化学溶液间歇冲洗样品系统，避免了细胞或细胞碎片快速堵塞过滤器和样品系统的污染。对表面积为 4.8 cm2 的过滤器进行间歇冲洗，可处理多达 40 个样品（总样品体积为 1.0 l）。在长时间培养过程中，采样系统和分析设备均无故障运行。培养温度在 34 至 40°C 之间变化。将温度从 34°C 升高到 37°C 会导致生长和特异性抗体生产率同时增加。在此温度范围内，葡萄糖、乳酸盐、谷氨酰胺和铵的特异性代谢率保持不变。进一步将温度升高到 40°C 会对生长速率产生负面影响，而特异性单克隆抗体生产率则略有增加。在 38 至 40°C 的温度范围内，其他特异性代谢率也有所增加。
