Cytotechnology. 2004 Jan;44(1-2):1-14. doi: 10.1023/B:CYTO.0000043395.36188.bc.
The kinetics and long-term stability of continuous production of monoclonal antibody IgG2b by hybridoma HD-24 cells immobilized in a fibrous-bed bioreactor (FBB) were studied for a period of approximately 8 months. The cells were immobilized in the fibrous bed by surface attachment of cells and entrapment of large cell clumps in the void space of the fibrous matrix. A high viable cell density of 1.01 x 10(8)/ml was attained in the bioreactor, which was about 63 times higher than those in conventional T-flask and spinner flask cultures. The continuous FBB produced IgG at a concentration of approximately 0.5 g/l, with reactor productivity of approximately 7 mg/h.l, which was about 23 times higher than those from conventional T-flask and spinner flask cultures. The IgG concentration can be further increased to approximately 0.67 g/l by using higher feed (glucose and glutamine) concentrations and running the reactor at a recycle batch or fed-batch mode. The long-term performance of this bioreactor was also evaluated. For a period of 36 days monitored, the MAb produced in the continuous well-mixed bioreactor at 50 h retention time (0.02/h dilution rate) was maintained at a steady concentration level of approximately 0.3 g/l with less than 8% drift. At the end of the study, it was found that approximately 25% of the cells were strongly attached to the fiber surfaces and the other approximately 75% entrapped or weakly immobilized in the fibrous matrix. The strongly attached cells had a high viability of approximately 90%, compared to approximately 75% for cells weakly immobilized and only approximately 1.4% for freely suspended cells, suggesting that the fibrous matrix preferentially retained and protected the viable (productive) cells. The FBB thus was able to maintain its long-term productivity because nonviable and dead cells were continuously washed off from the fibrous matrix. The high MAb concentration and production rate and excellent stability for continuous long-term production obtained in this study compare favorably to other bioreactor studies reported in the literature. The reactor performance can be further improved by providing better pH and aeration controls at higher feed concentrations. The FBB is easy to operate and scale-up, and thus can be used economically for industrial production of MAb.
将杂交瘤 HD-24 细胞固定在纤维床生物反应器 (FBB) 中连续生产单克隆抗体 IgG2b 的动力学和长期稳定性进行了约 8 个月的研究。细胞通过表面附着和大细胞团块在纤维基质的空隙中的包埋固定在纤维床上。在生物反应器中获得了 1.01x10(8)/ml 的高活细胞密度，比传统 T 瓶和搅拌瓶培养高约 63 倍。连续 FBB 以约 0.5g/l 的浓度生产 IgG，反应器生产率约为 7mg/h.l，比传统 T 瓶和搅拌瓶培养高约 23 倍。通过使用较高的进料（葡萄糖和谷氨酰胺）浓度并以循环分批或补料分批模式运行反应器，可将 IgG 浓度进一步提高至约 0.67g/l。还评估了该生物反应器的长期性能。在监测的 36 天期间，在 50h 保留时间（0.02/h 稀释率）下连续混合生物反应器中生产的单克隆抗体保持在约 0.3g/l 的稳定浓度水平，漂移小于 8%。研究结束时，发现大约 25%的细胞强烈附着在纤维表面上，其余大约 75%的细胞包埋或弱固定在纤维基质中。强烈附着的细胞具有约 90%的高存活率，而弱固定的细胞约为 75%，自由悬浮的细胞仅约为 1.4%，表明纤维基质优先保留和保护有活力（有生产力）的细胞。因此，FBB 能够保持其长期生产力，因为无活力和死亡的细胞不断从纤维基质中冲洗掉。与文献中报道的其他生物反应器研究相比，本研究中获得的高单克隆抗体浓度和生产速率以及连续长期生产的优异稳定性表现良好。通过在较高的进料浓度下提供更好的 pH 和曝气控制，可以进一步提高反应器性能。FBB 易于操作和放大，因此可以经济地用于单克隆抗体的工业生产。
