Johnson M, Lanthier S, Massie B, Lefebvre G, Kamen A A
Biomira Inc., Edmonton Research Park, Edmonton, Alberta, Canada.
Biotechnol Prog. 1996 Nov-Dec;12(6):855-64. doi: 10.1021/bp960072n.
As part of an effort to develop a suspension-culture perfusion-based process with high flow rate without the fouling and antibody retention inherent to filter-based cell-separation devices, we have evaluated and contributed to the development of the Centritech Lab centrifuge for the perfusion culture of hybridoma cells in protein-free medium. Culture start-ups showed that cell growth and monoclonal-antibody (MAb) production rates were similar in both a spinner flask and continuous centrifugation coupled to a bioreactor. The centrifuge efficiently separated viable cells from dead ones. Viable-cell recoveries were never below 98%, whereas dead-cell recoveries were usually around 80%. The cell content of the centrifuge supernatant and concentrate was strongly determined by the total amount of cells, viable and dead, in the culture broth, but an influence of the centrifugation parameters (feed rate, times of separation and discharge, and rotor speed) was observed. This understanding of the separation process inside the centrifuge is important and may apply to other similar devices. Monoclonal antibodies were not retained in the bioreactor during centrifugation perfusion. However, whereas similar growth rates were obtained in perfusion cultures using either continuous centrifugation or filtration, MAb concentrations were 35% lower in the former case. Utilization of the centrifuge in an intermittent fashion decreased the daily cell residence time outside the bioreactor, the daily pelleted-cell residence time in the centrifuge, and the frequency of cell passage to the centrifuge. This led to higher viable-cell numbers in the bioreactor and an accompanying increase in MAb concentrations, 225-250 mg of IgM L-1, equal to the performance of filter-based perfusion systems with the same cell line. It was hypothesized that having cells periodically packed at the bottom of the centrifuge insert (up to 800 x 10(6) cells mL-1) is deleterious to the culture by exposing the pelleted cells to prolonged nutrient limitations.
为了开发一种基于悬浮培养灌注的高流速工艺，避免基于过滤器的细胞分离装置固有的污染和抗体滞留问题，我们对Centritech实验室离心机进行了评估，并为其在无蛋白培养基中进行杂交瘤细胞灌注培养的开发做出了贡献。培养启动结果表明，在转瓶和与生物反应器相连的连续离心培养中，细胞生长和单克隆抗体（MAb）产生率相似。该离心机能够有效地将活细胞与死细胞分离。活细胞回收率从未低于98%，而死细胞回收率通常约为80%。离心机上清液和浓缩物中的细胞含量很大程度上取决于培养液中活细胞和死细胞的总量，但也观察到离心参数（进料速率、分离和排放时间以及转子速度）的影响。对离心机内部分离过程的这种理解很重要，可能适用于其他类似装置。在离心灌注过程中，单克隆抗体不会保留在生物反应器中。然而，尽管使用连续离心或过滤的灌注培养获得了相似的生长速率，但在前一种情况下，MAb浓度低35%。间歇性使用离心机减少了生物反应器外细胞的每日停留时间、离心机中细胞沉淀的每日停留时间以及细胞进入离心机的频率。这导致生物反应器中活细胞数量增加，同时MAb浓度也随之增加，达到225 - 250 mg IgM L-1，与使用相同细胞系的基于过滤器的灌注系统性能相当。据推测，使细胞定期堆积在离心机插入物底部（高达800×10⁶个细胞 mL⁻¹）会使沉淀细胞长时间处于营养限制状态，从而对培养有害。
