FILTRATION-BASED PERFUSION OF HYBRIDOMA CULTURES IN PROTEIN-FREE MEDIUM - REDUCTION OF MEMBRANE FOULING BY MEDIUM SUPPLEMENTATION WITH DNASE-I
FILTRATION-BASED PERFUSION OF HYBRIDOMA CULTURES IN PROTEIN-FREE MEDIUM - REDUCTION OF MEMBRANE FOULING BY MEDIUM SUPPLEMENTATION WITH DNASE-I
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DOI：
10.1002/bit.260430902
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发表时间：
1994-04-15
影响因子：
3.8
通讯作者：
MASSIE, B
中科院分区：
文献类型：
--
作者：
MERCILLE, S;JOHNSON, M;MASSIE, B
在这项研究中，一个基于过滤的灌注过程中开发的生产单克隆抗体（IgM）的悬浮杂交瘤细胞，生长在无蛋白的培养基。发现使用无蛋白质培养基进行灌注培养产生了许多可见的悬浮颗粒的形成，这些颗粒由聚集成纤维材料的死细胞和细胞碎片组成。在这样的无蛋白培养物中观察到令人惊讶的高表观活力。此外，膜污染发生更迅速地在无蛋白质的培养基比在传统的血清补充培养基。通过将脱氧核糖核酸酶I（DNase I）添加到无蛋白质培养基中，可以防止聚集体的形成，并更准确地跟踪总细胞群的进化。此外，DNA酶I显着减少污染的过滤膜，并且，对于两种不同类型的分离系统（错流和涡流过滤）和两种不同类型的膜（聚碳酸酯和磺化聚砜）。从这些结果可以清楚地看出，细胞死亡后释放的DNA片段的存在在膜污染中起着重要作用。发现使用涡流过滤模块的过滤膜的寿命比使用静态板框错流过滤模块的过滤膜的寿命大得多。结合DNA酶I使用涡旋流过滤允许维持灌注培养物超过1个月，而没有膜污染或抗体保留，并且具有恒定的渗透物IgM浓度为250 mg/L。在涡流过滤模块中，杂交细胞似乎逐渐适应增加的转速。(C)1994年，John Wiley and Sons，Inc.
In this study, a filtration-based perfusion process was developed for the production of monoclonal antibodies (IgM) by suspended hybridoma cells,grown in protein-free medium. It was found that the use of protein-free medium for perfusion culture generated the formation of numerous visible suspended particles consisting of dead cells and cellular debris aggregated into fibrous material. Surprisingly high apparent viabilities were observed in such protein-free cultures. In addition, membrane fouling occurred more rapidly in protein-free medium than in conventional serum-supplemented medium. By the addition of deoxyribonuclease I (DNase I) to the protein-free medium, it was possible to prevent the formation of aggregates and to follow the evolution of the total cell population more accurately. Moreover, DNase I significantly reduced the fouling of filtration membranes, and that, for two different types of separation systems (cross-flow and vortex-flow filtration) and two different types of membranes (polycarbonate and hydrophilized polysultone). From these results, it is clear that the presence of DNA fragments liberated following cellular death is playing an important role in membrane fouling. Longevity of filtration membranes was found to be considerably greater using a vortex-flow filtration module than with a static plate-and-frame cross-flow filtration module. The use of vortex-flow filtration in conjunction with DNase I allowed maintenance of perfusion cultures for more than 1 month without membrane fouling or antibody retention and with a constant permeate IgM concentration of 250 mg/L. Hybridoma cells appeared to gradually adapt to increasing rotational speed in the vortex-flow filtration module. (C) 1994 John Wiley and Sons, Inc.