Rapid automated selection of mammalian cell line secreting high level of humanized monoclonal antibody using Clone Pix FL system and the correlation between exterior median intensity and antibody productivity
Rapid automated selection of mammalian cell line secreting high level of humanized monoclonal antibody using Clone Pix FL system and the correlation between exterior median intensity and antibody productivity
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DOI：
10.2225/vol14-issue2-fulltext-7
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发表时间：
2011-03-15
影响因子：
2.7
通讯作者：
Kamal, Nazlee
中科院分区：
文献类型：
--
作者：
Dharshanan, Suba;Chong, Heilly;Kamal, Nazlee
高产哺乳动物细胞系的选择是生物制药生产工艺开发的关键步骤。以前，通过有限稀释法克隆用于分离分泌高水平人源化C2单克隆抗体的单克隆NS0细胞。然而，有限稀释法是耗时的，具有低概率的单克隆性，并显着限制的克隆数，可以切实可行的筛选。为了减少持续时间并增加获得具有高单克隆性的高产克隆的概率，使用自动菌落挑选器，克隆Pix FL系统来代替有限稀释法。我们能够筛选1 × 105个分泌人源化单克隆抗体的克隆，并且仅在7天内就选择了高产克隆。简而言之，使用半固体培养基分别培养单细胞并使其增殖成离散克隆。半固体培养基的高粘度性质将分泌产物保留在相关克隆附近。利用Clone Pix FL系统对所有克隆进行筛选，并自动分离具有不同外部荧光强度的生产克隆。我们能够分离罕见的高生产者（> 3000 FU），频率低至0.003%的人口。还进行定量ELISA以评估克隆的荧光强度与其相应的抗体生产率之间的相关性。与大于1000 FU的克隆相比，荧光强度<1000 FU的克隆显示出相对较低的抗体产率;然而，在此之上，产量与荧光强度的增加没有相关性。因此，尽管克隆Pix FL系统的高通量、快速和自动化性质允许在短时间内筛选大量细胞，同时也增加了获得稀有和珍贵的高产克隆的可能性，但下游分析对于确定"实际"和稳定的高产克隆仍然至关重要。
The selection of high-producing mammalian cell lines is a crucial step in process development for the production of biopharmaceuticals. Previously, cloning by limiting dilution method was used to isolate monoclonal NS0 cells secreting high levels of humanized-C2 monoclonal antibodies. However limiting dilution method is time consuming, has low probability of monoclonality and is significantly limited by the number of clones that can be feasibly screened. In order to minimize the duration and to increase the probability of obtaining high-producing clones with high monoclonality, an automated colony picker, Clone Pix FL system was used to replace limiting dilution method. We were able to screen 1 x 10(5) clones secreting humanized monoclonal antibodies and high producer clones were selected in just 7 days. Briefly, semi-solid media was used to immobilize single cells separately and allow them to proliferate into discrete clones. The high viscosity nature of the semi-solid media retains the secreted products in the vicinity of the associated clones. Using Clone Pix FL system, all clones were screened and the producer clones with different exterior fluorescent intensities were automatically isolated. We were able to isolate rare high-producers (> 3000 FU) with frequency of as low as 0.003% of the population. A quantitative ELISA was also performed to evaluate the correlation between the fluorescence intensity of clones with its corresponding antibody productivity. Clones with fluorescence intensity of < 1000 FU showed relatively low antibody productivity compared with those greater than 1000 FU; however above this there was no correlation of production with the increase in fluorescence intensity. Hence, although the high-throughput, rapid and automated nature of Clone Pix FL system allows the screening of large number of cells in a short period of time with also an increased in the probability of obtaining rare and precious high-producing clones, downstream analysis are still vital to determine the 'actual' and stable high producer clones.