Hartman Taymar E, Sar Nalin, Genereux Kimberly, Barritt Diana S, He Yimin, Burky John E, Wesson Mark C, Tso J Yun, Tsurushita Naoya, Zhou Weichang, Sauer Paul W
Process Sciences and Engineering, PDL BioPharma, Inc., 34801 Campus Drive, Fremont, California 94555, USA.
Biotechnol Bioeng. 2007 Feb 1;96(2):294-306. doi: 10.1002/bit.21099.
Presented is an antibody production platform based on the fed-batch culture of recombinant NS0-derived cell lines. NS0 host cells, obtained from the European Collection of Cell Cultures (ECACC, Salisbury, UK, Part No. 85110503), were first adapted to grow in a protein-free, cholesterol-free medium. The resulting host cell line was designated NS0-PFCF (protein-free, cholesterol-free). The five production cell lines presented here were generated using a common protocol consisting of transfection by electroporation and subcloning. The NS0-PFCF host cell line was transfected using a single expression vector containing the Escherichia coli xanthine-guanine phosphoribosyl transferase gene (gpt), and the antibody heavy and light chain genes driven by the CMV promoter. The five cell lines were chosen after one to three rounds of iterative subcloning, which resulted in a 19-64% increase in antibody productivity when four mother-daughter cell pairs were cultured in a fed-batch bioreactor process. The production cell lines were genetically characterized to determine antibody gene integrity, nucleotide sequences, copy number, and the number of insertion sites in the NS0 cell genome. Genetic characterization data indicate that each of the five production cell lines has a single stably integrated copy of the antibody expression vector, and that the antibody genes are correctly expressed. Stability of antibody production was evaluated for three of the five cell lines by comparing the early stage seed bank with the Working Cell Bank (WCB). Antibody productivity was shown to be stable in two of three cell lines evaluated, while one of the cell lines exhibited a 20% drop in productivity after passaging for approximately 4 weeks. These five NS0-derived production cell lines were successfully cultured to produce antibodies with acceptable product quality attributes in a standardized fed-batch bioreactor process, consistently achieving an average specific productivity of 20-60 pg/cell-day, and a volumetric productivity exceeding 120 mg/L-day (Burky et al., 2006). In contrast to the commonly available NS0 host cell line, which requires serum and cholesterol for growth, and the commonly used expression vector system, which uses a proprietary glutamine synthetase selection marker (GS-NS0), these NS0 cells are cholesterol-independent, grow well in a protein-free medium, use a non-proprietary selection marker, and do not require gene amplification for productivity improvement. These characteristics are advantageous for use of this NS0 cell line platform for manufacturing therapeutic antibodies.
本文介绍了一种基于重组NS0衍生细胞系补料分批培养的抗体生产平台。从欧洲细胞培养物保藏中心（ECACC，英国索尔兹伯里，编号85110503）获得的NS0宿主细胞，首先被适应在无蛋白、无胆固醇的培养基中生长。由此产生的宿主细胞系被命名为NS0-PFCF（无蛋白、无胆固醇）。这里展示的五个生产细胞系是使用由电穿孔转染和亚克隆组成的通用方案产生的。使用含有大肠杆菌黄嘌呤-鸟嘌呤磷酸核糖转移酶基因（gpt）以及由CMV启动子驱动的抗体重链和轻链基因的单个表达载体转染NS0-PFCF宿主细胞系。在进行一到三轮迭代亚克隆后选择了这五个细胞系，当在补料分批生物反应器过程中培养四对母-子细胞对时，抗体生产力提高了19%-64%。对生产细胞系进行基因特征分析以确定抗体基因的完整性、核苷酸序列、拷贝数以及在NS0细胞基因组中的插入位点数量。基因特征分析数据表明，五个生产细胞系中的每一个都有抗体表达载体的单个稳定整合拷贝，并且抗体基因正确表达。通过将早期种子库与工作细胞库（WCB）进行比较，对五个细胞系中的三个进行了抗体生产稳定性评估。在评估的三个细胞系中，有两个显示抗体生产力稳定，而其中一个细胞系在传代约4周后生产力下降了20%。这五个NS0衍生的生产细胞系在标准化的补料分批生物反应器过程中成功培养，以产生具有可接受产品质量属性的抗体，始终实现平均比生产力为20-60 pg/细胞·天，体积生产力超过120 mg/L·天（Burky等人，2006年）。与通常需要血清和胆固醇来生长的NS0宿主细胞系以及使用专有谷氨酰胺合成酶选择标记（GS-NS0）的常用表达载体系统相比，这些NS0细胞不依赖胆固醇，在无蛋白培养基中生长良好，使用非专有选择标记，并且不需要基因扩增来提高生产力。这些特性有利于使用该NS0细胞系平台来制造治疗性抗体。
