Regulation of recombinant monoclonal antibody production in Chinese hamster ovary cells: A comparative study of gene copy number, mRNA level, and protein expression
Regulation of recombinant monoclonal antibody production in Chinese hamster ovary cells: A comparative study of gene copy number, mRNA level, and protein expression
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DOI：
10.1021/bp0501524
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发表时间：
2006-01-01
影响因子：
2.9
通讯作者：
Sharfstein, ST
中科院分区：
文献类型：
--
作者：
Jiang, Z;Huang, Y;Sharfstein, ST
我们对具有不同重组单克隆抗体生产率（qAb）的CHO细胞进行了比较分析，并研究了MAb生产的调节。研究了两个家族的CHO细胞，每个家族由一个亲本和两个子代细胞系组成，通过逐步增加甲氨蝶呤（MTX）浓度产生。在中期生长期通过Southern和北方印迹法定量MAb重链（HC）和轻链（LC）基因拷贝数和mRNA水平。与亲本细胞系相比，我们观察到高产细胞系的基因拷贝数扩增2至3倍。然而，高生产者的mRNA水平高5至7倍，与qAb增加5至7倍密切相关。这些结果通过实时qPCR分析证实。我们的研究表明，MTX介导的单克隆抗体过表达的结果从两个基因拷贝数的增加和更有效的转录每个基因拷贝。我们还观察到HC与LC基因拷贝比可能影响单克隆抗体表达系统中的蛋白质产量。
We performed a comparative analysis on CHO cells with varying recombinant monoclonal antibody production rates (qAb) and investigated the regulation of MAb production. Two families of CHO cells each composed of one parental and two progeny cell lines, generated by stepwise increases in methotrexate (MTX) concentration, were studied. The MAb heavy chain (HC) and light (LC) gene copy numbers and mRNA levels were quantitated by Southern and northern blotting in midexponential growth phase. We observed 2- to 3-fold amplification in gene copy numbers in high producing cell lines when compared with parental cell lines. However, the mRNA levels in the high producers were 5- to 7-fold higher, correlating well with the 5- to 7-fold increase in qAb. These results were confirmed by real-time qPCR analysis. Our study reveals that the MTX-mediated MAb overexpression results from both an increase in the gene copy number and more efficient transcription of each gene copy. We also observed that the HC to LC gene copy ratio may affect the protein yield in MAb expression systems.