当前位置：
X-MOL 学术
›
Biotechnol. J.
›
论文详情
Our official English website, www.x-mol.net, welcomes your
feedback! (Note: you will need to create a separate account there.)
Comprehensive characterization of dihydrofolate reductase‐mediated gene amplification for the establishment of recombinant human embryonic kidney 293 cells producing monoclonal antibodies
Biotechnology Journal ( IF 3.2 ) Pub Date : 2020-12-13 , DOI: 10.1002/biot.202000351 Sang Yoon Lee 1 , Minhye Baek 1 , Gyun Min Lee 1
Biotechnology Journal ( IF 3.2 ) Pub Date : 2020-12-13 , DOI: 10.1002/biot.202000351 Sang Yoon Lee 1 , Minhye Baek 1 , Gyun Min Lee 1
Affiliation
|
Human embryonic kidney 293 (HEK293) cells with glycosylation machinery have emerged as an alternative host cell line for stable expression of therapeutic glycoproteins. To characterize dihydrofolate reductase/methotrexate (DHFR/MTX)‐mediated gene amplification in HEK293 cells, an expression vector containing dhfr and monoclonal antibody (mAb) gene was transfected into dhfr‐deficient HEK293 cells generated by knocking out dhfr and dhfrl1 in HEK293E cells. Due to the improved selection stringency, mAb‐producing parental cell pools could be generated in the absence of MTX. When subjected to stepwise selection for increasing MTX concentrations such as 1, 10, and 100 nM, there was an increase in the specific mAb productivity (qmAb) of the parental cell pool upon DHFR/MTX‐mediated gene amplification. High producing (HP) clones with a qmAb of more than 2‐fold of the corresponding cell pool could be obtained using the limiting dilution method. The qmAb of most HP clones obtained from cell pools at elevated MTX concentrations significantly decreased during long‐term culture (3 months) in the absence of selection pressure. However, some HP clones could maintain high qmAb during long‐term culture. Taken together, a stable HP recombinant HEK293 cell line can be established using DHFR/MTX‐mediated gene amplification together with dhfr− HEK293 host cells.
中文翻译：
全面表征二氢叶酸还原酶介导的基因扩增，用于建立产生单克隆抗体的重组人胚胎肾293细胞
具有糖基化机制的人类胚胎肾293（HEK293）细胞已经出现，成为稳定表达治疗性糖蛋白的替代宿主细胞系。为了表征二氢叶酸还原酶/氨甲蝶呤（DHFR / MTX）在HEK293细胞介导的基因扩增，将含有表达载体DHFR和单克隆抗体（mAb）的基因转染到DHFR缺陷型HEK293通过敲除生成的细胞的dhfr和dhfrl1在HEK293E细胞。由于提高了选择严格性，可以在不存在MTX的情况下生成产生mAb的亲本细胞库。进行逐步选择以增加MTX浓度（例如1、10和100 nM）时，比mAb生产率有所提高（DHFR / MTX介导的基因扩增后的亲本细胞池的单抗（q mAb）。使用有限稀释法可以得到q mAb超过相应细胞库2倍的高产（HP）克隆。在没有选择压力的情况下，长期培养（3个月）期间，从细胞池中以较高的MTX浓度获得的大多数HP克隆的q mAb显着降低。但是，某些HP克隆可以在长期培养过程中维持较高的q mAb。综上所述，可以使用DHFR / MTX介导的基因扩增以及dhfr - HEK293宿主细胞建立稳定的HP重组HEK293细胞系。
更新日期：2020-12-13
中文翻译：
全面表征二氢叶酸还原酶介导的基因扩增，用于建立产生单克隆抗体的重组人胚胎肾293细胞
具有糖基化机制的人类胚胎肾293（HEK293）细胞已经出现，成为稳定表达治疗性糖蛋白的替代宿主细胞系。为了表征二氢叶酸还原酶/氨甲蝶呤（DHFR / MTX）在HEK293细胞介导的基因扩增，将含有表达载体DHFR和单克隆抗体（mAb）的基因转染到DHFR缺陷型HEK293通过敲除生成的细胞的dhfr和dhfrl1在HEK293E细胞。由于提高了选择严格性，可以在不存在MTX的情况下生成产生mAb的亲本细胞库。进行逐步选择以增加MTX浓度（例如1、10和100 nM）时，比mAb生产率有所提高（DHFR / MTX介导的基因扩增后的亲本细胞池的单抗（q mAb）。使用有限稀释法可以得到q mAb超过相应细胞库2倍的高产（HP）克隆。在没有选择压力的情况下，长期培养（3个月）期间，从细胞池中以较高的MTX浓度获得的大多数HP克隆的q mAb显着降低。但是，某些HP克隆可以在长期培养过程中维持较高的q mAb。综上所述，可以使用DHFR / MTX介导的基因扩增以及dhfr - HEK293宿主细胞建立稳定的HP重组HEK293细胞系。
京公网安备 11010802027423号