Chemical Engineering Program, Cell Culture Engineering Laboratory (LECC), COPPE, Federal University of Rio de Janeiro (UFRJ), Cx. Postal 68502, Rio de Janeiro, RJ, 21941-972, Brazil.
Biochemistry Program, Federal University of Rio de Janeiro (UFRJ), IQ, Rio de Janeiro, 21941-909, Brazil.
Biotechnol Lett. 2020 Dec;42(12):2511-2522. doi: 10.1007/s10529-020-02952-8. Epub 2020 Jul 16.
To compare different approaches for the expression of an anti-PCSK9 biosimilar monoclonal antibody (mAb) in CHO cells using IRES-mediated tricistronic plasmid vectors combining different signal peptides, IRES elements and selection markers.
Transient transfection indicated a similar level of secreted mAb 48 h post-transfection for all constructs. However, transfections carried out with circular plasmids showed a higher expression than with linearized plasmids. After two months under selection pressure, only part of the transfected pools recovered. The cultures co-transfected using two antibiotics as selection markers for double selection did not recover. Growth, metabolism and mAb production profiles of the only part of the transfected pools recovered resulting stable pools were compared and the stable pool transfected with circular L1-LC-IRES-H7-HC-IRES-NEO plasmid was chosen for further studies, due to higher cell growth and mAb production. Critical quality attributes of the protein A-purified mAb such as purity, homogeneity, binding affinity to PCSK9, and amino acid sequence were assessed confirming the success of the approach adopted in this study.
The expression platform proposed showed to be efficient to produce a high-quality anti-PCSK9 mAb in stable CHO cell pools and provides benchmarks for fast production of different mAbs for characterization, formulation studies and pre-clinical investigation.
比较使用 IRES 介导的三顺反子质粒载体结合不同信号肽、IRES 元件和选择标记物在 CHO 细胞中表达抗 PCSK9 生物类似物单克隆抗体（mAb）的不同方法。
瞬时转染表明，所有构建体在转染后 48 小时内分泌的 mAb 水平相似。然而，与线性化质粒相比，圆形质粒的转染显示出更高的表达水平。在选择压力下两个月后，只有部分转染池得到恢复。使用两种抗生素作为双选择的选择标记物进行共转染的培养物没有恢复。比较了仅部分转染池恢复的稳定池的生长、代谢和 mAb 生产曲线，并选择了用圆形 L1-LC-IRES-H7-HC-IRES-NEO 质粒转染的稳定池进行进一步研究，因为该稳定池的细胞生长和 mAb 生产更高。对蛋白 A 纯化的 mAb 的关键质量属性（如纯度、均一性、与 PCSK9 的结合亲和力和氨基酸序列）进行了评估，证实了本研究中采用的方法的成功。
所提出的表达平台被证明能够有效地在稳定的 CHO 细胞池生产高质量的抗 PCSK9 mAb，并为不同 mAb 的快速生产提供了特征描述、配方研究和临床前研究的基准。
