With the increasing dominance of monoclonal antibodies (mAbs) in the biopharmaceutical industry and smaller antibody fragments bringing notable advantages over full-length antibodies, it is of considerable significance to choose the most suitable production system. Although mammalian expression system has been the preferred choice in recent years for mAbs production, E. coli could be the favorable host for non-glycosylated small antibody fragments due to the emergence of new engineered E. coli strains capable of forming disulfide-bonds in their cytoplasm.

In this study, non-glycosylated anti-TNF-α Fab’ moiety of Certolizumab pegol, produced by periplasmic expression in E. coli in previous studies, was produced in the cytoplasm of E. coli SHuffle strain. The results indicated that it is biologically functional by testing the antigen-binding activity via indirect ELISA and inhibition of TNF-α induced cytotoxicity using MTT test. Major factors affecting protein production and, optimized culture conditions were examined by analyzing growth characteristics and patterns of expression in 24 h of post-induction cultivation and, optimization of culture conditions by response surface methodology considering temperature, time of induction and concentration of inducer in small (tube) and shake-flask scale. Based on the results, temperature had the most significant influence on functional protein yield while exerting different impacts in small and shake-flask scales, which indicated that cultivation volume is also an important factor that should be taken into account in optimization process. Furthermore, richness of medium and slower cellular growth rate improved specific cellular yield of functional protein by having a positive effect on the solubility of Fab' antibody.

随着单克隆抗体（mAb）在生物制药行业的主导地位日益增强，更小的抗体片段相对于全长抗体具有显着优势，选择最合适的生产系统具有相当重要的意义。尽管近年来哺乳动物表达系统一直是单克隆抗体生产的首选，但由于新的工程大肠杆菌菌株的出现能够在其体内形成二硫键，因此大肠杆菌可能是非糖基化小抗体片段的有利宿主。细胞质。

在这项研究中，非糖基化的抗TNF-α赛妥珠单抗的的Fab”部分，通过周质表达中制备的大肠杆菌在以前的研究中，在的细胞质中产生大肠杆菌洗牌应变。结果表明，通过间接 ELISA 测试抗原结合活性和使用 MTT 测试抑制 TNF-α 诱导的细胞毒性，它具有生物学功能。通过分析诱导后培养 24 小时内的生长特征和表达模式，以及考虑温度、诱导时间和诱导剂浓度的响应面法优化培养条件，研究了影响蛋白质生产和优化培养条件的主要因素。 （管）和摇瓶秤。结果表明，温度对功能蛋白产量的影响最显着，而在小规模和摇瓶规模上的影响不同，这表明培养体积也是优化过程中应考虑的重要因素。