Aghdam Marjan Abri, Tohidkia Mohammad Reza, Ghamghami Elham, Ahmadikhah Asadollah, Khanmahamadi Morteza, Baradaran Behzad, Mokhtarzadeh Ahad
Department of Biological Science, Faculty of Basic Science, Higher Education Institute of Rab-Rashid, Tabriz, Iran.
Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences, Tabriz, Iran.
Adv Pharm Bull. 2022 May;12(3):583-592. doi: 10.34172/apb.2022.061. Epub 2021 Jul 3.
Production of functional recombinant antibody fragments in the periplasm of is a prerequisite step to achieve sufficient reagent for preclinical studies. Thus, the cost-effective and lab-scale production of antibody fragments demands the optimization of culture conditions. The culture conditions such as temperature, optical density (OD) at induction, induction time, and IPTG concentration were investigated to optimize the functional expression of a phage-derived scFv molecule using a design of experiment (DoE). Additionally, the effects of different culture media and osmolyte supplements on the expression yield of scFv were examined. The developed 2FI regression model indicated the significant linear effect of the incubation temperature, the induction time, and the induction OD on the expression yield of functional scFv. Besides, the statistical analysis indicated that two significant interactions of the temperature/induction time and the temperature/induction OD significantly interplay to increase the yield. Further optimization showed that the expression level of functional scFv was the most optimal when the cultivation was undertaken either in the TB medium or in the presence of media supplements of 0.5 M sorbitol or 100 mM glycine betaine. In the present study, for the first time, we successfully implemented DoE to comprehensively optimize the culture conditions for the expression of scFv molecules in a phage antibody display setting, where scFv molecules can be isolated from a tailor-made phage antibody library known as "Human Single Fold scFv Library I."
在周质中生产功能性重组抗体片段是为临床前研究获得足够试剂的前提步骤。因此，抗体片段的经济高效且实验室规模的生产需要优化培养条件。利用实验设计（DoE）研究了温度、诱导时的光密度（OD）、诱导时间和异丙基-β-D-硫代半乳糖苷（IPTG）浓度等培养条件，以优化噬菌体衍生的单链抗体片段（scFv）分子的功能性表达。此外，还研究了不同培养基和渗透剂补充剂对scFv表达产量的影响。所建立的二阶响应面（2FI）回归模型表明，培养温度、诱导时间和诱导OD对功能性scFv的表达产量具有显著的线性影响。此外，统计分析表明，温度/诱导时间和温度/诱导OD这两个显著的相互作用对提高产量有显著影响。进一步优化表明，当在TB培养基中或在添加0.5 M山梨醇或100 mM甘氨酸甜菜碱的培养基中进行培养时，功能性scFv的表达水平最为理想。在本研究中，我们首次成功实施DoE，全面优化了噬菌体抗体展示环境中scFv分子表达的培养条件，其中scFv分子可从一个名为“人类单折叠scFv文库I”的定制噬菌体抗体文库中分离得到。
