Behravan Aidin, Hashemi Atieh
Department of Pharmaceutical Biotechnology, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehan, I.R. Iran.
Res Pharm Sci. 2021 Mar 5;16(2):153-164. doi: 10.4103/1735-5362.310522. eCollection 2021 Apr.
The epithelial cell adhesion molecule (EpCAM), is one of the first cancer- associated markers discovered. Its overexpression in cancer stem cells, epithelial tumors, and circulating tumor cells makes this molecule interesting for targeted cancer therapy. So, in recent years scFv fragments have been developed for EpCAM targeting.
In this study, an scFv against EpCAM extracellular domain (EpEX) derived from 4D5MOC-B humanized mAb was expressed in k12 strain, and in order to obtain the optimum culture conditions in chemically defined minimal medium, response surface methodology (RSM) was employed. According to the RSM-CCD method, a total of 30 experiments were designed to investigate the effects of various parameters including isopropyl-b-D-thiogalactopyranoside (IPTG) concentration, cell density before induction, post-induction time, and post-induction temperature on anti EpEX-scFv expression level.
FINDINGS/RESULTS: At the optimum conditions (induction at cell density 0.8 with 0.8 mM IPTG for 24 h at 37 °C), the recombinant anti EpEX-scFv was produced at a titer of 197.33 μg/mL that was significantly consistent with the prediction of the model.
The optimized-culture conditions obtained here for efficient production of anti EpEX-scFv in shake flask cultivation on a chemically defined minimal medium could be applied to large- scale fermentation for the anti EpEX-scFv production.
上皮细胞粘附分子（EpCAM）是最早发现的癌症相关标志物之一。它在癌症干细胞、上皮肿瘤和循环肿瘤细胞中的过表达使得该分子成为靶向癌症治疗的研究热点。因此，近年来已开发出针对EpCAM的单链抗体片段（scFv）。
在本研究中，一种源自4D5MOC - B人源化单克隆抗体的抗EpCAM细胞外结构域（EpEX）的scFv在k12菌株中表达，为了在化学成分明确的基础培养基中获得最佳培养条件，采用了响应面法（RSM）。根据RSM - CCD方法，共设计了30个实验，以研究包括异丙基 - β - D - 硫代半乳糖苷（IPTG）浓度、诱导前细胞密度、诱导后时间和诱导后温度等各种参数对抗EpEX - scFv表达水平的影响。
在最佳条件下（细胞密度为0.8时，用0.8 mM IPTG在37°C诱导24小时），重组抗EpEX - scFv的产量为197.33μg/mL，与模型预测结果显著一致。
在此获得的用于在化学成分明确的基础培养基中摇瓶培养高效生产抗EpEX - scFv的优化培养条件可应用于抗EpEX - scFv生产的大规模发酵。
