Statistical optimization of culture conditions for expression of recombinant humanized anti-EpCAM single-chain antibody using response surface methodology

Aidin Behravan , Atieh Hashemi


Background and purpose: 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.

Experimental approach: In this study, an scFv against EpCAM extracellular domain (EpEX) derived from 4D5MOC-B humanized mAb was expressed in Escherichia coli 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.

Conclusion and implication: 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.


Anti EpEX; 4D5MOC-B humanized mAb; Response surface methodology; scFv.

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Patriarca C, Macchi RM, Marschner AK, Mellstedt H. Epithelial cell adhesion molecule expression (CD326) in cancer: a short review. Cancer Treat Rev. 2012;38(1):68-75.

DOI: 10.1016/j.ctrv.2011.04.002.

Rasooli F, Hashemi A. Efficient expression of EpEX in the cytoplasm of Escherichia coli using thioredoxin fusion protein. Res Pharm Sci. 2019;14(6):554-565.

DOI: 10.4103/1735-5362.272564.

Simon M, Stefan N, Plückthun A, Zangemeister-Wittke U. Epithelial cell adhesion molecule-targeted drug delivery for cancer therapy. Expert Opin Drug Deliv. 2013;10(4):451-468.

DOI: 10.1517/17425247.2013.759938.

Ahmad ZA, Yeap SK, Ali AM, Ho WY, Alitheen NBM, Hamid M. ScFv antibody: principles and clinical application. Clin Dev Immunol. 2012;2012:980250,1-16.

DOI: 10.1155/2012/980250.

Mohammadgholizad F, Hashemi A. Construction of recombinant Pichia pastoris expressing single-chain antibody fragment against extracellular domain of EpCAM. Koomesh. 2019;21:743-750.

Willuda J, Honegger A, Waibel R, Schubiger PA, Stahel R, Zangemeister-Wittke U, et al. High thermal stability is essential for tumor targeting of antibody fragments: engineering of a humanized anti-epithelial glycoprotein-2 (epithelial cell adhesion molecule) single-chain Fv fragment. Cancer Res. 1999;59(22):5758-5767.

Safdari Y, Ahmadzadeh V, Khalili M, Jaliani HZ, Zarei V, Erfani-Moghadam V. Use of single-chain antibody derivatives for targeted drug delivery. Mol Med. 2016;22:258-270.

DOI: 10.2119/molmed.2016.00043.

Rosano GL, Ceccarelli EA. Recombinant protein expression in Escherichia coli: advances and challenges. Front Microbiol. 2014;5:172-188.

DOI: 10.3389/fmicb.2014.00172.

Soheili S, Jahanian-Najafabadi A, Akbari V. Evaluation of soluble expression of recombinant granulocyte macrophage stimulating factor (rGM-CSF) by three different E. coli strains. Res Pharm Sci. 2020;15(3):218-225.

DOI: 10.4103/1735-5362.288424.

Jia B, Jeon CO. High-throughput recombinant protein expression in Escherichia coli: current status and future perspectives. Open Biol. 2016;6(8):160196,1-17.

DOI: 10.1098/rsob.160196.

Salehinia J, Sadeghi HMM, Abedi D, Akbari V. Improvement of solubility and refolding of an anti-human epidermal growth factor receptor 2 single-chain antibody fragment inclusion bodies. Res Pharm Sci. 2018;13(6):566-574.

DOI: 10.4103/1735-5362.245968.

Stanbury PF, Whitaker A, Hall SJ. Principle of Fermentation Thechnology. 3rd ed. Elsevier; 2016. pp: 769-777.

DOI: 10.1016/C2013-0-00186-7.

Zhang J, Greasham R. Chemically defined media for commercial fermentations. Appl Microbiol Biotechnol. 1999;51(4):407-421.

DOI: 10.1007/s002530051411.

Uhoraningoga A, Kinsella GK, Henehan GT, Ryan BJ. The goldilocks approach: a review of employing design of experiments in prokaryotic recombinant protein production. Bioengineering (Basel). 2018;5(4):89-115.

DOI: 10.3390/bioengineering5040089.

Papaneophytou CP, Kontopidis G. Statistical approaches to maximize recombinant protein expression in Escherichia coli: a general review. Protein Expr Purif. 2014;94:22-32.

DOI: 10.1016/j.pep.2013.10.016.

Soulari RN, Basafa M, Rajabibazl M, Hashemi A. Effective strategies to overcome the insolubility of recombinant ScFv antibody against EpCAM extracellular domain in E. coli. Int J Pept Res Ther. 2020;26:2465-2475.

DOI: 10.1007/s10989-020-10044-4.

Waegeman H, De Lausnay S, Beauprez J, Maertens J, De Mey M, Soetaert W. Increasing recombinant protein production in Escherichia coli K12 through metabolic engineering. N Biotechnol. 2013;30(2):255-261.

DOI: 10.1016/j.nbt.2011.11.008.

Bühning M, Friemel M, Leimkühler S. Functional complementation studies reveal different interaction partners of Escherichia coli IscS and human NFS1. Biochemistry. 2017;56(34):4592-4605.

DOI: 10.1021/acs.biochem.7b00627.

Tseng CL, Leng CH. Influence of medium components on the expression of recombinant lipoproteins in Escherichia coli. Appl Microbiol Biotechnol. 2012;93(4):1539-1552.

DOI: 10.1007/s00253-011-3516-8.

Palmer I, Wingfield PT. Preparation and extraction of insoluble (inclusion-body) proteins from Escherichia coli. Curr Protoc Protein Sci. 2004;6(6.3.):1-25.

DOI: 10.1002/0471140864.ps0603s38.

Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 2012;9(7):671-675.

DOI: 10.1038/nmeth.2089.

Kunert R, Reinhart D. Advances in recombinant antibody manufacturing. Appl Microbiol Biotechnol. 2016;100(8):3451–3461.

DOI: 10.1007/s00253-016-7388-9.

Rezaei M, Zarkesh-Esfahani SH, Gharagozloo M. The effect of different media composition and temperatures on the production of recombinant human growth hormone by CHO cells. Res Pharm Sci. 2013;8(3):211-217.

Shafiee F, Rabbani M, Jahanian-Najafabadi A. Optimization of the expression of DT386-BR2 fusion protein in Escherichia coli using response surface methodology. Adv Biomed Res. 2017;6(1):22-31.

DOI: 10.4103/2277-9175.201334.

Akbari V, Sadeghi HMM, Jafarian-Dehkordi A, Chou CP, Abedi D. Optimization of a single-chain antibody fragment overexpression in Escherichia coli using response surface methodology. Res Pharm Sci. 2015;10(1):75-83.

Volontè F, Piubelli L, Pollegioni L. Optimizing HIV-1 protease production in Escherichia coli as fusion protein. Microb Cell Fact. 2011;10(1):53-62.

DOI: 10.1186/1475-2859-10-53.

Sina M, Farajzadeh D, Dastmalchi S. Effects of environmental factors on soluble expression of a humanized anti-TNF-α scFv antibody in Escherichia coli. Adv Pharm Bull. 2015;5(4):455-461.

DOI: 10.15171/apb.2015.062.

Papaneophytou C, Kontopidis G. A comparison of statistical approaches used for the optimization of soluble protein expression in Escherichia coli. Protein Expr Purif. 2016;120:126-137.

DOI: 10.1016/j.pep.2015.12.014.

Papaneophytou CP, Rinotas V, Douni E, Kontopidis G. A statistical approach for optimization of RANKL overexpression in Escherichia coli: purification and characterization of the protein. Protein Expr Purif. 2013;90(1):9-19.

DOI: 10.1016/j.pep.2013.04.005.

Galloway CA, Sowden MP, Smith HC. Increasing the yield of soluble recombinant protein expressed in E. coli by induction during late log phase. Biotechniques. 2003;34(3):524-526.

DOI: 10.2144/03343st04.

Larentis AL, Nicolau JFMQ, Esteves GDS, Vareschini DT, De Almeida FVR, Dos Reis MG, et al. Evaluation of pre-induction temperature, cell growth at induction and IPTG concentration on the expression of a leptospiral protein in E. coli using shaking flasks and microbioreactor. BMC Res Notes. 2014;7(1):671-683.

DOI: 10.1186/1756-0500-7-671.


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