Implementation of a Design of Experiments to Improve Periplasmic Yield of Functional ScFv Antibodies in a Phage...

Production of functional recombinant antibody fragments in the periplasm of E. coli 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 (OD₆₀₀) 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 OD₆₀₀ 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 OD₆₀₀ 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."