Marjan Abri Aghdam

Purpose: Production of functional recombinant antibody fragments in the periplasm of E. coli isa prerequisite step to achieve sufficient reagent for preclinical studies. Thus, the cost-effectiveand lab-scale production of antibody fragments demands the optimization of culture conditions.Methods: The culture conditions such as temperature, optical density (OD600) at induction,induction time, and IPTG concentration were investigated to optimize the functional expressionof a phage-derived scFv molecule using a design of experiment (DoE). Additionally, the effectsof different culture media and osmolyte supplements on the expression yield of scFv wereexamined.Results: The developed 2FI regression model indicated the significant linear effect of theincubation temperature, the induction time, and the induction OD600 on the expression yieldof functional scFv. Besides, the statistical analysis indicated that two significant interactions ofthe temperature/induction time and the temperature/induction OD600 significantly interplay toincrease the yield. Further optimization showed that the expression level of functional scFvwas the most optimal when the cultivation was undertaken either in the TB medium or in thepresence of media supplements of 0.5 M sorbitol or 100 mM glycine betaine.Conclusion: In the present study, for the first time, we successfully implemented DoE tocomprehensively optimize the culture conditions for the expression of scFv molecules in aphage antibody display setting, where scFv molecules can be isolated from a tailor-made phageantibody library known as "Human Single Fold scFv Library I."

Single-chain variable fragments (scFvs) have gained increased attention among researchers in both academic and industrial fields owing to simple production in E. coli. The E. coli periplasm has been the site of choice for the expression of scFv molecules due to its oxidizing milieu facilitating correctly formation of disulfide bonds. Hence, the recovery of high-yield and biologically active species from the periplasmic space is a critical step at beginning of downstream processing. TES (Tris/EDTA/Sucrose) as a simple and efficient extraction method has been frequently used but under varied extraction conditions, over literature. This study, for the first time, aimed to interrogate the effects of four independent variables (i.e., Tris–HCl concentration, buffer’s pH, EDTA concentration, and incubation time) and their potential interactions on the functional extraction yield of an scFv antibody from the periplasmic space of E. coli. The results indicated that the Tris–HCl concentration and pH are the most significant variables in the TES method and displayed a positive effect at their lower values on the functional extraction yield. Besides, the statistical analysis revealed 4 significant interactions between different variables. Here is the first report on the successful application of a design of experiment based on a central composite design to establish a generic and optimal TES extraction condition. Accordingly, an optimal condition for TES extraction of scFv molecules from the periplasm of HB2151 at the exponential phase was developed as follows: 50 mM Tris–HCl at pH 7.2, 0.53 mM EDTA, and an incubation time of 60 min.