Recombinant Expression of Single-Chain Variable Fragment Antibody: A Comparative Study of Growth Medium Effects

Abstract

Background: Monoclonal antibodies (mAbs) play an important role in immunotherapy by targeting specific antigens and more effectively attacking cancer cells. Antibody fragments, such as single-chain variable fragments (scFvs), can offer several advantages over whole antibodies, including better tumor penetration and tissue clearance, fewer side effects, and easier recombinant production. Cell growth, protein production, and solubility in microbial cell factories have been found to be significantly affected by the composition of the growth medium. Improved solubility reduces aggregation challenges, leading to greater recovery of functional protein and reduced purification costs. Therefore, changing the type of culture medium can be considered a strategy to achieve higher yields of functional recombinant proteins. Objectives: Here, we investigated the influence of various culture media on the expression of scFvs derived from 4D5MOC-B, a mAb that binds to epithelial cell adhesion molecule (EpCAM), a biologically important marker for tumor immunotherapy. Methods: A genetically engineered Escherichia coli strain was employed to test the effect of four general media, including luria bertani (LB), terrific broth (TB), yeast tryptone 2x (TY2x), and minimal medium (M9), on cell density, scFv yield, and protein solubility. The experiments were conducted in duplicates for each medium. Data were presented as mean ± SD. Statistical analysis was conducted using one-way analysis of variance (ANOVA), followed by the Tukey post-test for pairwise comparisons. Results: The highest protein level was obtained in TB medium (215.19 ± 3.27 µg/mL), approximately 8-fold higher than that produced in M9 (26.83 ± 1.38 µg/mL). The maximum cell density of recombinant E. coli (10.89 ± 0.84) was achieved in TB after 22 hours. Shorter lag phases and higher cell densities in nutrient-rich media demonstrate how nutrient availability influences bacterial growth dynamics. Moreover, TY2x showed the highest soluble-to-insoluble protein ratio, 1.117 ± 0.048, followed by TB (0.926 ± 0.013), LB (0.883 ± 0.004), and M9 (0.662 ± 0.013). Conclusions: The present study highlights the importance of selecting a suitable culture medium for the promotion of recombinant protein synthesis in microbial systems. We also underline the potential industrial implications of using TB for high-density cultivation and reduced production costs.