With the increasing dominance of monoclonal antibodies (mAbs) in
the biopharmaceutical industry and smaller antibody fragments bringing
notable advantages over full-length antibodies, it is of considerable
significance to choose the most suitable production system. Although
mammalian expression system has been the preferred choice in recent
years for mAbs production, E. coli could be the favorable host for
non-glycosylated small antibody fragments due to the emergence of new
engineered E. coli strains capable of forming disulfide-bonds in their
cytoplasm.In this study, non-glycosylated anti-TNF-α Fab moiety of
Certolizumab pegol, produced by periplasmic expression in E. coli in
previous studies, was produced in the cytoplasm of E. coli SHuffle
strain. The results indicated that it is biologically functional by
testing the antigen-binding activity via indirect ELISA and inhibition
of TNF-α induced cytotoxicity using MTT test. Major factors affecting
protein production and, optimized culture conditions were examined by
analyzing growth characteristics and patterns of expression in 24 h of
post-induction cultivation and, optimization of culture conditions by
response surface methodology considering temperature, time of induction
and concentration of inducer in small (tube) and shake-flask scale.
Based on the results, temperature had the most significant influence on
functional protein yield while exerting different impacts in small and
shake-flask scales, which indicated that cultivation volume is also an
important factor that should be taken into account in optimization
process. Furthermore, richness of medium and slower cellular growth rate
improved specific cellular yield of functional protein by having a
positive effect on the solubility of Fab antibody.