Vitamin supplementation for enhanced production of therapeutic nanobody in E.coli

As low yields of nanobodies in E.coli. periplasm is a major concern for their large-scale production, this study explores the use of vitamins for enhanced production of a recombinant periplasmic nanobody product. Nanobody producing E.coli cells were cultivated in the modified Terrific-Broth supplemented with three different vitamins (biotin denoted as B, cyanocobalamin as C, and D-calcium pantothenate as P). Multiple combinations (B+P, B+C, P + C, and B+P + C) were examined, with B+P offering the highest specific growth rate, total biomass productivity, product accumulation and product expression. Densitometry analysis revealed 129.17 ± 1.1 mg/L nanobodies in case of B+P supplementation (24 % higher than control). B+P supplementation also offered 21 % better fermentation yield than control (as per RP-HPLC). The yield of purified nanobody product in case of B+P supplementation was at 35.0 ± 2.1 mg/L with purity levels of ≥ 80 %. Intact mass and peptide mapping analysis of purified nanobody revealed molecular weight of 15.2 kDa and 91 % sequence coverage with the theoretical nanobody sequence respectively. Spent media analysis revealed production of low acetate in case of B+P supplementation of media when compared to other conditions examined. Hence, the B+P supplementation could be used to produce improved amount of periplasmic nanobody in future.