Amino acid supplementation to achieve enhanced production of a fab fragment in Escherichia coli: rHu ranibizumab as a case study

Microbial host systems, specifically E. coli-based, are widely used hosts for the production of recombinant proteins and biotherapeutics. Overproduction of heterologous protein often causes excessive consumption of ATPs and other precursors, especially amino acids, and this can disrupt cell metabolism, thereby resulting in reduced cell growth and protein production. In this study, we demonstrate that supplementation of amino acids can reduce metabolic burden and thereby improve the biomass and protein yield. Ranibizumab, a fab fragment biotherapeutic, has been taken as a case study. Plackett-Burman Design of Experiments (DOE) based screening of all 20 amino acids was performed first to identify significant amino acids. Further, to confirm the finding of the screening study, consumption of amino acids was monitored and based on the pattern, the required amino acids were supplemented. This targeted amino acid supplementation resulted in a significant increase in biomass (28 % increase in dry cell weight), protein titre (40 % increase in g protein per g IBs), specific growth rate (by 23 %), and substrate uptake (by 37 %). The results demonstrate that optimal amino acid supplementation enhances process productivity.