This study focuses on optimization of the substrate feeding profile for enhanced production of ranibizumab, a recombinant therapeutic protein, in Escherichia coli. An established process analyzer, dielectric spectroscopy, was deployed to monitor the production process and to estimate real-time biomass. A validated mechanistic model was employed to formulate a multiobjective optimization (MOO) problem. The substrate flow rate during the fed-batch phase (F) was taken as the decision variable for MOO. The Pareto front resulting from MOO revealed that for a minimum broth volume (V) of 1.96 L, a maximum of 58.8 g of total biomass (XV) could be generated. The total biomass obtained from the optimal substrate feeding profile was 20.6% higher than the experimentally achieved total biomass. Enhanced productivity was achieved by the proposed MOO formulation, which facilitates the choice of any operating point from the Pareto front based on downstream expenses of the therapeutic product.