BACKGROUND Inactive aggregated protein deposits formed during fermentation of recombinant bacteria - inclusion bodies (IBs) - continue to be a significant source of active proteins. Understanding how the microenvironment, in particular growth medium, affects the quality and quantity of the IBs would be desirable for optimal production of proteins. In this study, we explore the effect of various chemically defined media components on IBs using a variety of analytical techniques. Expression of a biotherapeutic, ranibizumab, has been chosen as a case study. RESULTS A repertoire of nutrients, including but not limited to carbon and nitrogen source, vitamins, cofactors, buffering agents and minerals, were examined. Sucrose, biotin, pantothenate, glutathione, K2HPO4, K2SO4, cyanocobalamin and CaSO4 were shortlisted based on experimentation. A design of experiments (DOE) study was performed to identify optimal concentrations of and interactions between these nutrients. Higher concentrations of sucrose, biotin and pantothenate, and lower concentrations of cyanocobalamin, CaSO4 and glutathione, resulted in larger IBs and greater product expression. Further, K2HPO4 and K2SO4 concentrations did not seem to have a meaningful impact on product formation. CONCLUSIONS Medium composition affects size and quality of IBs. The size of IBs exhibits a positive correlation with the quantity of product expressed, as well as the purity of the product, but does not correlate with the total amount of host cell proteins produced. IB size could be used as a key process attribute during bioreactor optimization, and the otherwise difficult task of measuring product concentration in the fermentation broth could be avoided. (c) 2020 Society of Chemical Industry