BackgroundSerum have been traditionally used to support growth of animal cell cultures. However, the increasing growth of therapeutic biopharmaceuticals market, accelerated the high demand for the serum-free medium (SFM).ObjectiveThe main objective is to design a SFM for a stable rCHO cell line that produces a fully anti-human TNF-α monoclonal antibody (mAb) corresponding to HUMIRA® biosimilar.Materials and methodsDesign of Experiment (DoE) approaches were used to determine the key factors due to their effect on specific growth rate and mAb production. The production was carried out in T-flasks at different initial cell concentrations and then in Erlenmeyers with the developed SFM. mAb production was compared with commercial SFMs in terms of yield and productivity.ResultsRegarding to our findings, when the developed SFM-adapted cells were compared with the cells produced in commercial SFMs, the mAb productivity in developed SFM were higher (1.3–1.6 times) depending on higher mAb concentration and less (3–5 times) cell concentration. Additionally, the produced mAb in the developed SFM provided high conformational similarity with its originator HUMIRA®.ConclusionDoE approaches could be used to reduce cost and time in designing SFM for any commercially important cell line to produce high value biologics.