SummaryWe have developed a strategy for the optimization of plant cell suspension culture media using a combination of fractional factorial designs (FFDs) and response surface methodology (RSM). This sequential approach was applied to transformed tobacco BY-2 cells secreting a human antibody (M12) into the culture medium, in an effort to maximize yields. We found that the nutrients KNO 3 , NH 4 NO 3 and CaCl 2 and the hormones 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (BAP) had the most significant impact on antibody accumulation. The factorial screening revealed strong interactions within the nutrients group (KNO 3 , NH 4 NO 3 and CaCl 2 ) and also individually between 2,4-D and three other components (KNO 3 , NH 4 NO 3 and BAP). The RSM design resulted in a fivefold increase in the antibody concentration after 5 days and a twofold reduction in the packed cell volume (PCV). Longer cultivation in the optimized medium led to the further accumulation of antibody M12 in the culture medium (up to 107 lg/mL, day 10). Because the packed cell volume was reduced in the optimized medium, this enhanced the overall yield by 20-fold (day 7) and 31-fold (day 10) compared to the conventional MS medium.