A systematic approach was developed to identify and optimize the essential amino acids in defined minimal medium for the production of recombinant human interleukin-3 (rHuIL-3) by
. Starvation trials were carried out initially to narrow down the number of probable essential amino acids from an initial number of twenty to eight. Then a screening mixture experiment was designed and performed with the eight identified amino acids and distance-based multivariate analysis was employed to rank the probable essential amino acids regarding both growth and product formation. Following this procedure, the search was narrowed to four amino acids (Asp, Leu, Met, and Phe). Finally, a mixture design experiment known as the simplex lattice design was carried out and the composition of the optimum minimal medium was found using both statistical and neural network models.