A balanced supplementation method was applied to develop a serum and protein- free medium supporting hybridoma cell batch culture. The aim was to improve systematically the initial formulation of the medium to prevent limitations due to unbalanced concns. of vitamins and amino acids. In a first step, supplementation of the basal formulation with 13 amino acids, led to an increase of the specific IgA prodn. rate from 0.60 to 1.07 pg cell-1 h-1. The specific growth rate remained unchanged, but the supplementation enabled maintenance of high cell viability during the stationary phase of batch cultures for some 70 h. Since IgA prodn. was not growth- related, this resulted in an approx. 4-fold increase in the final IgA concn., from 26.6 to 100.2 mg l-1. In a second step, the liposol. vitamins E and K3 were added to the medium formulation. Although this induced a slightly higher maximal cell concn., it was followed by a sharp decline phase with the specific IgA prodn. rate falling to 0.47 pg cell-1 h-1. However, by applying a second cycle of balanced supplementation with amino acids this decline phase could be reduced and a high cell viability maintained for over 300 h of culture. In this vitamin- and amino acid- supplemented medium, the specific IgA prodn. rate reached a value of 1.10 pg cell-1 h-1 with a final IgA concn. of 129.8 mg l-1. The latter represents an increase of approx. 5-fold compared to the non- supplemented basal medium. [on SciFinder (R)]