Stability validation of seeding cell control parameters in large-scale hybridoma cell culture

Stability and reproducibility of seeding cell performance in large-scale hybridoma cell culture has been reported by controlling only initial cell seeding density. The aim of the current study was to integrate multiple seeding cell control parameters to maintain stable and consistent cell physiological status for HAb18 cell expansion. Three parameters and their ranges were investigated, including initial cell seeding density in the range of 0.075-0.5 x 10(6) cells ml(-1), "timepost" after cell passage between 8 and 36 h, and duration of subculture up to 6 months after cell revival. Cell performance was tested at the 1 L, 5 L, and 75 L scales. Desirable performance was found within the following parameter ranges: initial cell seeding density of 0.1-0.3 x 10(6) cells ml(-1), "timepost" after cell passage between 14 and 22 h, and duration of subculture within 3 months of cell revival. Our results showed that cell growth rate and antibody productivity of three batches at 1 L, 5 L, and 75 L scale were found to be stably maintained within a range of 0.036-0.047 h(-1) and 0.577-0.747 pg cell(-1) h(-1), with the positivity rate of antigen-binding activity within 97-99.75%, and the intensity of fluorescence around 200. This study may provide a simple but effective method to maintain seeding cell physiological status stable and consistent by combining seeding cell control parameters.