Mammalian Cell Culture Process Optimization

Investigations were made to study the process optimization of hybridoma cell culture, including cell weaning, protein-free medium development and fed-batch culture optimization, to improve the cell concentration and MAb production.First of all, mouse-mouse hybridoma cell line JJ1 cells were adapted from serum containing culture to protein-free culture by step-wise lowering of fetal bovine serum from 10% to zero.Then in the process of protein-free medium development, a protein-free medium with hydrolysate, PFW2, was obtained by testing four different hydrolysates to meet the needs of pre-clinical and clinical research, in which the development time was limited. A new high throughput method for medium component optimization including high throughput cell culture and high throughput endpoint viable cell assay estimation was developed. Using this method a new chemical defined protein-free medium, CDPF3, was obtained. The cell concentration and MAb production of JJ1 cell in PFW2 and CDPF3 was much higher than that in PFHM-II (Gibco), one of the major commercial protein-free medium for hybridoma cells.Furthermore,in the fed-batch optimization process, a growth-associated pattern between the production of MAb and growth of cells was identified through the kinetic studies of batch cultures, suggesting that the potential effectiveness of extending the duration of the exponential growth phase to improve MAb production. The concentration of glucose and glutamine in the initial medium was 1.1 and 0.3 mM, according to results of batch culture metabolism analysis and glucose, glutamine limiting culture. Compositions of amino acids in the feeding solution were optimized according to the stoichiometrical relationships between the consumptions of amino acids and glucose in batch and fed batch cultures to eliminate amino acid limitation. Ca²⁺/Mg²⁺ irons were found to be limiting by limiting factor screening.Finally, a fed-batch process was executed using a feeding medium supplemented with amino acids and a Ca²⁺/Mg²⁺ concentrate. The viable cell concentration, total cell concentration and the MAb production reached 7.04×10⁶ cells ml^-1, 9.12×10⁶ cells ml^-1 and 707 mg l^-1, 3 fold, 4 fold, 7.4 fold higher respectively than that in the serum containing batch culture.In this research, the importance of process optimization including cell weaning, medium optimization and fed-batch optimization to the nutrient limitation elimination in the culture, the cell concentration improvement and the MAb production improvement was highlighted.