Improving an on-line feeding strategy for fed-batch cultures of hybridoma cells by dialysis and 'Nutrient-Split'-feeding

The concept of the feeding strategy was to minimise the formation of inhibi ting metabolites and to increase the yield of monoclonal antibodies in fed- batch cultures of hybridoma cells by a balanced supply of substrates. A pro cess control system based on fieldbus technology was used for monitoring an d control. External program routines were implemented to control dissolved oxygen (DO) and to calculate the oxygen uptake rate (OUR) and cumulative ox ygen consumption (COC) simultaneously. A concentrated feed solution was sup plied according to the off-line estimated stoichiometric ratio between oxyg en and glucose consumption (GC). Feeding was initiated automatically when t he OUR decreased due to substrate limitation. The antibody concentration in creased three-fold compared to the conventional batch culture by applying t his strategy. But it was not possible to avoid inhibition by ammonia during the fed-batch phase. This was accomplished by the use of a dialysis membra ne. Dialysis fed-batch cultures were performed in a membrane dialysis react or with a 'nutrient-split' feeding strategy, where concentrated medium is f ed to the cells and toxic metabolites are removed into a buffer solution. T his resulted in a ten-fold increase of the antibody concentration compared to the batch. Amino acid concentrations were analysed to identify limiting conditions during the cultivation and to analyse the performance of the nut rient supply in the fed-batch and dialysis fed-batch.