An on-line feeding strategy based on oxygen uptake rate (OUR) was used to minimise the formation of inhibiting metabolites and to increase the yield of monoclonal antibodies in fed-batch cultures of hybridoma cells by a balanced supply of substrates. Concentrated feed was supplied according to the off-line estimated stoichiometric ratio between oxygen and glucose consumption (CC). The antibody concentration in fed-batch increased three-fold compared to the conventional batch culture by applying this strategy. Metabolites such as lactate and ammonia accumulated during fed-batch cultivation and it was not possible to avoid inhibition by ammonia. Fed-batch was efficiently improved by using the concept of dialysis and 'nutrient-split' feeding, where concentated medium is fed directly to the cells and toxic metabolites are removed over a dialysis membrane into a buffer solution. The 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 nutrient supply in the fed-batch and dialysis fed-batch. The potential of the dialysis fed-batch with 'nutrient-split' feeding is discussed with respect to substrate loss, efficiency and process stability in comparison to the conventional fed-batch.
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