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Effect of amino acid limitation and supplementation in Chinese hamster ovary fed-batch cultures Ghaffari, Navid


Fed-batch processes are the industrial norm for the production of recombinant proteins such as monoclonal antibodies (MAb) from Chinese hamster ovary (CHO) cells. Optimization of such processes is an important objective of industry process development groups. Amino acid availability is a key factor that is controlled to achieve the desired product yield and quality. In order to improve fed-batch productivity, the individual effects of limiting the three depleted amino acids were investigated for three antibody expressing CHO cell lines. Specifically, the effects of limiting glutamine, asparagine and cysteine on the cell growth, metabolism, antibody productivity and quality were investigated. Cysteine limitation was found to be detrimental to both the cell proliferation and the productivity for all three CHO cell lines. In contrast, asparagine limitation had no significant effect on either their growth or productivity. Glutamine limitation resulted in a reduction in growth but not in cell specific productivity, again for all three cell lines. Neither glutamine nor asparagine limitation significantly affected the MAb glycosylation. However, the fucosylation ratio was reduced in the absence of cysteine. It was confirmed that cysteine is a rate limiting factor for the productivity and growth of the three CHO cell lines. Replenishing cysteine after 1 day of the limitation allowed the cells to regain their growth and productivity; however, this was not observed after 2 days of cysteine limitation. Under cysteine limitation there was increased oxidative damage to the mitochondria, possibly caused by reduced synthesis of co-enzyme A which is essential for functionality of the TCA cycle. Finally, a fed-batch protocol was developed to improve the MAb productivity of CHO-DXB11 cells and the results were compared to the results with a commercial feed. Although use of the commercial feed resulted in higher maximum cell and final MAb concentrations, maintaining the levels of cysteine yielded cell specific production rates that were comparable to the commercial feed culture. Overall, the results of this study showed that amino acid limitations have varied effects on the performance of CHO cell cultures, such that it is important to focus process development efforts on the critical amino acids.

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