Amino Acid Metabolism of a High Density Perfusion Culture

Abstract

Using an external cell separator based on cell sedimentation and recycling, the density of viable cells reached a level around 10*10⁶ cells ml^-1 with a perfusion rate of 1.0 day^-1. This is five times higher density than achievable in batch cultivations. The specific antibody productivity decreased by a factor two in the middle period of the cultivation from the value 0.4 µg 1*10⁶ cells^-1 h^-1 observed initially, when the culture was operated in batch mode. However, the productivity recovered in the final high density stage of the cultivation. In the late period of the cultivation when the viable cell concentration increased to 10*10⁶ cells ml^-1 a shift in metabolism was observed. The two most important substrates for energy generation, glucose and glutamine, were reduced from feed concentrations of 25 mM respective 10 mM to 1.5 mM and 1 mM. At the same time the specific glycine production increased so the glycine concentration ended at 1.5 mM and aspartate was produced giving a concentration of 0.02 mM. It is suggested that this shift in metabolism leads to enhanced ATP production on glutamine consumption. Calculations of specific glucose consumption and lactate production also indicate that the glucose utilization when necessary, changes to be more energy economical. Anyhow this investigation shows a transition in metabolic state in high cell density perfusion culture, and that this transition may be correlated to a positive effect on monoclonal antibody production. High cell density cultures may have another amino acid metabolism than low cell density cultures, and such metabolic changes may have a beneficial influence on specific antibody productivity. The combination of these two factors caused a volumetric productivity of monoclonal antibody of 100 mg 1^-1 d^-1 for the high cell density culture.