Great cell density perfusion process of antibody producing CHO cells was developed in disposable WAVE Bioreactor? using external hollow fiber filter as cell separation device. with the present settings, maximal cell densities of 2.14 108 cells/mL, accomplished for the first time inside a wave-induced bioreactor, and 1.32 108 cells/mL were reached using TFF and ATF systems, respectively. Using TFF, the cell denseness was limited by the membrane capacity for the experienced high viscosity and by the pCO2 level. Using ATF, the Ulipristal acetate cell denseness was limited by the vacuum capacity failing to pull the highly viscous fluid. Ulipristal acetate Therefore, the TFF system allowed reaching higher cell densities. The TFF inlet pressure was highly correlated to the viscosity leading to the development of a model of this pressure, which is a useful tool for hollow dietary fiber design of TFF and ATF. At very high cell denseness, the viscosity launched physical limitations. This led us to Nrp2 recommend cell densities under 1.46 108 cell/mL based on the analysis of the theoretical range between the cells for the present cell collection. ? 2013 American Institute of Chemical Engineers was determined as (5) where and are the flow rate in the fiber lumen and the lumen radius. Results and Discussion Development of the perfusion process set-up The bioreactor was either connected to an ATF or a TFF system (see set-up in Figure 1a). Pictures of the systems in operation at KTH’s lab are given in Figures 1b,c. The Cellbag? was connected to the HF of the ATF or the TFF via two dip tubes piercing the upper surface of the bag (in the vicinity of the center) and anchored on the bag bottom at ports with large opening allowing cell broth circulation. The dip tube length was as short as possible for optimal operation of the ATF system and prevention of bubble occurrence in the dip pipe. Bubbles in the drop tube were vunerable to impair the purification function and trigger shear harm to the cells. The new air occurrence was preliminary studied utilizing a water model in the ATF system. We determined that at operating quantity 4.5 L in presence of 50 ppm AF no air gathered in the drop tube even at high rocking rates and angles, see Figure 2. AF presence partially prevented the air occurrence however not completely even though no foam was formed on the liquid surface. Open in a separate window Figure 1 (a) Process set-up for TFF- and ATF-based perfusion; 2 cells/mL, maintained during 6 days, also using a wave-induced perfusion system equipped with an internal membrane. It is the first time that data of cell densities of 0.9C1.3 108 cells/mL maintained during 18 days are presented. Although used in industry, ATF-based perfusion and concentrated fed-batch processes are seldom published5, 17 and are not disclosed even if very high Per C6 cell densities have been announced.23 To our knowledge, it is the first time that CHO cell density above 108 cells/mL is published in a Ulipristal acetate disclosed ATF process. Cell metabolism and medium cell metabolism and medium effect The concentrations of glucose, glutamine, lactate, and ammonium in the bioreactor are given in Figures 5cCf. Except for the first days in all the runs and the last days of TFF#10, the residual concentrations of glucose and glutamine were often very low (6 and 0.7 mM, respectively, sometimes measured as 0); however, these energy sources were present in the fresh medium and supplementary added as well. The lactate and ammonium concentrations were mostly 46 and 6 mM, respectively. The lactate range was comparable to the range observed in fed-batch runs while the ammonia concentration reached up to 18 mM in fed-batch (see Part II24). The cell specific rates of consumption and production of these metabolites are represented as a function of the cell density in Figures 6aCd. The is the volume fraction of spheres or solid in the mixture, that is, sphere volume cell density. At high cell density, the.