Very high density of CHO cells in perfusion by ATF or TFF in WAVE bioreactor™. Part I. Effect of the cell density on the process

Abstract

High cell density perfusion process of antibody producing CHO cells was developed in disposable WAVE Bioreactor™ using external hollow fiber filter as cell separation device. Both “classical” tangential flow filtration (TFF) and alternating tangential flow system (ATF) equipment were used and compared. Consistency of both TFF- and ATF-based cultures was shown at 20–35 × 106 cells/mL density stabilized by cell bleeds. To minimize the nutrients deprivation and by-product accumulation, a perfusion rate correlated to the cell density was applied. The cells were maintained by cell bleeds at density 0.9–1.3 × 108 cells/mL in growing state and at high viability for more than 2 weeks. Finally, with the present settings, maximal cell densities of 2.14 × 108 cells/mL, achieved for the first time in a wave-induced bioreactor, and 1.32 × 108 cells/mL were reached using TFF and ATF systems, respectively. Using TFF, the cell density was limited by the membrane capacity for the encountered high viscosity and by the pCO2 level. Using ATF, the cell density was limited by the vacuum capacity failing to pull the highly viscous fluid. Thus, 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 fiber design of TFF and ATF. At very high cell density, the viscosity introduced physical limitations. This led us to recommend cell densities under 1.46 × 108 cell/mL based on the analysis of the theoretical distance between the cells for the present cell line. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:754–767, 2013

Perfusion Processes

“…Maiorella et al (1991) observed no cell damage up to a shear rate of 3,000 s À1 in cell separation for harvest from Sf-9, hybridoma and myeloma cultures using crossflow filtration. Clincke et al applied shear rates of 1,000, 2,400 and 3,400 s À1 in CHO cell perfusion without observing an adverse effect (Clincke et al 2013b). During this study, blockage of the hollow fibers was never observed.…”

“…At very high cell densities, the cells occupy a non-negligible fraction of the liquid, e.g. around 45 % at 2 Â 10 8 CHO cells/mL (Clincke et al 2013b). Hence larger cells occupy a larger fluid fraction for the same cell density.…”

“…Applying the CSPR method Clincke et al obtained more than 2 Â 10 8 cells/mL (Clincke et al 2013b). In this process glucose and glutamine were fed separately from the medium at a stoichiometric rate such that their residual concentrations in the bioreactor were maintained at low levels.…”

“…A graphical representation of the effect of lactate or ammonia concentrations on the growth rate or the cell specific production rate can provide guidelines for the selection of limits of these by-products in the process (Clincke et al 2013b). Finally more sophisticated approaches such as metabolomics can provide a deeper understanding of the production processes (Vernardis et al 2013). 13.4.5 Cell Density: Target, Monitoring and Control 13.4.5.1 Selection/Optimization of the Cell Density Two decades ago, the cell density in perfusion processes was around a few millions cells/mL in many cases.…”

The future of industrial bioprocessing: Batch or continuous?

“…In comparison, the continuous MAb processes are still far from technological maturity, but rapidly evolving. Recent results demonstrate high cell density of >100e6 cells/mL and productivity of 3.5 g/l-day (Clincke, 2013;Yin, 2014). This suggests a significant upward potential, which will challenge and likely outperform the established fed-batch paradigm.…”