Veronique Chotteau

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

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.

Very high density of Chinese hamster ovary cells in perfusion by alternating tangential flow or tangential flow filtration in WAVE bioreactor™—part II: Applications for antibody production and cryopreservation

A high cell density perfusion process of monoclonal antibody (MAb) producing Chinese hamster ovary (CHO) cells was developed in disposable WAVE Bioreactor™ using external hollow fiber (HF) filter as cell separation device. Tangential flow filtration (TFF) and alternating tangential flow (ATF) systems were compared and process applications of high cell density perfusion were studied here: MAb production and cryopreservation. Operations by perfusion using microfiltration (MF) or ultrafiltration (UF) with ATF or TFF and by fed‐batch were compared. Cell densities higher than 108 cells/mL were obtained using UF TFF or UF ATF. The cells produced comparable amounts of MAb in perfusion by ATF or TFF, MF or UF. MAbs were partially retained by the MF using ATF or TFF but more severely using TFF. Consequently, MAbs were lost when cell broth was discarded from the bioreactor in the daily bleeds. The MAb cell‐specific productivity was comparable at cell densities up to 1.3 × 108 cells/mL in perfusion and was comparable or lower in fed‐batch. After 12 days, six times more MAbs were harvested using perfusion by ATF or TFF with MF or UF, compared to fed‐batch and 28× more in a 1‐month perfusion at 108 cells/mL density. Pumping at a recirculation rate up to 2.75 L/min did not damage the cells with the present TFF settings with HF short circuited. Cell cryopreservation at 0.5 × 108 and 108 cells/mL was performed using cells from a perfusion run at 108 cells/mL density. Cell resuscitation was very successful, showing that this system was a reliable process for cell bank manufacturing.

Can We Identify Biotechnological Processes

Abstract The identification problem of biotechnological processes is threefold : (i) determination of the number of biological reactions, (ii) identification of the underlying reaction network, (iii) identification of the kinetics. In most practical cases, these three parts of the identification problem can be completely decoupled from one another.

Study of a recombinant CHO cell line producing a monoclonal antibody by ATF or TFF external filter perfusion in a WAVE Bioreactor

Major advantages of perfusion are high cell numbers and high total production in a relatively small size bioreactor. Moreover, perfusion is optimal when the product of interest is unstable or if th ...

Very high cell density perfusion of CHO cells anchored in a non-woven matrix-based bioreactor.

Recombinant Chinese Hamster Ovary (CHO) cells producing IgG monoclonal antibody were cultivated in a novel perfusion culture system CellTank, integrating the bioreactor and the cell retention function. In this system, the cells were harbored in a non-woven polyester matrix perfused by the culture medium and immersed in a reservoir. Although adapted to suspension, the CHO cells stayed entrapped in the matrix. The cell-free medium was efficiently circulated from the reservoir into- and through the matrix by a centrifugal pump placed at the bottom of the bioreactor resulting in highly homogenous concentrations of the nutrients and metabolites in the whole system as confirmed by measurements from different sampling locations. A real-time biomass sensor using the dielectric properties of living cells was used to measure the cell density. The performances of the CellTank were studied in three perfusion runs. A very high cell density measured as 200 pF/cm (where 1 pF/cm is equivalent to 1 × 10(6)viable cells/mL) was achieved at a perfusion rate of 10 reactor volumes per day (RV/day) in the first run. In the second run, the effect of cell growth arrest by hypothermia at temperatures lowered gradually from 37 °C to 29 °C was studied during 13 days at cell densities above 100 pF/cm. Finally a production run was performed at high cell densities, where a temperature shift to 31 °C was applied at cell density 100 pF/cm during a production period of 14 days in minimized feeding conditions. The IgG concentrations were comparable in the matrix and in the harvest line in all the runs, indicating no retention of the product of interest. The cell specific productivity was comparable or higher than in Erlenmeyer flask batch culture. During the production run, the final harvested IgG production was 35 times higher in the CellTank compared to a repeated batch culture in the same vessel volume during the same time period.

Identification of a Reaction Mechanism for a Class of Animal Cell Cultures

Abstract A reaction mechanism for an animal cell culture of adherent cells (VERO cells) is proposed. This model is validated in different experimental conditions (including batch and renewed cultures) with a model-based estimator of the biomass. An important original feature of the model is to take a dozen of amino acids into account instead of glutamine only as usual in animal cell cultures modeling.

On dynamically generating relevant elementary flux modes in a metabolic network using optimization

Elementary flux modes (EFMs) are pathways through a metabolic reaction network that connect external substrates to products. Using EFMs, a metabolic network can be transformed into its macroscopic counterpart, in which the internal metabolites have been eliminated and only external metabolites remain. In EFMs-based metabolic flux analysis (MFA) experimentally determined external fluxes are used to estimate the flux of each EFM. It is in general prohibitive to enumerate all EFMs for complex networks, since the number of EFMs increases rapidly with network complexity. In this work we present an optimization-based method that dynamically generates a subset of EFMs and solves the EFMs-based MFA problem simultaneously. The obtained subset contains EFMs that contribute to the optimal solution of the EFMs-based MFA problem. The usefulness of our method was examined in a case-study using data from a Chinese hamster ovary cell culture and two networks of varied complexity. It was demonstrated that the EFMs-based MFA problem could be solved at a low computational cost, even for the more complex network. Additionally, only a fraction of the total number of EFMs was needed to compute the optimal solution.

Development of a Large Scale Process for the Production of Recombinant Truncated Factor VIII in CHO Cells under Cell Growth Arrest Conditions

A large scale biopharmaceutical process has been developed for the production of a genetically engineered variant of coagulation factor VIII. Factor VIII is the coagulation protein that is defective or missing in hemophilia A. Two challenging techniques, ceil growth arrest and perfusion, have been applied to ensure a high production yield. Key development aspects of this process will be presented. A B-domain deleted variant of the factor VIII gene, the factor VIII SQ gene, has been constructed and integrated in Chinese Hamster Ovary CHO DG44 DHFR− cells. The cells are cultivated in serum-free medium which has been developed to optimize the factor VIII SQ production. The cultivation and purification processes have been selected to reduce the deleterious actions of proteases present in the cultivation process. Factor VIII SQ has structural and functional characteristics comparable to full length factor VIII and has been licensed in Europe and USA under the name ReFacto (Genetics Institute).

Effect of Peptones and Study of Feeding Strategies in a CHO Based Fed-batch Process for the Production of a Human Monoclonal Antibody

Eight commercial peptones, derived from plants, were studied for their ability of improving the cell growth and the productivity of a CHO cell line producing a human monoclonal antibody. They were also compared to yeast, lactalbumin and meat derived peptones. Seven plant peptones were selected and further studied in combination by Design of Experiment. The best three peptones were then tested in combinations in fed-batch cultivation. The fed-batch process was based on low concentrations of glucose and glutamine with feeding of amino acids, peptones and feed medium including vitamins, metal traces and biosynthesis precursors. This process was based on Biovitrum protein-free proprietary medium for the base medium and the feeding medium. Different feeding strategies, different peptone combinations and phosphate feeding were studied for their ability to improve the cell density, the cell specific productivity and the cultivation longevity.

High Cell Density Perfusion Culture has a Maintained Exoproteome and Metabolome

The optimization of bioprocesses for biopharmaceutical manufacturing by Chinese hamster ovary (CHO) cells can be a challenging endeavor and, today, heavily relies on empirical methods treating the bioreactor process and the cells as black boxes. Multi-omics approaches have the potential to reveal otherwise unknown characteristics of these systems and identify culture parameters to more rationally optimize the cultivation process. Here, the authors have applied both metabolomic and proteomic profiling to a perfusion process, using CHO cells for antibody production, to explore how cell biology and reactor environment change as the cell density reaches ≥200 × 106  cells mL-1 . The extracellular metabolic composition obtained in perfusion mode shows a markedly more stable profile in comparison to fed-batch, despite a far larger range of viable cell densities in perfusion. This stable profile is confirmed in the extracellular proteosome. Furthermore, the proteomics data shows an increase of structural proteins as cell density increases, which could be due to a higher shear stress and explain the decrease in cell diameter at very high cell densities. Both proteomic and metabolic results shows signs of oxidative stress and changes in glutathione metabolism at very high cell densities. The authors suggest the methodology presented herein to be a powerful tool for optimizing processes of recombinant protein production.