Cell Bleed Rate Research Articles

Establishment and optimization of a high-throughput mimic perfusion model in ambr® 15.

To establish an automated high-throughput mimic perfusion scale-down model (SDM) in ambr® 15 system. An optimized SDM for mimic perfusion was developed in ambr® 15 system. Cell retention in ambr® 15 was realized by sedimentation and supernatant removal with a retention rate > 95%. Although the SDM couldn't reach the viable cell density (VCD) at a bench scale bioreactor (BR), it maintained VCD at approximately 30 × 106 cells/mL with a cell bleeding rate estimated theoretically and predicted the cell specific perfusion rate (CSPR). A base-feeding strategy was developed to alleviate the pH drop during sedimentation which would adversely have an impact on cell growth, and showed an apparent cell viability improvement from 79.6% (control) to 90.1% on Day 18. The optimized SDM for mimic perfusion was employed for media screening in two cell lines. A small-scale high-throughput perfusion model in ambr® 15 was developed, optimized to improve cell viability, and as a result, utilized for media screening in two cell lines.

Model simulation and analysis of perfusion culture of mammalian cells at high cell density.

Rate equations recently proposed by the authors for growth, death, consumption of nutrients, and formation of lactic acid, ammonium, and monoclonal antibody of hybridoma cells are used to simulate and analyze the behavior of perfusion cultures. Model simulations are in good agreement with experimental results from three different cell lines under varied perfusion and cell bleed rates except for cultures with very low viability. Analysis of simulations and experimental results indicates that in perfusion cultures with a complete cell separation cell bleed rate is a key parameter that strongly affects all the process variables, whereas the perfusion rate mainly affects the total and viable cell concentrations and the volumetric productivity of monoclonal antibody. Growth rate, viability, and specific perfusion rate of cells are only a function of the cell bleed rate. This also applies to cultures with partial cell separation in the permeate if the effective cell bleed rate is considered. It is suggested that the (effective) cell bleed rate of a perfusion culture should be carefully chosen and controlled separately from the perfusion rate. In general, a low cell bleed rate that warrants a reasonable cell viability appears to be desirable for the production of antibodies. Furthermore, model simulations indicate the existence of an optimum initial glucose concentration in the feed. For the cell lines considered, the initial glucose concentration used in normal cell culture media is obviously too high. The initial glutamine concentration can also be reduced to a certain extent without significantly impairing the growth and antibody production but considerably reducing the ammonia concentration. The mathematical model can be used to predict these optimum conditions and may also be used for process design.

Effect of Cell Purge on the Stability of Microfiltration Cell Recycle Fermentation Systems

Fermentation with complete cell recycle on microfiltration tubular membranes was studied and its effect upon broth rheology evaluated. The increase in cell concentration was found to be responsible for a continuous rise in viscosity leading to changes of broth rheology and consequent operating problems. A strategy to control the broth rheological behaviour with the aim of achieving long-term stable fermentations is proposed. This strategy involves the use of a cell purge in order to maintain cell concentration at a manageable level. The results obtained show that the total cell concentration in the fermenter can be stabilised if the cell bleed rate is calculated to remove exactly the amount of cells produced. To obtain long-term stable fermentations, the time to initiate the bleeding procedure and the cell bleed rate need to be determined and optimised. © 1997 SCI.

Partial and total cell retention in a filtration-based homogeneous perfusion reactor.

Suspended mammalian cells can be cultivated in a variety of operational modes (pure chemostat, total cell retention, or partial cell retention) in a homogeneous perfusion bioreactor by varying the cell bleed rate. Hybridomas were grown in the reactor at a perfusion rate of 2.0 day-1 for over 10 weeks at different specific growth rates and viable cell densities achieved by varying the extent of cell retention. Cell metabolism in the reactor was found to vary with the extent of cell retention, which determined both cell density and specific growth rate. With partial cell retention, the nutrient consumption and metabolite production rates decreased with both increasing growth rate and increasing cell density. The specific and volumetric antibody production rates, however, increased dramatically with cell density (and to a lesser extent with decreasing growth rate). The specific MAb production rate was lower with total cell retention than with partial retention at the same growth rate. Since the reactor can be operated over a range of perfusion rates and extents of cell retention, the system can be used to culture cell lines with widely different productivity patterns.

Kinetic study on HBs-MAb production in continuous cultivation

Continuous cultivation of mouse-mouse hybridoma, T0405 cells producing an IgG monoclonal antibody (MAb) against hepatitis B surface antigen, was carried out in a membrane reactor with partial cell bleeding. By changing cell bleeding rate, viable cell concentrations were maintained at a high level in the range of 1.0 × 10 6 to 4.0 × 10 6 ml −1, which is difficult to attain in a conventional continuous culture. The characteristics of MAb production, and metabolism of glucose and glutamine at high cell density were investigated under steady state. This cell line's MAb production was found to be growth associated. The calculation of ammonia yield on glutamine showed that ammonia was mainly produced when glutamine was used for cell maintenance, while lactate yield on glucose used for cell growth was higher than that for cell maintenance.

Tangential flow filtration for continuous cell recycle culture of acidogenic bacteria

The operation of cell recycle systems by membrane filtration is discussed, stressing the importance of the mutual interaction between the biological and the physical characteristics of the fermentation process. Broth circulation flow rate, dilution rate, and cell bleed rate are identified as being the most important process variables. Their effects on process performance and criteria for its adequate selection are analysed. Fermentation results for different acidogenic microorganisms are presented and the kinetic modelling of high cell density recycling systems is discussed.

Ultrafiltration membrane cell recycle for continuous culture of Propionibacterium

A high-cell-concentration culture of Propionibacterium acidi-propionici in a fermenter with recycling through a tangential membrane filtration unit is described. Circulation rate and fermentation start-up procedures are discussed in order to optimize the system performance and the operation at high dilution rates. Maximum cell concentrations up to 140 g dry weight/l were obtained, with a propionic acid productivity of 10.3 g/l hr and a product yield of 0.56 g propionic acid/g glucose. Rheological studies showed that at cell mass concentrations above 90-100 g/1 the fermentation broth viscosity increased rapidly and changed its behaviour from Newtonian to pseudoplastic. The use of an adequate cell bleed rate allowed the cell concentration to be maintained at a desirable value and consequently to stabilize the reactor operation.