Kinetic studies on hybridoma cells immobilized in fixed bed reactors.

Cultures with immobilized hybridoma cells were performed in fixed bed systems. "Steady state" values for volume-specific substrate uptake and metabolite production rates were determined at various perfusion rates and superficial flow velocities of the medium within the carrier matrix. Data from fixed bed volumes between 50 and 600 ml did not show any difference. The volume-specific glutamine and glucose uptake rate turned out to be independent of the superficial flow velocity, but decreased with decreasing glutamine and glucose concentration. The volume-specific oxygen uptake rate increased with increasing superficial flow velocity and substrate concentration, respectively. A similar behavior was observed for the ratio between oxygen and glucose uptake rate. The production rate for monoclonal antibodies was neither affected by the substrate concentration nor by the superficial flow velocity. The metabolic parameters of the immobilized cells were put into kinetic equations and compared to those of suspended cells. It could be concluded that the metabolism of the immobilized cells is determined by the oxygen supply within the macroporous carriers.

Dialysis cultures with immobilized hybridoma cells for effective production of monoclonal antibodies.

UNLABELLED: An industrial scale reactor concept for continuous cultivation of immobilized animal cells (e.g. hybridoma cells) in a radial-flow fixed bed is presented, where low molecular weight metabolites are removed via dialysis membrane and high molecular products (e.g. monoclonal antibodies) are enriched. In a new "nutrient-split" feeding strategy concentrated medium is fed directly to the fixed bed unit, whereas a buffer solution is used as dialysis fluid. This feeding strategy was investigated in a laboratory scale reactor with hybridoma cells for production of monoclonal antibodies. A steady state monoclonal antibody concentration of 478 mg l(-1) was reached, appr. 15 times more compared to the concentration reached in chemostat cultures with suspended cells. Glucose and glutamine were used up to 98%. The experiments were described successfully with a kinetic model for immobilized growing cells. Conclusions were drawn for scale-up and design of the large scale system. ABBREVIATIONS: c(Glc) - glucose concentration, mmol l(-1); c(Gln) - glutamine concentration, mmol l(-1); c(Amm) - ammonia concentration, mmol l(-1); c(Lac) - lactate concentration, mmol l(-1); c(MAb) - MAb concentration, mg l(-1); D - dilution rate, d(-1); D(i) - dilution rate in the inner chamber of the membrane dialysis reactor, d(-1); D(0) - dilution rate in the outer chamber of the membrane dialysis reactor, d(-1); q*(FB,Glc) - volume specific glucose uptake rate related to the fixed bed volume, mmol l(FB) (-1) h(-1); q*(FB,Gln) - volume specific glutamine uptake rate related to the fixed bed volume, mmol l(FB) (-1) h(-1).

Improvement of the culture stability of non-anchorage-dependent animal cells grown in serum-free media through immobilization.

A murine hybridoma cell line producing a monoclonal antibody against penicillin-G-amidase and a murine transfectoma cell line secreting a monovalent chimeric human/mouse Fab-antibody fragment were cultivated in three different media (serum-containing, low protein serum-free, and iron-rich protein-free) in flask cultures, stirred reactors and a fixed bed reactor. In static batch cultures in flasks both cell lines showed similar good growth in all three media.In suspension in a stirred reactor, the hybridoma cell line could be cultivated satisfactory only in serum-containing medium. In low protein serum-free medium, Pluronic F68 had to be added to protect the hybridoma cells against shear stress. But even with this supplement only batch, not chemostat mode was possible. In iron-rich protein-free medium the hybridoma cells grew also in continuous chemostat mode, but the stability of the culture was low. The transfectoma cell line did not grow in stirred reactors in any of the three media.Good results with both cell lines were obtained in fixed bed experiments, where the cells were immobilized in macroporous Siran(®)-carriers. The media, which were optimized in flask cultures, could be used without any further adaptation in the fixed bed reactor. Immobilization improved the stability and reliability of cultures of non-adherent animal cells in serum-free media tremendously compared to suspension cultures in stirred reactors. The volume-specific glucose uptake rate, an, indicator of the activity of the immobilized cells, was similar in all three media. Deviations in the metabolism of immobilized and suspended cells seem to be mainly due to low oxygen concentrations within the macroporous carriers, where the cells are supplied with oxygen only by diffusion.

Estimation of specific glucose uptake rates in cultures of hybridoma cells.

The specific glucose uptake rate of a hybridoma cell line was determined in batch and continuous suspension cultures. For high density cultivation a membrane dialysis reactor was used. The aim was to obtain data, which can be used for process design and control. A decrease of the specific glucose uptake rate was observed during the course of batch cultures, even when the specific growth rate remained constant. These results are valid for cell densities between 2 x 10(5) and 2 x 10(6) ml-1. At cell densities between 2 x 10(6) and 1.5 x 10(7) ml-1 during continuous cultivation the specific glucose uptake rate remained constant. A relationship between the specific glucose uptake rate and the cell number was found and formulated in a mathematical equation. Literature data for different hybridoma cell lines fit in this plot very well. Applying this relationship, a correct estimation of the course of the glucose concentration in batch and continuous cultivation is possible.

Effect of NH3 on the cell growth of a hybridoma cell line.

Ammonia often has been reported to inhibit cell growth. The aqueous ammonia equilibrium between the un-ionized from (NH3) and the ammonium ion (NH4+) depends on the pH of the solution. Extensive studies in batch and continuous cultivation by varying pH and total ammonia concentration were carried out to investigate whether a kinetic model describing growth inhibition by ammonia has to be based on the total ammonia concentration, or the concentration of NH3. A significant relationship between the specific growth rate and death rate, respectively, and the NH3 concentration, but not the total ammonia concentration, was detected. An adaptation of the cells to high ammonia levels was not observed. Based on these results a new kinetic model for ammonia mediated growth inhibition is suggested. For high density cultivation it is recommended to control the pH at the lower limit of the growth optimum to keep the NH3 level low.