On-line immunoanalysis of monoclonal antibodies during a continuous culture of hybridoma cells.

The monoclonal-antibody production of an immobilized hybridoma cell line cultivated in a fluidized-bed reactor was monitored on-line for nearly 900 h. The monoclonal antibody concentration was determined by an immuno affinity-chromatography method (ABICAP). Antibodies directed against the product, e.g. IgG, were immobilized on a micro-porous gel and packed in small columns. After all IgG present in the sample was bound to the immobilized antibodies, unbound proteins were removed by rinsing the column. Elution of the bound antibodies followed and the antibodies were determined by fluorescence. The analytical procedure was automated with a robotic device to enable on-line measurements. The correlation between the on-line determined data and antibody concentrations measured by HPLC was linear.A sampling system was constructed, which was based on a pneumatically actuated in-line membrane valve integrated into the circulation loop of the reactor. Separation of the cells from the sample stream was achieved by a depth filter made of glass-fibre, situated outside the reactor. Rapid obstruction of the filter by cells or cell debris and contamination of the sample system was avoided by intermittent rinsing of the sample system with a chemical solution. The intermittent rinsing of the filter, which had a surface of 4.8 cm(2), resulted in an operational capacity of up to 40 samples (1.0 l total sample volume). Both the sampling system and the analytical device functioned without failure during this long-term culture.The culture temperature was varied between 34 and 40 °C. Raising the temperature from 34 up to 37 °C resulted in a simultaneous increase of growth and specific antibody production rate. Specific metabolic rates of glucose, lactate, glutamine and ammonium stayed constant in this temperature range. A further enhancement of temperature up to 40 °C had a negative effect on the growth rate, whereas the specific monoclonal antibody production rate showed a small increase. The other specific metabolic rates also increased in the temperature range between 38 to 40 °C.

Automation of selective assays for on-line bioprocess monitoring by flow-injection analysis.

On-line analysis of one component in a complex media used for bioprocesses requires the application of selective tests such as enzymes assays. Because these assays are susceptible to interference by other medium components and have a limited detection range, automatic sample pretreatment is a prerequisite. The progress made with automatic sample pretreatment in flow-injection analysis makes this technique particularly suitable for on-line monitoring of bioprocesses. Moreover, newly developed software control systems may improve the necessary robustness of flow-infection analysis systems.

On-line control of an immobilized hybridoma culture with multi-channel flow injection analysis.

An immobilized hybridoma cell line was cultivated at controlled glucose and glutamine concentrations. On-line analysis of the substrates was carried out with a multi-channel flow injection analysis system. The analysis system also determined on-line the lactate and ammonium concentration. The substrate concentrations were controlled using an adaptive-control strategy. This strategy consisted of the estimation of the real-time concentrations and volumetric substrate consumption rates by an Extended Kalman Filter, and a minimum variance controller, which used the estimated parameters to set the feed rates of the substrates. The closed-loop control was used to start-up two cultures with either glucose or glutamine as control-substrate for the medium feed rate. The controller kept the concentration of the control-substrate constant by enhancing the medium feed rate simultaneously to the increasing volumetric consumption rate of the substrate. When glutamine was used as control-substrate, the glucose concentration remained relatively constant, whereas the glutamine concentration decreased during the start-up at a constant glucose concentration. This indicates that glutamine is consumed faster than glucose and will be a better control-substrate to avoid limitation during the start-up of a culture with the applied hybridoma cell line. During the colonization of the microcarriers, the yield of ammonium on glutamine decreased from 0.80 to 0.55 (mol mol-1), indicating a change in the glutamine metabolism. The yield of lactate on glucose stayed constant for both experiments. During long-term culture of more than 800 h, the controller kept both the glucose and glutamine concentrations constant at perfusion rates between 0.50 h-1 and 0.15 h-1. The medium, glucose and glutamine feed rate were independently controlled. Both the specific glutamine and glucose consumption rates remained constant for all perfusion rates, which was probably as a result of the constant concentrations. The specific monoclonal antibody production rate decreased with the perfusion rate decreasing from 0.40 h-1 to 0.20 h-1. The immobilized-cell concentration decreased only at the lowest perfusion rate. Both effects could not be explained directly by the increasing ammonium and lactate concentrations nor by the decreasing amino-acid concentrations.

Sublethal sensitivity index as an ecotoxicity parameter measuring energy allocation under toxicant stress: application to cadmium in soil arthropods.

Toxic substances may affect the life history of a species in a variety of ways. Different species maintain different priorities in coping with the physiological consequences of toxicant-induced stress. This is expressed by changes in energy allocation to different life-history characteristics which may have great consequences for the response at the population level. In this study the terrestrial invertebrates Platynothrus peltifer (Oribatida), Orchesella cincta, Folsomia candida (Collembola), and Porcellio scaber (Isopoda) were chosen to evaluate species-specific sensitivity of life histories. Effects on reproduction and weight increase under exposure to cadmium in the food were analyzed. The answer to the question of which species is the most sensitive depends on the parameter chosen. A comparison of sensitivity on the basis of sublethal effects showed P. peltifer to be the most sensitive species; on the basis of lethal effects however, the species O. cincta was the most sensitive. This discrepancy between effect parameters resulted in differences between the distance of the concentrations at which lethal and sublethal effects occur for different species. The ratio between the lethal effect concentration and the sublethal effect concentration is called the sublethal sensitivity index (SSI) and is proposed as a parameter expressing maintenance of sublethal functions under toxicant stress. The SSI seems to be a valuable parameter for evaluating the likelihood of population-level effects under toxicant stress. To extrapolate effects found in the laboratory to the field situation, more attention should be paid to the relationships between effects on life-history parameters and effects on population growth.

On-line monitoring of an animal cell culture with multi-channel flow injection analysis.

A multi-channel flow injection analysis system was used for on-line monitoring of a continuous animal cell culture with high cell density. With this system, the glucose, lactate and glutamine concentration were determined using immobilized dehydrogenases, ammonium using an aqueous o-phthaldialdehyde solution. Glutamine concentration was determined on the basis of the difference between a glutamine and a glutamate measurement. To prevent disturbance of the measurement and pollution of the system, the analytes in the sample were separated from high molecular compounds by on-line dialysis. On-line gas dialysis was used to avoid interference of other amino groups with the ammonium determination. In addition, dialysis was used as a dilution step. The measurement time for all four components was 42 min. This time included a final washing period after the analysis cycle. The system was calibrated once a day. Two continuous cultivations of a hybridoma cell line immobilized in open-porous glass carriers were monitored, using a fluidized bed reactor as cultivation system. The concentration of glutamine, glucose and ammonium determined with the on-line FIA system were in good agreement with the off-line data determined once a day. Only the lactate data showed some deviation. The immobilized enzyme reactors could be used for up to 3000-5000 injections. During the first cultivation, lasting 200 h, the start up period of the reactor was monitored. The on-line measurements described much better the time-course of the concentrations than the off-line data. It was possible to estimate the growth rate of the cells in the micro-carriers by the on-line data. In the course of the second cultivation, which lasted almost 1000 h, the influence of the dissolved oxygen concentration on the cell metabolism was monitored. It was noted that a sudden change of the glutamine concentration in the feed caused a fast change of the consumption and production rate of the measured metabolites.