ONLINE IMMUNOANALYSIS OF MONOCLONAL-ANTIBODIES DURING A CONTINUOUS-CULTURE OF HYBRIDOMA CELLS

The monoclonal-antibody production of an immobilized hybridoma cell li ne cultivated in a fluidized-bed reactor was monitored on-line for nea rly 900 h. The monoclonal antibody concentration was determined by an immune affinity-chromatography method (ABICAP). Antibodies directed ag ainst the product, e.g, IgG, were immobilized on a micro-porous gel an d packed in small columns. After all IgG present in the sample was bou nd to the immobilized antibodies, unbound proteins were removed by rin sing the column. Elution of the bound antibodies followed and the anti bodies were determined by fluorescence. The analytical procedure was a utomated with a robotic device to enable on-line measurements. The cor relation between the on-line determined data and antibody concentratio ns measured by HPLC was linear. A sampling system was constructed, whi ch was based on a pneumatically actuated in-line membrane valve integr ated into the circulation loop of the reactor. Separation of the cells from the sample stream was achieved by a depth filter made of glass-f ibre, situated outside the reactor. Rapid obstruction of the filter by cells or cell debris and contamination of the sample system was avoid ed by intermittent rinsing of the sample system with a chemical soluti on. 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 dev ice functioned without failure during this long-term culture. The cult ure temperature was varied between 34 and 40 degrees C. Raising the te mperature from 34 up to 37 degrees C resulted in a simultaneous increa se 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 degrees C had a negative effect on the growth rate, whereas the specif ic monoclonal antibody production rate showed a small increase. The ot her specific metabolic rates also increased in the temperature range b etween 38 to 40 degrees C.