GROWTH AND MONOCLONAL ANTIBODY PRODUCTION OF FREE AND IMMOBILISED HYBRIDOMAS

Abstract

The growth and monoclonal antibody (Ig) production of free and immobilised hybridoma cells, 6BB and 2G3 cells were studied. The former secretes IgM for blood typing whereas the latter produces IgG raised against liver cancer cells. The free cells were cultured flask under controlled batchwise conditions. Quantitative metabolic and Ig production rates the integral method. It was found in a spinner were analyzed that using growth, metabolic, and Ig production rates were dissimilar in the two cell lines studied. Maximum viable cell densities of 8 x 105 and 7 x 105 cells/ml and overall Ig production rates of 15.65 µg-IgM/ml and 5.48 µg-IgG/ml-day were obtained for the 6BB and 2G3 cells respectively. These results suggest that the rates of substrate utilization and Ig production are not directly related to each other and that they are cell-line dependent. A mixed-growth associated Ig production model describes well the results obtained. Immobilised cells Ca-alginate, gel were prepared by beads. Optimal entrapment in immobilisation conditions with respect to gel stability and cell growth were found to be 1.5% Na-alginate prepared in PBS, a gelation time of 6 min and an inoculum size of 2x105 cells/ml-gel. Under these conditions, inoculum total cells population up to 50 times the size has been observed. Apart from higher metabolic rates in immobilised 2G3 cells compared to immobilised 6BB, growth and Ig production rates were higher in 6BB than in 2G3. For both cell lines, the semi-continuous cultures were maintained at viable cell densities above about 5-6 x 106 cells/ml-gel, with a concomitant maintenance of the Ig production rates of 205.2 µg-IgM/ml-gel-day and 80.4 µg-IgG/ml-gel-day for 6BB and 2G3 cells respectively over a period of about 2-3 weeks. Similar amino acid uptake and production patterns for free and immobilised hybridoma cultures were observed for the cell lines examined. The observed deficiency in some essential amino acids at the end of growth or stationary phase suggests that one or more of the amino acids may be linked to growth cessation as well as Ig production. Compared to the immobilised cells corresponding free generally exhibited growth and other metabolic transfer limitation of the rates due cells, the lower specific mainly to mass nutrients and through the gel phase. Significant amounts particular, IgM, were found to accumulate phase. However, the gel matrix appears to metabolites of Ig, in in the gel provide a conducive environment for the growth of. hybridoma cells. The immobilised cells were well protected and high cell density with good viability could be maintained for continual Ig production.