HYPER-STIMULATION OF MONOCLONAL-ANTIBODY PRODUCTION BY HIGH OSMOLARITY STRESS IN ERDF MEDIUM

An earlier study (Chua et al., 1994) showed that hybridoma 2HG11 culti vated in a basal medium called eRDF, which is enriched in amino acids, enabled higher immunoglobulin (Ig) production with and without serum, when compared to two other traditional media RPMI and DMEM/F12. A fur ther enhancement of Ig productivity was achieved when the osmolarity o f the culture medium was increased from 300 mOsm to 350 mOsm (Oh et al ., 1993). To determine whether the eRDF media was indeed better, three other cell lines, two IgG producers (TB/C3 and 113/17) and an IgM pro ducer (B10), were tested. The results showed that maximum viable cell densities in eRDF medium were up to 3-times higher than in RPMI and ma ximum Ig titres were 2-8-times higher than in DMEM/F12 and RPMI. The t hree cell lines were similarly subjected to osmotic increases from 300 mOsm to 350 and 400 mOsm by addition of NaCl. There was an increase i n Ig titres of between 30% to 100% compared to the control medium, alt hough cell growth was reduced. Thus, hyper-stimulation by osmolarity s tress was found to be generally effective in eliciting higher Ig produ ction; the extent of enhancement being more pronounced for certain cel l lines. Other osmolytes such as sucrose and KCl demonstrated similar effects of increasing Ig productivity. Study on the mechanism of actio n of osmotic stress on hybridoma 2HG11 revealed that hyper-stimulation of Ig productivity was fundamentally related to a greater availabilit y of amino acids to cells as the cells actively accumulated more salt and amino acids to compensate for the higher medium osmolarity. Uptake of the amino acid analogues C-14-aminoisobutyric acid and H-3-methyla minoisobutyric acid into cells increased to 2.34 X 10(3) cpm per cell per min and 6.35 x 10(3) cpm per cell per min, respectively, under osm otic stress. This corresponds to an 85% increase in uptake via the Na-dependent symport and a 50% increase in uptake via the Na+-independen t and Na+-dependent symports. In the 350 mOsm medium, hybridomas also demonstrated an increase in metabolic activities of 5-10% compared to the control. This, together with the reduced specific growth rate in c ells under osmotic stress, suggests that more energy was channelled in to the biosynthetic pathway of Ig production.