Effect of cloned gene dosage on the expression level and stability of chimeric antibody producing CHO cells

Recombinant Chinese hamster ovary (CHO) cells expressing high level of chimeric antibody against S surface antigen of hepatitis B virus (HBV) were obtained using stepwise methotrexate (MTX)-dihydrofolate reductase (dhfr) mediated gene amplification procedure and subsequent cell cloning by limiting dilution method. Specific growth rate of amplified clone was inversely proportional to the MTX level used in gene amplification. Specific antibody productivity (q(Ab)) of amplified clones rapidly increased with MTX level up to 0.32 mu M and thereafter, q(Ab) became saturated at 20 mu g/10(6)cells/day, The heavy chain gene copy number of amplified clones directly correlated with q(Ab). The q(Ab) of all tested clones gradually decreased during the long-term culture in the absence of MTX, which resulted from the loss of antibody cDNA. Despite the decreased q(Ab), the 1 mu M amplified clone could maintain high volumetric antibody productivity over three months because cell growth improved during long-term culture in the absence of MTX.