Recombinant Chinese hamster ovary (rCHO) cells expressing a high level of chimeric antibody were obtained by cotransfection of heavy- and light-chain cDNA expression vectors into dihydrofolate reductase-deficient CHO cells and subsequent gene amplification in medium containing stepwise increments in methotrexate (MTX) level up to 1.0 mu M. To determine the clonal variability within the amplified cell population in regard to antibody production stability, 20 subclones were randomly isolated from the amplified cell population at 1.0 mu M MTX (CS13-1.0 cells). Clonal analysis showed that CS13-1.0 cells were heterogeneous with regard to specific growth rate (mu) and specific antibody productivity (q(Ab)), although they were derived from a single clone. The mu and q(Ab) of 20 subclones were in the range of 0.51 to 0.72 day(-1) and 10.9 to 19.1 mu g/10(6) cells/day, respectively. During 8 weeks of cultivation in the absence of selective pressure, the mu of most subclones did not change significantly. On the other hand, their q(Ab) decreased significantly. Furthermore, the relative decrease in q(Ab) varied among subclones, ranging from 30% to 80%. Southern and Northern blot analyses showed that this decreased q(Ab) resulted mainly from the loss of amplified immunoglobulin (Ig) gene copies and their respective cytoplasmic mRNAs. For the sake of screening convenience, an attempted was made to correlate the initial properties of subclones (such as mu, q(Ab), and Ig gene copies) with their antibody production stability during long-term culture. Among these initial properties examined, only q(Ab) of subclones could help to predict their stability to some extent. The subclones with high q(Ab) were relatively stable with regard to antibody production during long-term culture in the absence of selective pressure (P < 0.005, ANOVA). Taken together, the clonal heterogeneity in an amplified CHO cell population necessitates clonal analysis for screening stable clones with high q(Ab). (C) 1998 John Wiley & Sons, Inc.