Recombinant Chinese hamster ovary (CHO) cells expressing a humanized antibody were obtained by transfection of an antibody expression vector (pKC-GS-HC-huS) into CHO-K1 cells and subsequent glutamine synthetase (GS)-mediated gene amplification in media containing different concentrations of methionine sulfoximine (MSX). Concentrations consisted of 25, 200, 500, and 1000 microM of MSX. The highest producer (HP) subclones were isolated from each MSX level by the limiting dilution method and were characterized with respect to antibody production. No positive relationship was observed between specific antibody productivity (q(Ab)) and MSX concentration. Furthermore, it was found that the antibody production stability of these subclones was very poor even in the presence of selection pressure. During long-term cultures in the presence of the corresponding concentrations of MSX, q(Ab) of all HP subclones significantly decreased for the first six passages and thereafter stabilized. Southern and slot blot analyses showed that the loss of antibody gene copies was only partially responsible for the decreased q(Ab). Fluorescence in situ hybridization (FISH) analysis revealed some cytogenetic features indicative of antibody production instability. Unstable chromosomal structures including dicentrics, rings, and extremely long chromosomes were observed. Amplified sequences enclosed in nuclear projections were often observed. The telomeric repeat sequence, which may be involved in the stabilization of amplified arrays, was found to be absent at the ends of most marker chromosomes. Furthermore, FISH analysis revealed that the overall chromosome content was duplicated in some HP subclones. When metaphase of 12 high producing parental clones was examined, the frequency of occurrence of the polyploidy was 25%. Taken together, the data obtained here suggests that instability could be a concern in the development of CHO cells with GS-mediated gene amplification.