Heterogeneity within dihydrofolate reductase-mediated gene amplified population of chinese hamster ovary cells producing recombinant antibody

Abstract

Recombinant Chinese hamster ovary cells expressing high level of chimeric antibody against S surface antigen of hepatitis B virus were obtained by cotransfection of heavy and light chain cDNA expression vectors and subsequent stepwise methotrexate (MTX)-dihydrofolate reductase (dhfr)-mediated gene amplification. In order to determine the heterogeneity within amplified cell population, twenty subclones were randomly isolated from amplified cell pool at 1.0 μM MTX and their stability with respect to antibody production was characterized. For 8 weeks of cultivation in the absence of selective pressure, the specific growth rates(μ) of most subclones did not change significantly. On the other hand, their specific antibody productivity ($q_{Ab}$) decreased significantly. However, the relative extent of decrease in $q_{Ab}$ was varied among subclones, ranging from 30 % to 80 %. Southern and northern blot analyses showed that the loss of antibody productivity resulted mainly from those of amplified immunoglobulin (Ig) gene copies and their respective cytoplasmic mRNA. Furthermore, the stability of subclones regarding antibody productivity was not related with either their $q_{Ab}$s or Ig gene copies at initial point of culture. Accordingly, the antibody production of each subclone during long-term culture cannot be predicted based on its initial $q_{Ab}$s or Ig gene copies.