Analysis of changes during subclone development and ageing of human antibody-producing heterohybridoma cells by Northern blot and flow cytometry

The economic importance of obtaining high-producing subclones for large sca le production of pharmaceutical proteins is self-evident. However, few pape rs have studied the changes that occur during subclone development. This in formation would be important for further improvement of screening and subcl oning protocols. We have therefore compared subclones of a human-mouse hete rohybridoma cell line producing a human antibody against HIV-1. Three subcl ones with low, medium and high specific production rates were selected for this study and their light and heavy chain mRNA content, the intracellular content of light and heavy chain and the specific secretion rates compared. In addition the long time stability of antibody expression in the highest producing subclone was analysed for one year. For the three subclones a cor relation between the intracellular content in light chain and the secretion rate was found, while the intracellular content in heavy chain was the sam e for all three subclones. These results indicate that the assembly in the endoplasmic reticulum (ER) is one of the major rate limiting factors in ant ibody production. During long time cultivation of the heterohybridoma cell line a continuous decrease in light and heavy chain production was seen wit hout the appearance of a non producing sub-population. (C) 1999 Elsevier Sc ience B.V. All rights reserved.