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 scale production of pharmaceutical proteins is self-evident. However, few papers have studied the changes that occur during subclone development. This information would be important for further improvement of screening and subcloning protocols. We have therefore compared subclones of a human-mouse heterohybridoma cell line producing a human antibody againt HIV-1. Three subclones 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 correlation between the intracellular content in light chain and the secretion rate was found, while the intracellular content in heavy chain was the same for all three subclones. These results indicate that the assembly in the endoplasmic reticulum (ER) is one of the major rate limiting factors in antibody production. During long time cultivation of the heterohybridoma cell line a continuous decrease in light and heavy chain production was seen without the appearance of a non producing sub-population.
Borth
N
N
Institute for Applied Microbiology, University of Agriculture, Vienna, Austria. borth@mail.boku.ac.at
Strutzenberger
K
K
Kunert
R
R
Steinfellner
W
W
Katinger
H
H
D000818:Animals; D000906:Antibodies / Q000235:genetics; D000917:Antibody Formation / Q000502:physiology*; D015152:Blotting, Northern; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000276:immunology; D002999:Clone Cells; D005434:Flow Cytometry / Q000379:methods*; D015497:HIV-1 / Q000276:immunology; D006801:Humans; D006825:Hybridomas / Q000166:cytology*; D007143:Immunoglobulin Heavy Chains / Q000032:analysis; D007147:Immunoglobulin Light Chains / Q000032:analysis; D051379:Mice; D012333:RNA, Messenger / Q000032:analysis*; D013997:Time Factors