Changes during subclone development and ageing of human antibody-producingrecombinant CHO cells

Some of the problems encountered with human or human-mouse heterohybridomas , such as low growth rates and high serum requirements, have led to the inc reased use of recombinant cell lines for production of human antibodies. To evaluate the suitability of such alternative cell lines for the production of human antibodies we have analysed several subclones with differing spec ific production rates of a recombinant CHO cell line. Gene copy number and site of chromosomal integration for the light and heavy chain and the dhfr gene were determined by in-situ hybridisation. Specific mRNA content was an alysed by Northern blot. In addition the intracellular content in light and heavy chain was measured by flow cytometry and the specific secretion rate s were determined. The stability of gene expression was followed in the hig hest producing subclone for over a year. As previously seen in heterohybrid oma cells a high expression rate of light chain is beneficial in speeding u p secretion rates of whole antibody. When grown in the presence of G418 and methotrexate the amplified gene copies in the genome of recombinant CHO ce lls were stable over more than 100 passages. However, the expression of lig ht chain, and with it the secretion rate, decreased with time. The low intr acellular concentration of light chain resulted in accumulation of heavy ch ain in the endoplasmic reticulum due to retention by chaperones. The specif ic secretion rate decreased by 50% after 100 passages. When no G418 or meth otrexate were present 75% of the gene copies were lost after 100 passages. (C) 1999 Elsevier Science B.V. All rights reserved.