Changes during subclone development and ageing of human antibody-producing recombinant CHO cells
- 作者：Strutzenberger ; Karl ; Borth ; Nicole ; Kunert ; Renate ; Steinfellner ; Willi ; Katinger ; Hermann
- 关键词：Antibody production ; Stability ; Recombinant Chinese hamster ovary cells ; Light and heavy chain ; CHO ; Chinese hamster ovary cells ; dhfr/DHFR ; dihydrofolate reductase gene/protein ; G418 ; neomycin ; MTX ; methotrexate
- 刊名：Journal of Biotechnology
- 出版年：1999
- 期刊代码：53_01681656
- 类别：imm
- 出版时间：April 15, 1999
- 卷：69
- 期：2-3
- 页码：215-226
- 文件大小：668 K
摘要
Some of the problems encountered with human or human-mouse heterohybridomas, such as low growth rates and high serum requirements, have led to the increased 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 specific 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 analysed by Northern blot. In addition the intracellular content in light and heavy chain was measured by flow cytometry and the specific secretion rates were determined. The stability of gene expression was followed in the highest producing subclone for over a year. As previously seen in heterohybridoma cells a high expression rate of light chain is beneficial in speeding up secretion rates of whole antibody. When grown in the presence of G418 and methotrexate the amplified gene copies in the genome of recombinant CHO cells were stable over more than 100 passages. However, the expression of light chain, and with it the secretion rate, decreased with time. The low intracellular concentration of light chain resulted in accumulation of heavy chain in the endoplasmic reticulum due to retention by chaperones. The specific secretion rate decreased by 50%after 100 passages. When no G418 or methotrexate were present 75%of the gene copies were lost after 100 passages.