Development of Stable Cell Line for the production of Recombinant anti-Her2 Antibody
Project no. 1306 · Status: Kapanmış · 2016
Summary
The biological products used in cancer treatment are some of the most expensive drugs on the market and are a growing financial burden on health-care systems. By 2020, numerous major biological cancer drugs will lose their patent protection. Nonetheless competition will occur in the pharmaceutical market with an increase in the number of biosimilars that will produce an alternative to the original product and quite expensive original drugs will be substituted biosimilar drugs priced lower. The aim of the project is to develop a Chinese hamster ovary (CHO) cell line that stably secretes herceptin biosimilar targeting the HER2 extracellular domain in the treatment of human epidermal growth factor receptor 2 (HER2) overexpressing breast cancer patients. The mammalian expression vector called pIRES2/LC/HC containing heavy and light chains of anti-HER2 monoclonal antibody were developed by gene optimization, cloning and transformation and was verified by restriction enzyme digestion, multiplex PCR and DNA sequencing. Since the pIRES2/LC/HC vector contains the gentamicin resistance gene, the geneticin sensitivity to be used for the selection of stable cells was determined as 500 µg/mL. Transfection of the pIRES2/LC/HC vector into CHO cells was performed using lipofectamine. Stable cells have been selected in the presence of selective agent (growth medium containing 500 µg/ml) for 21 days. The single cell colonies obtained from the mixed population were obtained using the limited dilution technique