Optimization of humanized monoclonal antibody against human tumor necrosis factor-α production

Tumor necrosis factor alpha (TNF-α) is an important cell-signaling factor of the immune system. Anti-TNF-α monoclonal antibodies (mAbs) are frequently used as therapeutic agents in chronic immune-mediated inflammatory diseases. The aim of this study is to investigate the effects of the different production systems and different feeding strategies on the growth parameters of this cell line, product yield/productivity and quality of the product and accordingly to design a feasible process for anti-TNF-α mAb manufacturing. In this thesis, glutamine synthetase (GS) - Chinese hamster ovary (CHO) cells that constantly produce humanized anti-TNF-α mAb, were cultivated in different culture conditions including different commercially available chemically defined (CD) media in batch culture. To evaluate the effect of low culture temperature on mAb production, the cells were cultivated at 33, 35, and 37°C. Thereafter, different fed-batch cultivation approaches were used to cultivate the cells. pH, dissolved oxygen (DO), aeration rate and stirring speed were optimized in 1L stirred tank bioreactor. mAb production process was successfully scaled up to 4L. In order to assess the growth parameters of the cells in different production systems glucose consumption, lactate production were determined by HPLC and mAb productivities of the systems were determined by indirect ELISA. mAbs were partially purified by protein A affinity chromatography. The supernatants from the 4L bioreactor were concentrated by cross flow filtration system. SDS- PAGE was applied to prove the both purity of the product and mAbs' reduced size. Products were also characterized according to their conformational structure. Approximately 2 g/L mAb was observed in CD2 medium with FB5 cultivation mode by using biphasic 35°C culturing temperature in 1L bioreactor. The bioreactor was controlled at 40±10% DO and the culture pH was kept at 7,2±0,5. The produced mAb under those conditions gave high conformational similarity with its originator molecule (HUMIRA®).