Conference Dates

May 6-11, 2018


Tumor necrosis factor alpha (TNFα) is a proinflammatory cytokine that mediates the homeostasis of immune responses; its exacerbated production is associated with the pathogenesis of autoimmune and chronic inflammatory diseases. Anti-TNFα drugs have revolutionized the treatment of inflammatory conditions such as rheumatoid arthritis and Crohn’s disease. Currently, a worldwide race is on stage for the production of biosimilars moved by patent expiration of monoclonal antibodies (mAbs), such as anti-TNFα adalimumab. This project is based on the first development stage for an adalimumab biosimilar candidate with potential for national production through the generation of a productive and stable cell line and assessment of its functionality. The robotic system ClonePix was used for screening and isolation of colonies from transfected CHO-S stable pools plated in semisolid medium. Selected clones were expanded based on growth and productivity. Purified mAbs from different clones were tested for binding and functional activity. The binding affinity of the denominated adabut clones to TNFα and FcRs, tested by surface plasmon resonance, did not differ statistically when compared to reference adalimumab. One functional activity assay demonstrated the antibody neutralization capacity of the cytotoxicity induced by TNFα in L929 murine fibroblasts. A second assay confirmed adabut as an antagonist of the TNFα activity by the inhibition of the cell adhesion molecule expression in HUVEC cultures. The binding kinetics and functional analysis performed suggest a potential for further development of adabut as a biosimilar. The process of the cell line development for adabut generated data consisting of cell growth/viability, volumetric and specific productivity, antigen binding kinetics, functional assays and long-term stability, from which 3 clones were selected. One clone (123) was top ranked and 2 others (70 and 225) showed equivalent performance. To increase the basal productivity of clone 123, 0.6g/L, we run 3 fed-batch experiments to evaluate 6 basal media, 8 supplements and a temperature shift to 32 C on day 6 on some conditions. The experiments were conducted in 250 and 500mL-shaker flasks along 14 days or while cell viability was above 60%. The flasks were sampled daily for cell counting/viability by Vi-Cell XR cell counter and glucose monitoring. Samples were stored for additional metabolites and antibody concentration assessment. By testing 24 different fed-batch strategies we were able to increase up to 6 times the volumetric clone productivity, while maintaining product quality. No significant differences were observed between the mAbs purified from days 14, 16 or 17 fed-batch cultures and the reference commercial product by SDS-PAGE, SEC-HPLC, IEX-HPLC and IEF analyses.

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