Development of a novel system for the expression and production of recombinant monoclonal antibody, tocilizumab,...

The development of anti-interleukin-6-receptor (IL6R) recombinant monoclonal antibody Tocilizumab (TCZ) production in a methylotrophic yeast Pichia pastoris was the focus of this research work. The molecular weight of full-length TCZ is approximately 148 kDa. A bicistronic expression vector with both heavy and light chain genes was constructed within individual inducible alcohol oxidase promoters and secretory signal peptides. The recombinant strains were subcultured several times to maintain the stability of both heterologous antibody genes. A gene copy number ratio (light to heavy chain) of 3:2 was observed to be the best transformant for the secretory expression of Tocilizumab. The expression of TCZ was confirmed with Western blot and ELISA techniques. The structural analysis of full-length TCZ was carried out by circular dichroism spectroscopy and alpha fold predictions. The extracellular expression of the full-length TCZ is achieved at ~0.33 mg/L over 96 h of batch fermentation. The antibody was further purified using Protein A resin-based affinity chromatography, and various comparative cell-free and cell-based functional assays were performed with marketed TCZ (ACTEMRA™, Cipla) and Pichia-derived TCZ to establish the interaction with its cognate receptor IL6R and functional efficacy, respectively.