Modulation of Mannose levels in N-linked glycosylation through cell culture process conditions in order to increase ADCC activity for an antibody Biosimilar
May 6-11, 2018
Glycosylation of recombinant proteins is one of the key product quality attribute that is closely monitored throughout the development life cycle of the biologic starting from cell line development to process development and commercial manufacturing. Glycosylation profiles of the recombinant proteins can be influenced by cell line, cell culture media components, and cell culture process parameters. Glycosylation often plays a critical role in biological activity, efficacy and stability of recombinant protein therapeutics. Specifically, glycosylation has been associated with serum half-life of the product as well as the immunogenicity of the molecule. In addition, the glycoform make-up of the molecule can influence effector function activities such as antibody dependent cell cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) of the molecule. In this study, we describe our efforts to modulate N-glycan profile for a CHO cell line producing IgG biosimilar to match the innovator molecule product quality profile. After the initial process development efforts, the ADCC activity of the biosimilar was observed to be lower than the original innovator molecule. The decrease in ADCC activity was attributed to lower levels of mannose content in comparison to the innovator molecule. An extensive screening study was conducted to investigate several factors that are known to modulate mannose content in the N-glycan of the protein.1 The factors tested included modifying cell culture process parameters such as osmolality, glutamine levels, temperature and pH, supplementing feed with alternate sugars and amino sugars (mannose, fructose, glucosamine and galactosamine), changing intracellular pH using ammonium chloride and addition of small molecule ionophores like monensin (a sodium-hydrogen ionophore capable of integrating into biological membranes and thus disturbing sodium-hydrogen gradients across those membranes). All these conditions demonstrated different levels of regulation in total mannose level, however, the supplementation of feed medium with alternate sugars and amino sugars was the most viable process means to independently alter glycosylation while maintaining other aspects of product quality, cell growth and productivity. The results from this screening study were further optimized using a response surface DOE bioreactor study. Significant increase in mannose levels was observed with respect to the control condition. The increase in ADCC activity was observed with increasing level of mannose and comparable ADCC activity to the innovator was achieved. However, a scale based effect was observed during scale-up of the process. Upon investigation, it was observed that the combination of sugar/amino sugar supplementation levels interacted with absolute osmolality levels which varied slightly between scales and in turn dictated absolute level of increase in N-glycan mannose content. Additional studies were performed in the established scale-down model, 3L bench-top bioreactor, to confirm the optimized process. The results from these studies will be used to collectively present a model based on sugar/amino sugar concentrations and osmolality to predictively increase mannose content in glycosylated proteins.
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Shahid Rameez, Yogender Gowtham, Dustin Costello, Srivatsan Gopalakrishnan, Sigma Mostafa, and Abhinav Shukla, "Modulation of Mannose levels in N-linked glycosylation through cell culture process conditions in order to increase ADCC activity for an antibody Biosimilar" in "Cell Culture Engineering XVI", A. Robinson, PhD, Tulane University R. Venkat, PhD, MedImmune E. Schaefer, ScD, J&J Janssen Eds, ECI Symposium Series, (2018). http://dc.engconfintl.org/ccexvi/125