An integrated modular framework for modeling the effect of ammonium on the sialylation process of monoclonal antibodies produced by CHO cells

Background: Monoclonal antibodies (mABs) have emerged as one of the most important therapeutic recombinant proteins in the pharmaceutical industry. Their immunogenicity and therapeutic efficacy are influenced by post-translational modifications, specifically the glycosylation process. Bioprocess conditions can influence the intracellular process of glycosylation. Among all the process conditions that have been recognized to affect the mAB glycoforms, the detailed mechanism underlying how ammonium could perturb glycosylation remains to be fully understood. It was shown that ammonium induces heterogeneity in protein glycosylation by altering the sialic acid content of glycoproteins. Hence, understanding this mechanism would aid pharmaceutical manufacturers to ensure consistent protein glycosylation. Methods: Three different mechanisms have been proposed to explain how ammonium influences the sialylation process. In the first, the inhibition of CMP-sialic acid transporter, which transports CMP-sialic acid (sialylation substrate) into the Golgi, by an increase in UDP-GlcNAc content that is brought about by the augmented incorporation of ammonium into glucosamine formation. In the second, ammonia diffuses into the Golgi and raises its pH, thereby decreasing the sialyltransferase enzyme activity. In the third, the reduction of sialyltransferase enzyme expression level in the presence of ammonium. We employed these mechanisms in a novel integrated modular platform to link dynamic alteration in mAB sialylation process with extracellular ammonium concentration to elucidate how ammonium alters the sialic acid content of glycoproteins. Results: Our results show that the sialylation reaction rate is insensitive to the first mechanism. At low ammonium concentration, the second mechanism is the controlling mechanism in mAB sialylation and by increasing the ammonium level (< 8 mM) the third mechanism becomes the controlling mechanism. At higher ammonium concentrations (> 8 mM) the second mechanism becomes predominant again. Conclusion: The presented model in this study provides a connection between extracellular ammonium and the monoclonal antibody sialylation process. This computational tool could help scientists to develop and formulate cell culture media. The model illustrated here can assist the researchers to select culture media that ensure consistent mAB sialylation.