Glycosylation plays important roles in maintaining the structure and function of glycoproteins and is considered as a critical quality attribute of biopharmaceuticals. However, it is challenging to control the glycan profile of a protein product with limited understanding of the relationship between culture process and glycosylation. Here, we report a combined method integrating medium components and process parameters to manipulate the glycan profile of an antibody fusion protein. First of all, the glycan extension and degradation process were evaluated. Then, the effects of individual medium supplements and process parameters on glycosylation were explored. In the end, the method was validated by combining the effectors of glycan precursor supply, key enzyme regulators and cofactors to manipulate the glycosylation for achieving a desired glycan profile.<br/><br/>The target antibody fusion protein is a highly sialylated glycoprotein containing six N-glycan sites and numerous O-glycan sites. Significant loss of sialic acids in the antibody fusion protein during cell culture process was observed, the mechanism of the phnomenon was studied. Dynamic calculation has been carried out to evaluate the contribution of intracelluar glycan extension and extracellular sialic acid degradation. The results showed the proportion was almost 1:1. Intracelluar glycan extension was hampered because of limited nucleotide-sugars supply and down-regulation of some glycotransferases. Extracellular sialic acid degradation was caused by sialidase, released to the media when cell death, and it was sensitive to some process parameters. Then, hormones and glycan precursors were chosen to enhance both N-glycan and O-glycan sialylation levels by enhancing glycan extension steps in combination with process parameter optimization leading to lower amount of sialic acid degradation. Finally, both sialic acid content and N-glycan profile of the antibody fusion protein were kept consistent throughout the optimized cell culture process. Further, the process has been successfully scaled up to a 1500L production bioreactor without changing glycosylation of the antibody fusion protein.<br/><br/>This study lays a solid foundation for better understanding of the glycosylation process, leading to the potential manipulation of the glycan profile of a protein product in a cell culture process.

Manipulating sialylation of antibody fusion protein in cell culture process

253rd National Meeting (2017)

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