Monitoring sialylation levels of Fc-fusion protein using size-exclusion chromatography as a process analytical technology tool.

Abstract

To develop a rapid process analytical technology (PAT) tool that can measure sialic acid content of an Fc-fusion protein from cell culture samples. A statistical significant correlation between the sialic acid content and size-exclusion chromatography (SEC)-HPLC retention time of an Fc-fusion protein was observed when analyzing the titer of the samples. Using linear fitting analysis, the data fit the model well with R (2) = 0.985. Based on the SDS-PAGE and oligosaccharide analysis, we speculate that the amounts of the glycans could expand the structure of the Fc-fusion protein. This was manifested by the SEC-HPLC method in which proteins were separated based on its molecular size. In order to development a robust PAT method, an internal standard was used to improve the precision of the method by reducing systematic errors. We found the change of SEC retention time (delta t) and sialic acid content were highly correlated (R (2) = 0.992). This method was further validated by a 1500 l production process. SEC-HPLC is a promising PAT tool to monitor the sialic acid content of Fc-fusion protein during biomanufacturing or medium optimization processes.