Zhejiang Hisun Pharmaceutical Hangzhou Co.

Hangzhou, China

Zhejiang Hisun Pharmaceutical Hangzhou Co.

Hangzhou, China

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Liu J.,East China University of Science and Technology | Wang J.,East China University of Science and Technology | Fan L.,East China University of Science and Technology | Chen X.,East China University of Science and Technology | And 6 more authors.
World Journal of Microbiology and Biotechnology | Year: 2015

Sialic acid levels of therapeutic glycoprotein play an important role in plasma half-life. An undesirable decrease of sialic acid content was observed when we increased Fc-fusion protein productivity fourfold in a GS-CHO cell line by bioprocess optimization. We investigated the potential mechanism for the sialic acid content reduction. We found that limited nucleotide sugar precursor and the extracellular sialidase were not responsible for the reduction of the sialic acid content after titer improvement. Oligosaccharide analysis revealed that the lack of protein galactosylation was the potential cause for the reduction of sialic acid content. Thus we validated this notion by evaluated galactose supplementation in 2 L bioreactors. Cell culture performance was not impacted by addition of up to 40 mM galactose except for the glucose consumption rate. Addition of 20 mM galactose to the bioreactor resulted in the increase of 44 % for total sialic acid content and 20.3 % for sialylated glycans. These data were further validated when the process was run on 200 L scaled bioreactor. These data together show that the galactosylation plays an apparent role in sialylation in our current system. © 2015, Springer Science+Business Media Dordrecht.


Liu J.,East China University of Science and Technology | Chen X.,East China University of Science and Technology | Chen X.,Zhejiang Hisun Pharmaceutical Hangzhou Co. | Fan L.,East China University of Science and Technology | And 5 more authors.
Biotechnology Letters | Year: 2015

Objective: To develop a rapid process analytical technology (PAT) tool that can measure sialic acid content of an Fc-fusion protein from cell culture samples. Results: 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 R2 = 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 (deltat) and sialic acid content were highly correlated (R2 = 0.992). This method was further validated by a 1500 l production process. Conclusion: SEC–HPLC is a promising PAT tool to monitor the sialic acid content of Fc-fusion protein during biomanufacturing or medium optimization processes. © 2015, Springer Science+Business Media Dordrecht.

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