title: Monitoring sialylation levels of Fc-fusion protein using size-exclusion chromatography as a process analytical technology tool.
abstract: 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 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.
  
  
  CONCLUSIONS
  SEC-HPLC is a promising PAT tool to monitor the sialic acid content of Fc-fusion protein during biomanufacturing or medium optimization processes.
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, 200237, China.
authors.forename: Jintao
authors.initials: J
authors.lastname: Liu
authors.forename: Xinning
authors.initials: X
authors.lastname: Chen
authors.forename: Li
authors.initials: L
authors.lastname: Fan
authors.forename: Xiancun
authors.initials: X
authors.lastname: Deng
authors.forename: H
authors.initials: H
authors.lastname: Fai Poon
authors.forename: Wen-Song
authors.initials: WS
authors.lastname: Tan
authors.forename: Xuping
authors.initials: X
authors.lastname: Liu
mesh_terms: D000818:Animals; D016466:CHO Cells; D002240:Carbohydrate Sequence; D002850:Chromatography, Gel / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D003412:Cricetulus; D006031:Glycosylation; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry* / Q000378:metabolism; D019158:N-Acetylneuraminic Acid / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D007141:Immunoglobulin Fc Fragments; D011994:Recombinant Proteins; D019158:N-Acetylneuraminic Acid