Quantitative analysis and process monitoring of site-specific glycosylation microheterogeneity in recombinant human interferon-gamma from Chinese hamster ovary cell culture by hydrophilic interaction chromatography.
A chromatographic method was developed for quantitative analysis of site-specific microheterogeneity of the two N-linked glycosylation sites in recombinant human interferon-gamma produced from Chinese hamster ovary (CHO) cell culture. After the interferon-gamma was harvested by affinity chromatography, the tryptic digestion was carried out. The two glycopeptide pools, isolated from reversed-phase chromatography of tryptic digestion of interferon-gamma, were subjected to further separation by hydrophilic interaction chromatography. Each peak in the chromatograms was identified by matrix-assisted laser desorption ionization and time-of-flight mass spectrometry (MALDI-TOF-MS). The overall elution order of the glycopeptides was the following: neutral glycopeptides, monosialylated glycopeptides, bisialylated glycopeptides, trisialylated glycopeptide and tetrasialylated glycopeptides. Based on the integrated peak area for each compound in the chromatograms, the percentage for each glycan was utilized to quantify the glycosylation pattern of the interferon-gamma. Finally, sialylation and antennarity structure percentages at the two glycosylation sites were chosen as the quality indicators in process monitoring of interferon-gamma production from a serum-free suspension-batch CHO culture.
Zhang
J
J
Biotechnology Process Engineering Center, Massachusetts Institute of Technology, Cambridge 02139, USA.
Wang
D I
DI
D000818:Animals; D001216:Asparagine / Q000737:chemistry; D016466:CHO Cells; D002240:Carbohydrate Sequence; D002845:Chromatography / Q000379:methods; D006224:Cricetinae; D006020:Glycopeptides / Q000032:analysis; D006031:Glycosylation; D006801:Humans; D007371:Interferon-gamma / Q000032:analysis / Q000096:biosynthesis* / Q000737:chemistry; D008969:Molecular Sequence Data; D011994:Recombinant Proteins; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D006020:Glycopeptides; D011994:Recombinant Proteins; D001216:Asparagine; D007371:Interferon-gamma