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Cellosaurus publication CLPUB00344

Publication number CLPUB00344
Authors Ogorek C.
Title Characterization of the N-glycosylation profile of the new avian cell line AGE1.CR and the modification of fucosylation.
Citation Thesis PhD (2012), Freien Universitat Berlin, Germany
Web pages http://www.diss.fu-berlin.de/diss/receive/FUDISS_thesis_000000037547
Abstract In the present dissertation the N-glycosylation profile of a new avian cell line, AGE1.CR, has been characterised. This cell line has been developed from retina cells of the Muscovy duck (Cairina moschata) by immortalization via stable transfection with E1 genes from human adenovirus type 5 and was designed by ProBioGen AG to replace primary chicken cells in the field of vaccine production. AGE1.CR.pIX is a modified CR cell line transfected for stable expression of the pIX-gene that encodes a structural protein of human adenovirus type 5. This modification further increases productivity of the cell line for certain viral vectors. However the AGE1.CR.pIX cell line showed an increased glucose consumption and propensity to cell-cell aggregation. Given that cell contacts and glycosylation are closely related, the N-glycosylation profile of both avian cell lines (AGE1.CR and AGE1.CR.pIX) was characterised. With regard to functionality the focus of the characterisation was on cell-specific sialylation and fucosylation. In addition, native cell membrane glycoproteins and recombinant glycoproteins were analysed by specific lectin staining, chromatography and mass spectrometry. Sole expression of N-acetylneuraminic acid (Neu5Ac) instead of typical N-glycolylneuraminic acid (Neu5Gc) was determined by RP-HPLC for the AGE1.CR and AGE1.CR.pIX cell line as in human cells. Surprisingly, specific lectin staining coupled with FACS-analysis showed an alpha2-3-and alpha2-6-glycosidic linked Neu5Ac for avian cell lines. In comparison, the common used producer cell line Chinese hamster ovary (CHO) exclusively expresses alpha2-3-linked Neu5Ac. Due to the ability of expressing a2-6-linked Neu5Ac the avian cell lines are useful to produce anti-inflammatory recombinant human IgGs (rec.IgG). The analysed N-glycosylation profile of avian cell membrane glycoproteins exhibited predominantly complex type tri-antennary N-glycans. Similar to the analysed human neuronal cell line (AGE1.HN, ProBioGen), avian cells showed a higher antennary composition in contrast to the CHO cell line with predominantly complex type bi-antennary N-glycans. By chromatography and mass spectrometry, a decreased fucosylation state could be determined for avian cell membrane glycoproteins in contrast to CHO. Complex type N-glycans were found to be predominantly single fucosylated in the alpha1-6-linked state also known as core-fucose. In contrast to a decreased fucosylation state of avian cell membrane glycoproteins rec.IgG was produced with a IgG-typical high core-fucosylation. Furthermore, in comparison of avian and CHO cell lines avian cell membrane glycoproteins and rec.IgGs showed a decreased galactosylation state. The AGE1.CR showed further a decreased galactosylation state in contrast to the AGE1.CR.pIX cell line, as the only detected difference in between of the two avian N-glycosylation profiles. Because galactosylation is one of the final processing steps of well-folded proteins this supports the model where pIX in the AGE1.CR.pIX activates heat shock responses that potentially improve protein folding and maturation. It is also demonstrated that the avian cells, as opposed to CHO, express rec.IgG with bisecting GlcNAc structures similar to IgG in human serum. Furthermore a new strategy for stable reduction of fucosylation was successfully implemented in avian and CHO cell lines expressing rec.IgG. This method inhibits the de novo synthesis of GDP-fucose and demonstrates a novel alternative to common strategies such as the alpha1-6-fucosyltransferase (FUT8)-knockout. rec.IgG with demonstrated reduction in fucosylation showed an increased Fc-mediated effector function. The avian cell lines, were further used to produce the modified vaccinia virus Ankara (MVA), an important strain for vectorial vaccine applications. The N-glycosylation profile of MVA produced in avian cells reflected host properties. However, MVA-particles exhibited an increased sialylation state as compared with the cell membrane glycoproteins. We speculate that the virus promotes this state as MVA-particles with higher sialylation are protected against early clearing out of serum by asialoglycoprotein receptors. With the help of analysed cell membrane glycoproteins and rec. IgGs, elementary characteristics of the avian N-glycosylation profile could be determined. Thus the AGE1.CR and AGE1.CR.pIX cell lines showed an alpha2-3-and alpha2-6-linked Neu5Ac, bisecting-GlcNAc and core- fucosylation. Finally, in the avian N-glycosylation pro le no for human potent immunogenic monosaccharides, such as Xylose or Neu5Gc, could be found. Recombinant human IgG from AGE1.CR resembled low amounts of mannose rich structures as detected with CHO cells. Thus the new avian cell lines (AGE1. CR and AGE1.CR.pIX) exhibit a human-like N-glycosylation profile and appear suitable not only for virus production but also for production of therapeutic proteins.
Cell lines CVCL_S509; AGE1.CR