CARB 75 |
| Glycosylation of recombinant proteins is of particular importance because it can play significant roles in the clinical properties of the glycoprotein, such as enzyme activity, protein stability, pharmacokinetics, and immunogenicity. In this work, we determined the structure of the N-glycans of recombinant human Factor IX and protein C produced in the transgenic pig mammary gland. Using a combination of capillary electrophoresis, HPLC, and Electrospray Ionization Ion Trap Mass Spectrometry (ESI-IonTrap MS), we have found that the majority of N-glycans of transgenic pig-derived Factor IX and protein C(over 95%) are complex bi- and tri-antennary with one or two terminal N-acetylneuraminic acid(NeuAc) moieties. Using these highly sensitive techniques, we were unable to detect any high mannose or hybrid N-glycan structures. We also found that the N-glycan structures of the glycoproteins produced in the porcine mammary epithelial cells differed with respect to N-glycans from glycoproteins produced in porcine thyroid, B-cells, and cell-surface glycoproteins. The sialic acid found in transgenic Factor IX was NeuAc; we were unable to detect N-glycolylneuraminic acid(NeuGc) moieties that are commonly found in porcine glycoproteins that are produced in other tissues and cell types. Additionally, we were unable to detect any glycans that had a terminal Galα(1,3)Gal disaccharide sequence, which is strongly antigenic in humans. Collectively, these data indicate that there are significant species-specific and tissue/cell-specific differences in N-glycan structures among animals used for transgenic animal bioreactors. The results show that the porcine mammary gland can be a viable candidate bioreactor for production of recombinant human glycoproteins that require complex, sialylated N-linked glycans. |
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Chemical Glycobiology Symposium
7:00 PM-9:00 PM, Tuesday, 12 September 2006 Moscone Center -- Hall D, Poster
Sci-Mix
Division of Carbohydrate Chemistry |