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Analysis of the asparagine-linked oligosaccharides of recombinant human glycoproteins produced in the porcine mammary gland

Geun-Cheol Gil, University of Nebraska - Lincoln

Abstract

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, the N-glycan structures of recombinant human Factor IX (tg-FIX) and Protein C (tg-PC) produced in the transgenic pig mammary gland were determined. It has been found that the majority of N-glycans of tg-FIX and tg-PC are complex bi- and tri-antennary with one or two terminal N-acetylneuraminic acid (Neu5Ac) moieties. We also found that the N-glycan structures of tg-FIX and tg-PC produced in the porcine mammary epithelial cells differed with respect to N-glycans from endogenous glycoproteins produced in porcine thyroid, and B-cells. Tg-FIX and tg-PC contain no Neu5Gc moiety that is commonly found in porcine glycoproteins that are produced in porcine thyroid and B-cells. Additionally, tg-FIX and tg-PC do not contain any glycans that has a terminal Galα(1,3)Gal disaccharide sequence, which is strongly antigenic in humans. N-glycan structures of tg-FIX and tg-PC are also compared to the published N-glycan structures of recombinant human glycoproteins produced in different transgenic animals. While tg-FIX and tg-PC contain only complex structures, the transgenic animal derived antithrombin III, C1 inhibitor, and lactoferrin have both high mannose and complex structures. In addition, terminal Neu5Gc and Neu5Ac moieties are found in the transgenic animal derived antithrombin III, but only Neu5Ac in the other proteins. Collectively, these data indicate that there may be significant species-specific and tissue/cell-specific differences in N-glycan structures among animals used for transgenic animal bioreactors. We also have found that N-glycan profiles of tg-FIX are consistent during lactation with respect to the overall distribution of sialylated vs. neutral oligosaccharides. 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. ^

Subject Area

Engineering, Chemical

Recommended Citation

Gil, Geun-Cheol, "Analysis of the asparagine-linked oligosaccharides of recombinant human glycoproteins produced in the porcine mammary gland" (2006). ETD collection for University of Nebraska - Lincoln. AAI3216336.
http://digitalcommons.unl.edu/dissertations/AAI3216336

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