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The study of culture redox potential’s effect on glycosylation and production of monoclonal antibodies in mammalian cell culturesDionne, Benjamin 14 January 2015 (has links)
The glycosylation patterns of monoclonal antibodies (Mabs) have become very important in determining therapeutic abilities of many drugs. The thesis studied 3 cell lines producing humanized Mabs in the presence of variable concentrations of the reducing agent dithiothreitol (DTT) to artificially lower the CRP and affect glycan patterns. A new high-throughput hydrophilic interaction chromatography (HILIC) method was developed and used to show a decrease in the Galactosylation Index (GI) of NS0 IgG1 by as much as 50% in cultures with CRP values lower than -100 mV. The shift in GI was unique to NS0 cultures; CHO DP-12 indicated no significant change in GI but did have a 7% increase in fucosylated species in cultures with higher [DTT]. Furthermore no DTT related shifts were observed in any of the CHO EG2-hFc glycans. EG2-hFc did however have an exceptionally high GI of 0.625 compared to GIs of 0.245 in DP-12 and 0.314 in NSO. Another component of the trials determined, using S35 radiolabeling, that the assembly pathway of IgG1 progressed via HC→HC2→HC2LC→HC2LC2 and that the ratio of heavy chain dimer to heavy chain monomer increased greatly over time for cultures with higher DTT concentrations. The increase in heavy chain dimers and lower GI appear to be correlated, possibly due to disruption of the disulfide bonds between LC and HC within the Golgi. This disruption in disulfide bonds affecting galactosyltransferase (GalT) activity is supported by the findings that the partially reduced fragments of IgG1; HC and HC2, are less galactosylated than the HC2LC and whole IgG1 when treated with GalT. When native and agalactosylated EG2-hFc and IgG1 were treated with GalT in vitro, EG2-hFc exhibited an almost 10 fold higher activity level. The cause for the higher activity may be due to overall size difference or point mutations in the Fc region of EG2-hFc. Through the manipulation of CRP, glycan patterns can be influenced however the effect is not universal and must be determined on a per cell line basis. Furthermore, EG2-hFc’s higher GI value may translate into better in vivo activity as a therapeutic and determination of reasons for the high GI may lead to better means for future glycoengineering. / February 2015
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Total Synthesis of the Tumor-Associated Carbohydrate Antigen Lewis A Lewis X Hexasaccharide and Selected FragmentsMickael, Guillemineau 13 August 2012 (has links)
Carbohydrates constitute the most abundant class of natural products in the living world and they play various roles. They are notably involved in cell-cell interactions and immune reactions. It has been observed that tumor cells express, on their surface, unusual oligosaccharides named Tumor-Associated Carbohydrate Antigen (TACA). One TACA of interest to our research group is the Lewis A Lewis X hexasaccharide that is displayed on the surface of squamous lung carcinoma cells. Since carbohydrates are involved in immune reactions and can be recognized by antibodies, it becomes possible to design a carbohydrate-based vaccine against these tumor cells. This thesis describes the total synthesis of the TACA Lewis A Lewis X hexasaccharide and the preparation of two fragments: one tetra- and one pentasaccharide. These molecules were prepared as hexyl and aminohexylglycosides. In addition, the hexasaccharide was synthesized as a disulfide. This diversity of these synthons will allow conjugation to a protein, analysis by nuclear magnetic resonance techniques, and immobilization on gold of the antigen. Without doubt, this work is a significant contribution to the development of an anti cancer vaccine as it constitutes the first stage of the process.
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Inženýrství mikrobiálních glykosidáz pro změnu syntetického potenciálu / Engineering of microbial glycosidases for modifying synthetic potentialHovorková, Michaela January 2020 (has links)
Glycosidases (EC 3.2.1.) alias glycoside hydrolases are enzymes that catalyze the cleavage of a glycosidic bond between two carbohydrates or between a carbohydrate and an aglycone. Under suitable conditions (especially reduction of water activity in the reaction mixture), these enzymes are also able to synthesize a glycosidic bond. By targeted mutagenesis of the catalytic centre of the enzymes, it is possible to suppress or completely abolish their hydrolytic activity. Enzyme synthesis using glycosidases makes it possible to prepare bioactive galactosides, for example galectin ligands. The present work deals mainly with β-galactosidase from Bacillus circulans, its recombinant expression and mutagenesis. In the first part of the work, the commercially prepared plasmid of -galactosidase from B. circulans isoform A that I designed was used for recombinant expression in E. coli. It was necessary to optimize the conditions of the enzyme production. As it is a large protein (189 kDa), the expression vector pCOLD II and cold production at 15 ř C were used. The enzyme is specific for the formation of the β-1,4 glycosidic bond and has been used to synthesize complex tri- and tetrasaccharide ligands that cannot be prepared with a crude commercial preparation containing undesirable enzyme activities....
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Biochemical Characterization of β-galactosidase from Enterobacter sp. YSUMotari, Fred Ondabu 02 May 2023 (has links)
No description available.
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