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
| Identifer | oai:union.ndltd.org:MANITOBA/oai:mspace.lib.umanitoba.ca:1993/30224 |
| Date | 14 January 2015 |
| Creators | Dionne, Benjamin |
| Contributors | Butler, Michael (Microbiology), Court, Deborah (Microbiology) Perreault, Helene (Chemistry) Kamen, Amine (Bioengineering, McGill) |
| Source Sets | University of Manitoba Canada |
| Detected Language | English |
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