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- Kenny, Diarmuid T., et al.
(författare)
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Mass spectrometric analysis of O-linked oligosaccharides from various recombinant expression systems
- 2013
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Ingår i: Methods in Molecular Biology. - Totowa, NJ : Humana Press. - 1064-3745 .- 1940-6029. ; 988, s. 145-167, s. 145-167
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Tidskriftsartikel (refereegranskat)abstract
- Analysis of O-linked glycosylation is one of the main challenges during structural validation of recombinant glycoproteins. With methods available for N-linked glycosylation in regard to oligosaccharide analysis as well as glycopeptide mapping, there are still challenges for O-linked glycan analysis. Here, we present mass spectrometric methodology for O-linked oligosaccharides released by reductive β-elimination. Using LC-MS and LC-MS2 with graphitized carbon columns, oligosaccharides are analyzed without derivatization. This approach provides a high-throughput method for screening during clonal selection, as well as product structure verification, without impairing sequencing ability. The protocols are exempli fied by analysis of glycoproteins from mammalian cell cultures (CHO cells) as well as insect cells and yeast. The data shows that the method can be successfully applied to both neutral and acidic O-linked oligosaccharides, where sialic acid, hexuronic acid, and sulfate are common substituents. Further characterization of O -glycans can be achieved using permethylation. Permethylation of O-linked oligosaccharides followed by direct infusion into the mass spectrometer provide information about oligosaccharide composition, and subsequent MS n experiments can be carried out to elucidate oligosaccharide structure including linkage information and sequence.
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| 2. |
- Chotteau, Veronique, 1963-, et al.
(författare)
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Development of a fed-batch process for the production of a recombinant protein X in CHO-GS system : Case study from the cell to reactor process ready for pilot scale cultivation
- 2010
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Ingår i: Cells and Culture. - Dordrecht : Springer Science+Business Media B.V.. - 9789048134182 ; , s. 723-725
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- A new cell line was created using CHO-GS system. The most promising clones were adapted to different base cultivation media leading to the selection of one medium. The fed-batch process development was performed in spinner, shake flask and bioreactor scale. It included the selection of a feed medium, the choice of the feed strategy and the optimisation of the glucose feeding. The process was then simplified by using a single feed including the feed medium and the glucose feed. Finally up-scaling parameters like aeration and CO2 stripping were studied in 3 L and 15 L bioreactors in preparation for pilot scale operation. This process proved to be robust, reproducible and suitable for large and commercial scale operation.
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| 3. |
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| 4. |
- Sjöblom-Hallén, A, et al.
(författare)
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Gene expression profiling identifies STAT3 as a novel pathway for immunomodulation by cholera toxin adjuvant.
- 2010
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Ingår i: Mucosal immunology. - : Elsevier BV. - 1935-3456 .- 1933-0219. ; 3:4, s. 374-386
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Tidskriftsartikel (refereegranskat)abstract
- Earlier studies have reported on both proinflammatory and anti-inflammatory activities of cholera toxin (CT). As CT is a powerful adjuvant, we were interested in identifying genes with a possible involvement in these functions. A global gene expression analysis in mouse B cells showed that CT regulated <100 annotated genes, which encoded transcription factors, G proteins, cell-cycle regulators, and immunoregulating molecules. Interestingly, CT regulated the expression of the signal transducer and activator of transcription (STAT)3 gene and influenced the level and activation of both isoforms STAT3alpha and STAT3beta, in vitro in a B-cell line and in Peyer's patch (PP) B cells and in vivo in freshly isolated splenic B cells from CT-treated mice. This effect was cAMP dependent and was not seen with CTB. B cells pre-exposed to CT were significantly more susceptible to the activation of STAT3 by interleukin (IL)-6 and IL-10. This exerted a stronger inhibitory effect of IL-10 on lipopolysaccharide (LPS)-stimulated B-cell proliferation and cytokine production (IL-6). Moreover, IgG1 and IgA production induced by LPS and IL-10 were enhanced by the addition of CT to cultures of PP or splenic B cells. This is the first study to provide a molecular mechanism that can reconcile previous findings of proinflammatory and anti-inflammatory effects by CT adjuvant.Mucosal Immunology advance online publication 7 April 2010. doi:10.1038/mi.2010.16.
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