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In the present work the production of the human monoclonal Anti-HIV Antibody 2G12 in Nicotiana tabacum L. cv. BY-2 cell cultures has been optimised. Two independent cell lines have been used. The antibody produced from the cell line GFD was secreted into the apoplast, whereas the antibody produced from the cell line GFERD was localized in the endoplasmic reticulum. The main topics studied in this work were the analysis and characterisation of the cell lines, the development of a production medium by design of experiments, the modulation of the antibody production with mathematic modells and the process development and optimisation. In the first part the method for the selection of stable transgenic cell lines was investigated in more detail. The results showed that the the reporter protein DsRed can be used initially for identifying promising cell lines, however, a subsequent screening under production conditions should be done. The analysis of the cell line GFD#5 showed that this line was heterogeneous, i.e. oligoclonal, and that monoclonalization would be important to further increase the production yields. During the work, the cell line showed a drop in productivity, probably resulting from a stress response. The reduction of expression was epigenetic due to promoter methylation because productivity has been restored after cultivating exponentially growing cells in the presence of aza-cytidine. A new production medium called MSNP for Nicotiana tabacum L. cv. BY-2 cell cultures have been developed. The use of this medium led to an increase of the antibody yields in shaking flask as well as in bioreactor experiments: Cell line GFD#5, secreted mAb 2G12• 20-x higher concentration compared to the standard MS-medium • 2-x higher concentration compared to the already optimised MSN-Medium• No impact on the product quality: high activity, homogeneous N glycan-patternCell line GFERD#17, ER-retarded mAb 2G12• 2,6-x higher volumentic product yield compared to the standard MS-Medium• No impact on the product quality: high activity, homogeneous N glycan-patternFurthermore the use of design of experiments enabled the generation of mathematic models for predicting the extracellular 2G12-concentration and the ceell biomass. Process analytical technologies (PAT) have successfully been implemented within this work for Nicotiana tabacum L. cv. BY 2 suspension cell cultures. Dielectric spectroscopy was established as an inline biomass measurement and linear correlations between the conventional offline parameters such as the packed cell volume, the fresh weight and the dry weight and the corresponding conversation factor have been determined.The use of a fed-batch strategy with a mixed feed of sucrose and potassium nitrate enabled the successful uncoupling of the growth and production phase. |
