PhD Thesis
Design of Optimal CHO Protein N-glycosylation Profiles
The amount of marketed therapeutic glycoproteins is increasing steadily and so does the knowledge about the importance and effects of N-glycosylation for patient safety, drug efficacy and pharmacokinetics. Unlike many other expression platforms, chinese hamster ovary (CHO) cell lines possess the ability to provide biopharmaceuticals with N-glycans similar to humans and are therefore the preferred expression host for the majority of glycoproteins.
However, the N-glycan profile of CHO is very heterogeneous and only human-similar, but not human-identical. For some therapeutic products a more homogeneous sugar profile with certain human-identical N-glycan structures is desired. Therefore, it is a fundamental aim to re-design the N-glycan machinery of CHO to produce tailored homogeneous N-glycan structures.
The overall purpose of the thesis was to engineer CHO cells towards specific N-glycan structures. To save time during the cell line development, we examined CRISPR/Cas9 multiplexing to target several genes simultaneously. By this we aimed to provide cell lines for the production of biopharmaceuticals with homogeneous product quality and human-identical Nglycan structures.
The first part of the thesis is a review introducing to the topic and displays how genetic engineering tools as CRISPR are widely used for N-glycan engineering in CHO but also other expression platforms. Following this is an explorative study of CRISPR/Cas9 multiplexing ten gene targets in CHO presenting observed advantages and limitations of the applied protocol.
In the main part of the thesis, the successful production of non-galactosylated glycoproteins (mAb and EPO) after generating cell lines with disruption of B4GALT1, 2, 3 and 4 are depicted. The decrease of galactosylation and heterogeneity of N-glycans was also found on total secreted proteins of the developed cell lines.
Furthermore, the generation of a cell line with ten gene disruptions and overexpression of a human glycosyltransferase allowed the production of recombinant A1AT and C1INH with human-like N-glycosylation and in vitro activity. The generated cell lines allow the study of possibly novel applications for non-galactosylated glycoproteins and a sustainable and safe production platform to provide recombinant A1AT and C1INH.
Finally, we conclude and discuss future perspectives of the obtained results in this thesis.
Language: | English |
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Publisher: | Technical University of Denmark |
Year: | 2018 |
Types: | PhD Thesis |
ORCIDs: | Amann, Thomas |