Design of Optimal CHO Protein N-glycosylation Profiles

Thomas Amann

Research output: Book/ReportPh.D. thesisResearch

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Abstract

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.
Original languageEnglish
Place of PublicationKgs. Lyngby
PublisherTechnical University of Denmark
Number of pages142
Publication statusPublished - 2018

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