Native and artificial miRNAs as versatile tools for glycoengineering in CHO production cells
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Date
2024-12-19
Authors
Schlossbauer, Patrick
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Dissertation
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Abstract
Market analysis clearly shows that the biopharmaceutical industry is growing rapidly, and with the increasing demand for biosimilars, and the introduction of tailor-made therapies the need for new technologies to modulate complex molecules emerges simultaneously. Monoclonal antibodies (mAbs) produced in Chinese hamster ovary (CHO) cells still reflect approximately 50 % of newly approved biologicals in the last decade. As the effector function of mAbs is highly sensitive to changes in the glycosylation pattern attached to the protein, this modification has been subject to numerous engineering approaches. Standard methods like radical gene knockout or energy consuming overexpression, however, often fail to meet moderate effector functions. Therefore, the present work aimed to elucidate the possibilities to use microRNAs (miRNAs) as fine-tuning regulators of mAb glycosylation in CHO cells. MiRNAs were identified regulating mAb fucosylation and galactosylation in transient experiments and in stable miRNA overexpressing CHO production cell lines. Here, strategies for the overexpression of miRNAs were tested with subsequent analysis of regulation on transcript and glycosylation level and potentially bioprocessing relevant parameters. Furthermore, artificial miRNAs (amiRNAs) which reflect improved synthetic variants of their native counterparts, were designed and applied in stable cell line development. Thereby, basic principles of efficient amiRNA regulation and potential pitfalls were identified. The experiments were accompanied by miRNA target prediction, which served as a valuable tool to hint at potential regulations and was evaluated by the generated data. Summarized, numerous cell lines with varying fucosylation and galactosylation properties could be generated which clearly demonstrated the applicability of miRNAs or tailor-made amiRNAs as versatile engineering tools in cell line development to generate moderate phenotypic changes required in biosimilar production.
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Universität Ulm
Institut für Pharmazeutische Biotechnologie
Institut für Pharmazeutische Biotechnologie
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DFG Project uulm
EU Project THU
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Klingler F., Naumann L., Schlossbauer P., Dreyer L., Burkhart M., Handrick R., Junker H.D., Hesse F., Neusüß C., Otte K., Metabolic Engineering (2023): A novel system for glycosylation engineering by natural and artificial miRNAs. https://doi.org/10.1016/j.ymben.2023.03.004
Schlossbauer P., Klingler F., Naumann L., Burkhart M., Handrick R., Korff K., Neusüß C., Otte K., Hesse F., Engineering in Life Sciences (2024): Stable overexpression of native and artificial miRNAs for the production of differentially fucosylated antibodies in CHO cells. https://doi.org/10.1002/elsc.202300234
Klingler F., Schlossbauer P., Naumann L., Handrick R., Hesse F., Neusüß C., Otte K., Biotechnology and Bioengineering (2023): Developing microRNAs as engineering tools to modulate monoclonal antibody galactosylation. https://doi.org/10.1002/bit.28616
Schlossbauer P., Klingler F., Burkhart M., Leroux A.-C., Hesse F., Otte K., Heterologous Protein Production in CHO Cells: Methods and Protocols, Second Edition (2024): MiRNA chaining for efficient stable overexpression to improve protein quantity and quality in CHO cells. https://doi.org/10.1007/978-1-0716-4104-0_7
Schlossbauer P., Klingler F., Otte K., Hesse, F., in Re:GEN Open (2024): Predicting potential targets of miRNAs affecting monoclonal antibody glycosylation in CHO cells using a simplified evaluation workflow. https://doi.org/10.1089/regen.2023.0027
Schlossbauer P., Klingler F., Naumann L., Burkhart M., Handrick R., Korff K., Neusüß C., Otte K., Hesse F., Engineering in Life Sciences (2024): Stable overexpression of native and artificial miRNAs for the production of differentially fucosylated antibodies in CHO cells. https://doi.org/10.1002/elsc.202300234
Klingler F., Schlossbauer P., Naumann L., Handrick R., Hesse F., Neusüß C., Otte K., Biotechnology and Bioengineering (2023): Developing microRNAs as engineering tools to modulate monoclonal antibody galactosylation. https://doi.org/10.1002/bit.28616
Schlossbauer P., Klingler F., Burkhart M., Leroux A.-C., Hesse F., Otte K., Heterologous Protein Production in CHO Cells: Methods and Protocols, Second Edition (2024): MiRNA chaining for efficient stable overexpression to improve protein quantity and quality in CHO cells. https://doi.org/10.1007/978-1-0716-4104-0_7
Schlossbauer P., Klingler F., Otte K., Hesse, F., in Re:GEN Open (2024): Predicting potential targets of miRNAs affecting monoclonal antibody glycosylation in CHO cells using a simplified evaluation workflow. https://doi.org/10.1089/regen.2023.0027
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DFG Project THU
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Keywords
CHO, Chinese hamster ovary, miRNA, miRNS, Glykosidierung, Monoklonaler Antikörper, CHO-Zelle, Biopharmaceutics, Cricetulus; Genetics, MicroRNAs, Antibodies, Monoclonal, Glycosylation, DDC 570 / Life sciences