Chinese hamster ovary cells (CHO) are the most commonly used cell lines in biopharmaceutical manufacturing. Their popularity arises from the ability to express complex therapeutic proteins possessing human-like post-translational modifications in high amounts. The number of newly approved innovative biologics has been increasing tremendously in recent years, together with the number of approved biosimilars, representing a highly similar versions an already approved biologics. Difficulties in obtaining and maintaining the desired level of the critical quality attributes of therapeutic proteins, the pace of development as well as meeting the increasing demands for therapeutic proteins, are major challenges of current biopharmaceutical development. Therapeutic proteins, both innovative and biosimilars, are mostly glycosylated. Glycans directly influence the stability, efficacy, plasma half-life, immunogenicity, and effector functions of therapeutics. Hence, glycosylation is widely recognized as one of the main critical quality attributes of therapeutic glycoproteins. Due to the typically high heterogeneity of glycoforms, controlling glycosylation represents one of the most challenging aspects of biopharmaceutical development. Here, we tested and developed genetic tools enabling target modulation of the expression of endogenous genes involved in glycosylation in CHO cell lines. The implementation of a specific genetic tool based on synthetic transcription factors, i.e. based on CRISPR technology, into CHO production cell lines enabled us to achieve the targeted glycosylation profile of the model therapeutic protein. The efficiency of the CRISPR tool was compared to traditional technologies for gene expression modulation, such as shRNA technology and gene overexpression. Additionally, we have developed a multiplex tool allowing simultaneous regulation of multiple target genes involved in glycosylation. We demonstrated the applicability of the developed glycoengineering approaches for addressing challenges in the development of biosimilars. Lastly, we believe that developed glycoengineering approaches apply to a wide range of biosimilar projects and speed up cell line development timelines.
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