20.500.12556/RUL-138877 Coupling CRISPR interference with FACS enrichment new approach in glycoengineering of CHO cell lines for therapeutic glycoprotein production Difficulties in obtaining and maintaining the desired level of the critical quality attributes (CQAs) of therapeutic proteins as well as the pace of the development are major challenges of current biopharmaceutical development. Therapeutic proteins, both innovative and biosimilars, are mostly glycosylated. Glycans directly influence the stability, potency, plasma half-life, immunogenicity, and effector functions of the therapeutic. Hence, glycosylation is widely recognized as a process-dependent CQA of therapeutic glycoproteins. Due to the typically high heterogeneity of glycoforms attached to the proteins, control of glycosylation represents one of the most challenging aspects of biopharmaceutical development. Here, we explored a new glycoengineering approach in therapeutic glycoproteins development, which enabled us to achieve the targeted glycoprofile of the Fc-fusion protein in a fast manner. Coupling CRISPRi technology with lectin-FACS sorting enabled downregulation of the endogenous gene involved in fucosylation and further enrichment of CHO cells producing Fc-fusion proteins with reduced fucosylation levels. Enrichment of cells with targeted glycoprofile can lead to time-optimized clone screening and speed up cell line development. Moreover, the presented approach allows isolation of clones with varying levels of fucosylation, which makes it applicable to a broad range of glycoproteins differing in target fucosylation level. CHO cells cell line development fucosylation glycoengineering therapeutic glycoproteins celice CHO razvoj celične linije fukozilacija glikoinženiring terapevtski glikoproteini true false true Angleški jezik Slovenski jezik Članek v reviji 2022-08-24 09:47:05 2022-08-24 09:47:11 2022-09-15 04:17:20 0000-00-00 00:00:00 2022 0 0 13 str. iss. 7, art. e2100499 Vol. 17 2022 0000-00-00 Zaloznikova Objavljeno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 542:547.96 1860-7314 10.1002/biot.202100499 112903939 Biotechnology_Journal_-_2022_-_Glin_ek_-_Coupling_CRISPR_interference_with_FACS_enrichment__New_approach_in.pdf Biotechnology_Journal_-_2022_-_Glin_ek_-_Coupling_CRISPR_interference_with_FACS_enrichment__New_approach_in.pdf 1 4B651091A7F5C8A144D4DA574380CDAA 83af66106a7b01ea69cf963bdf610dcb7d173dce0e50bb5f36d542c50882e23b 63b3a9b6-2381-11ed-92af-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=159362 https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.202100499 1 f102afed-2380-11ed-92af-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=159361 Fakulteta za farmacijo 0 0 0