Coupling CRISPR interference with FACS enrichment : new approach in glycoengineering of CHO cell lines for therapeutic glycoprotein production

Glinšek, Katja (author), Kramer, Lovro (author), Krajnc, Aleksander (author), Krajnc, Eva (author), Pirher, Nina (author), Marušič, Jaka (author), Hellmann, Leon (author), Podobnik, Barbara (author), Štrukelj, Borut (author), Ausländer, David (author), Gaber, Rok (author)

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.

• Language: English
• Keywords: CHO cells, cell line development, fucosylation, glycoengineering, therapeutic glycoproteins
• Work type: Article
• Typology: 1.01 - Original Scientific Article
• Organization: FFA - Faculty of Pharmacy
• Publication status: Published
• Publication version: Version of Record
• Year: 2022
• Number of pages: 13 str.
• Numbering: Vol. 17, iss. 7, art. e2100499
• PID: 20.500.12556/RUL-138877
• UDC: 542:547.96
• ISSN on article: 1860-7314
• DOI: 10.1002/biot.202100499
• COBISS.SI-ID: 112903939
• Publication date in RUL: 24.08.2022

GLINŠEK, Katja, KRAMER, Lovro, KRAJNC, Aleksander, KRAJNC, Eva, PIRHER, Nina, MARUŠIČ, Jaka, HELLMANN, Leon, PODOBNIK, Barbara, ŠTRUKELJ, Borut, AUSLÄNDER, David and GABER, Rok, 2022, Coupling CRISPR interference with FACS enrichment : new approach in glycoengineering of CHO cell lines for therapeutic glycoprotein production. Biotechnology journal [online]. 2022. Vol. 17, no. 7,  e2100499. [Accessed 19 September 2025]. DOI 10.1002/biot.202100499. Retrieved from: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=eng&id=138877

Record is a part of a journal

• Title: Biotechnology journal
• Shortened title: Biotechnol. j.
• Publisher: Wiley-VCH
• ISSN: 1860-7314
• COBISS.SI-ID: 522332953

Licences

• License: CC BY 4.0, Creative Commons Attribution 4.0 International
• Link: http://creativecommons.org/licenses/by/4.0/
• Description: This is the standard Creative Commons license that gives others maximum freedom to do what they want with the work as long as they credit the author.
• Licensing start date: 24.08.2022

Secondary language

• Language: Slovenian
• Keywords: celice CHO, razvoj celične linije, fukozilacija, glikoinženiring, terapevtski glikoproteini

Projects

• Funder: Other - Other funder or multiple funders
• Project number: OP20.04327(C3330-19-952017)

• Funder: Other - Other funder or multiple funders
• Project number: OP20.04327(C3330-19-952017)