Generation of Long-Lived CHO Cells Suitable for Production of Afucosylated Antibodies and Fc-Fusion Proteins

Abstract— Using genome editing, we created a homozygous α-(1,6)-fucosyltransferase (FUT8⁻/⁻) knockout in apoptosis-resistant CHO 4BGD cells, yielding the new 4BGD-F cell line. Combining CRISPR/Cas9 with paired gRNAs and non-specific puromycin selection yielded a cell population with an exceptionally high FUT8 knockout frequency, obviating the need for metabolic enrichment with lentil lectin (Lens culinaris agglutinin, LCA). Despite impaired clonogenicity of the knockout cells, we successfully isolated multiple clonal cell lines harboring extensive biallelic FUT8 deletions. Isolated clones with biallelic deletions retained key parental line characteristics: viability >90% in 17-day fed-batch cultures at high densities (>15 × 10⁶ cells/mL), and rapid selectability using both dihydrofolate reductase and glutamine synthetase systems. Mass spectrometric analysis of the test protein GLP1-Fc secreted by 4BGD-F cells confirmed the absence of N-glycan fucosylation. The CHO 4BGD-F cell line provides a valuable platform for producing afucosylated antibodies with enhanced antibody-dependent cellular cytotoxicity.