Gomez Aquino, I.; Ghahremanzamaneh, M.; Tsopanoglou, A.; Blanco, A.; Carillo, S.; Bones, J.; Jimenez del Val, I.

{beta}4-galactosylation is a critical quality attribute of therapeutic monoclonal antibodies (mAbs), enhancing complement-dependent cytotoxicity, antibody-dependent cytotoxicity, and antibody-dependent cellular phagocytosis. Despite its therapeutic importance, galactosylation remains the most variable glycosylation motif due to its sensitivity to cell culture conditions. Here, we describe a dual genetic engineering strategy applied to two mAb-producing CHO cell lines, DP12 and VRC01, to simultaneously overcome the cellular machinery and metabolic bottlenecks that limit {beta}4-galactosylation. The first engineering event knocks out COSMC, the chaperone required for core 1 {beta}-1,3-galactosyltransferase 1 activity, to redirect UDP-Gal consumption from O-linked {beta}3-galactosylation towards mAb Fc N-linked {beta}4-galactosylation. The second event overexpresses {beta}-1,4-galactosyltransferase 1 ({beta}4GalT1) to augment cellular galactosylation machinery. Each modification was characterised individually (COSMC- and GalT+) and in combination (C-/GT+) across both cell lines in batch and fed batch cultures. The combined C-/GT+ strategy consistently achieved greater than 90% mAb Fc {beta}4-galactosylation, irrespective of host cell line or culture mode. Metabolic characterisation confirmed that both engineering events alleviate their respective bottlenecks: COSMC knockout redirects UDP-Gal flux and {beta}4GalT1 overexpression increases N-galactosylation capacity. The C-/GT+ strategy also reduced production of Man5 glycans, which accelerate serum clearance and pose immunogenicity risks. Metabolic profiling suggests that the COSMC knockout attenuates UTP consumption and contributes to reduced Man5 production. C-/GT+ glycoengineering had no negative impact on mAb titre. Our results establish the C-/GT+ dual glycoengineering strategy as a robust approach for consistently achieving high mAb galactosylation across diverse cell culture conditions, with the additional benefit of reduced Man5 glycans.