Unmasking Glycoforms: Lectin‐Based Profiling and Functional Implications of Targeted Glycosylation Knockouts in Chinese Hamster Ovary Cells

Monoclonal antibody (mAb) glycosylation is a critical quality attribute that is difficult to rationally engineer and rapidly assess during cell line development. Here, we investigate whether cell-surface glycosylation can serve as a surrogate readout of mAb product glycosylation following targeted glycogene engineering in CHO cells. Five key glycogenes (COSMC, FUT8, β4GALT1, ST3GAL4, and ST6GAL1) were investigated in two mAb-producing CHO cell lines. Product glycan analysis revealed consistent, gene-specific effects across hosts, including loss of core fucosylation, and tunable galactosylation and sialylation. Lectin-based surface profiling reliably reflected product outcomes for COSMC and FUT8 modifications but showed limited correspondence with product galactosylation and α2,3-sialylation phenotypes, highlighting glycosylation pathway redundancy and context dependence. These findings demonstrate that lectin-based profiling is most effective for identifying glycoengineering outcomes associated with nonredundant pathways rather than as a universal predictor of product glycosylation.