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.