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bioRxiv · 10.64898/2026.05.13.724788

Unmasking Glycoforms: Lectin-Based Profiling and Functional Implications of Targeted Glycosylation Knockouts in CHO Cells

Abascal Ruiz, C.·Lim, S. L. Y.·Brink, J.·Carillo, S.·Casey, E.·Bones, J.·Jimenez del Val, I.
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Abstract

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 predictive indicator of mAb product glycosylation following targeted glycogene engineering in CHO cells. Five key glycogenes (COSMC, FUT8, B4GALT1, ST3GAL4, 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 tuneable galactosylation and sialylation. Lectin-based surface profiling reliably reflected product outcomes for COSMC and FUT8 modifications but showed limited predictive power for galactosylation and 2,3-sialylation, highlighting glycosylation pathway redundancy and context dependence. This study provides the first systematic, cross-cell line evaluation of lectin-based cell-surface glycan profiling as a predictor of mAb product glycosylation, establishing its practical utility and inherent limitations for CHO glycoengineering workflows. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/724788v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@6d5cfborg.highwire.dtl.DTLVardef@1f38e0aorg.highwire.dtl.DTLVardef@f25fa2org.highwire.dtl.DTLVardef@64a0dc_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Abascal Ruiz, C., Lim, S. L. Y., Brink, J., Carillo, S., Casey, E., Bones, J., Jimenez del Val, I.. 2026-05-13. Unmasking Glycoforms: Lectin-Based Profiling and Functional Implications of Targeted Glycosylation Knockouts in CHO Cells. https://doi.org/10.64898/2026.05.13.724788

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