Selective control of glycosylation is crucial in the development and production of therapeutic glycoproteins, as glycan structures significantly influence protein stability, efficacy, immunogenicity, and pharmacokinetic properties. Modulation of N-linked galactosylation has emerged as a promising strategy for glycoengineering of recombinant antibodies and other biopharmaceuticals. However, selective control of N-linked galactosylation remains challenging in therapeutic glycoprotein production. In this doctoral thesis, we investigated small-molecule modulation of galactosylation by targeting β-1,4-galactosyltransferase 1, the key enzyme that catalyzes galactose transfer during N-glycan maturation. Our work began with structure-based virtual screening using the crystallographic structure of β-1,4- galactosyltransferase 1. We filtered large libraries of commercially available compounds for drug-like physicochemical properties, generated multiple ligand conformations, and docked the resulting structures into the UDP-binding site using constrained molecular docking. This workflow identified a set of top-ranked candidates based on predicted binding modes, chemical diversity, and commercial availability. We then evaluated selected hits experimentally using an in vitro UDP-based luminescence assay to quantify enzymatic inhibition. This approach identified several micromolar inhibitors of the isolated enzyme, confirmed the validity of the computational screening strategy, and revealed novel non-sugar inhibitor chemotypes. Subsequent dose response experiments allowed direct comparison of inhibitory potency among the most promising compounds. To determine biological relevance, we tested these inhibitors in mammalian cell cultures producing recombinant IgG antibodies. Fed-batch cultivations in deep-well plates and small-scale stirred bioreactors enabled systematic assessment of cell growth, viability, productivity, and antibody N-glycan profiles after compound addition. Several compounds reduced galactosylation in a concentration-dependent manner without impairing bioprocess performance. However, cellular outcomes did not consistently correlate with enzymatic potency as one compound increased galactosylation despite strong on-target inhibition in vitro. We quantified intracellular compound concentrations using liquid chromatography–tandem mass spectrometry after time-resolved cell lysis, to assess compound permeability and intracellular exposure. These measurements revealed pronounced differences in cellular uptake and showed that direct intracellular inhibition alone could not account for the observed glycosylation changes. The data instead support contributions from indirect, membrane-associated, or metabolism-dependent mechanisms. The doctoral thesis demonstrates the potential and limitations of direct galactosyltransferase inhibition and establishes an integrated workflow connecting computational design with bioprocess-relevant cell culture evaluation. The findings provide a foundation for the rational development of next-generation glycoengineering strategies and contribute to a deeper understanding of the complex mechanisms governing therapeutic protein glycosylation.
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