β-1,4-galactosyltransferase 1 inhibitors modulate cellular N-glycan profiles

Area of Science:

• Biotechnology
• Biochemistry
• Cell Biology

Background:

• N-glycosylation is a critical post-translational modification impacting protein function.
• Beta-1,4-galactosyltransferase 1 (B4GALT1) is a key enzyme in the N-glycan synthesis pathway.

Purpose of the Study:

• To evaluate the in vivo efficacy of four B4GALT1 inhibitors in recombinant IgG-producing cell cultures.
• To assess the impact of these inhibitors on cell culture performance and N-glycan profiles.
• To investigate the correlation between in vitro inhibitor potency and in vivo cellular activity.

Main Methods:

• Fed-batch bioprocesses were employed to culture cells producing recombinant IgG antibodies.
• Dose-dependent effects of B4GALT1 inhibitors on cell growth, viability, and productivity were monitored.
• N-glycan profiles were analyzed using InstantPC labeling and UHPLC-FLD.
• Intracellular inhibitor concentrations were quantified using LC-MS/MS.

Main Results:

• Inhibitors exhibited diverse in vivo activities, diverging from their on-target potency.
• Cellular permeability, metabolic stability, and off-target effects influenced inhibitor performance.
• Significant impact on N-glycan profiles and cell culture parameters was observed, varying by inhibitor.

Conclusions:

• In vitro potency does not always predict in vivo efficacy for B4GALT1 inhibitors in cell culture.
• Cellular properties are critical determinants for successful glycosylation pathway modulation.
• Findings inform the development of novel glycoengineering tools for biopharmaceutical production.