A low redox potential affects monoclonal antibody assembly and glycosylation in cell culture

Area of Science:

• Biotechnology
• Biopharmaceutical Manufacturing
• Glycobiology

Background:

• Monoclonal antibody (MAb) glycosylation and intracellular assembly are critical for consistent glycan profiles.
• Understanding factors influencing these processes, such as cellular redox potential, is essential for biopharmaceutical production.

Purpose of the Study:

• To investigate the impact of altered cellular redox potential on MAb glycosylation and intracellular assembly.
• To determine how varying concentrations of dithiothreitol (DTT) affect the glycan profile and assembly pathway of IgG1 MAbs.

Main Methods:

• An IgG1-producing NS0 cell line was cultured with varying DTT concentrations to modulate culture redox potential (CRP).
• Growth, CRP, and glycan heterogeneity (using HILIC-HPLC) were monitored.
• MAb assembly pathways were analyzed using ³⁵S-labeled nascent IgG1 molecules and autoradiography.

Main Results:

• Macroheterogeneity remained unchanged, but the Galactosylation Index (GI) decreased by up to 50% at lower CRP or higher DTT levels.
• Reduced GI correlated with increased production of agalactosylated and asialylated MAb species.
• The IgG1 assembly pathway (HC→HC₂→HC₂LC→HC₂LC₂) was consistent across conditions, but higher DTT increased the heavy chain dimer to monomer ratio.

Conclusions:

• Lowering cellular redox potential via DTT significantly impacts MAb galactosylation, leading to altered glycan profiles.
• Disruption of disulfide bonds in higher assembly stages may contribute to reduced galactosylation.
• These findings highlight the potential for redox control mechanisms to influence MAb glycan patterns during production.