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

- Dithiothreitol reduced redox potential & decreased galactosylation in a secreted Mab.
- Intermediates of the Mab assembly pathway were analyzed by radioactive labelling.
- Under reducing conditions the proportion of the heavy chain dimer increased.
- The rate of galactosylation of heavy chain dimers was lower than other intermediates.
- The altered assembly pathway is the most likely cause of low level galactosylation.

Glycosylation and intracellular assembly of monoclonal antibodies (MAbs) is important for glycan profile consistency. To better understand how these factors may be influenced by a lower redox potential, an IgG1-producing NS0 cell line was grown in the presence of varying concentrations of dithiothreitol (DTT). Cultures were monitored for growth and culture redox potential (CRP) with glycan heterogeneity determined using a HILIC-HPLC method. Macroheterogeneity was unchanged in all conditions whereas the Galactosylation Index (GI) decreased by as much as 50% in cultures with lower CRP or higher dithiothreitol levels. This shift in GI is reflected in more agalactosylated and asialylated species being produced. The MAb assembly pathway was determined using radioactive isotope 35S incorporated into nascent IgG1 molecules. The assembly pathway for this IgG1 was shown to progress via HC → HC2 → HC2LC → HC2LC2 in all conditions tested and autoradiographs highlighted that the ratio of heavy chain dimer to heavy chain monomer increased over time with increasing DTT concentrations. This increase and correspondingly lower GI values may be due to disruption of the disulfide bonds at higher levels of assembly. A change in the assembly pathway may alter the final IgG glycan pattern and lead to control mechanisms that influence glycan profiles of MAbs.