Evaluation of effects of Fc domain high-mannose glycan on antibody stability.

Elevated levels of CH2 domain N-linked high-mannose (HM) glycans are commonly observed in therapeutic monoclonal antibodies at various stages of the development. The effect of HM glycans on antibody stability was evaluated by using two approaches. In the first approach, immunoglobulin G (IgG) 1 material containing 21% HM was incubated at 29°C for 6 weeks and fractionated into monomeric and aggregate species by using size-exclusion chromatography (SEC). These fractions were analyzed for the levels of HM. No significant difference was observed in the amount of HM in aggregate and monomer fractions indicating that the HM-containing fractions did not have a greater tendency to form aggregates. In the second approach, both IgG1 material and IgG2 material were separated by Concanavalin-A affinity chromatography into a HM-enriched fraction and a HM-depleted fraction, respectively. Real-time and accelerated stability studies were carried out with these fractions together with untreated samples under standard formulation conditions. The stability of these fractions over time was monitored using SEC and cation-exchange chromatography. No significant difference was observed in rates of aggregation or charge variant formation. These data indicate that HM glycans had no effect on the IgG1 and IgG2 product's stability under the formulation conditions studied.