Abstract
BACKGROUND/OBJECTIVES: Monoclonal antibodies have successfully been used for a variety of indications. Many therapeutic antibodies are IgG1 and elicit effector functions as part of their mechanism of action. It is well known that aggregate levels should be controlled for therapeutic antibodies. Although there are several reports describing the impact of antibody aggregates on FcγR binding, most of these have been performed with surface plasmon resonance in an avidity-based format. What is less well known is which Fcγ receptor is most impacted by antibody aggregation and how antibody aggregates impact binding to Fcγ receptors in solution-based formats and in cell-based assays. METHODS: An effector-competent IgG1 (mAb1) was forcibly degraded and fractionated by size exclusion chromatography to enrich for aggregates. The fractions were examined for FcγR binding by SPR with different formats and in solution. The fractions were also analyzed with cell-based FcγR reporter assays. RESULTS: All Fcγ receptors displayed increased binding to enriched mAb1 aggregates in the avidity-based SPR methods and in solution, with FcγRIIa impacted the most. When examined with an antibody-down SPR format that is not usually susceptible to avidity, FcγRIIa did not show increased binding with mAb1 aggregation. Although activity for mAb1 aggregates increased slightly in an FcγRIIa cell-based reporter assay, it decreased in the FcγRIIIa reporter assay (most likely due to differences in fucosylation from the reference standard). CONCLUSIONS: Monoclonal antibody aggregation can impact FcγR binding for avidity-based binding formats. Even at low levels of antibody aggregation, FcγRII binding increases substantially.