Rapid screening of IgG quality attributes - effects on Fc receptor binding

The interactions of therapeutic antibodies with fragment crystallizable gamma (Fc gamma) receptors and neonatal Fc receptors (FcRn) are measured in vitro as indicators of antibody functional performance. Antibodies are anchored to immune cells through the Fc tail, and these interactions are important for the efficacy and safety of therapeutic antibodies. High-throughput binding studies on each of the human Fc receptor classes (Fc gamma RI, Fc gamma RIIa, Fc gamma RIIb, Fc gamma RIIIa, and Fc gamma RIIIb) as well as Fc gamma Rn have been developed and performed with human IgG after stress-induced modifications to identify potential impact in vivo. Interestingly, we found that asparagine deamidation (D-N) reduced the binding of IgG to the low-affinity Fc receptors (Fc gamma RIIa, Fc gamma RIIb, Fc gamma RIIIa, and Fc gamma RIIIb), while Fc gamma RI and FcRn binding was not impacted. Deglycosylation completely inhibited binding to all Fc gamma receptors, but showed no impact on binding to FcRn. On the other hand, afucosylation only impacted binding to Fc gamma RIIIa and Fc gamma RIIIb. Methionine oxidation at levels below 7%, multiple freeze/thaw cycles and short-term thermal/shake stress did not influence binding to any of the Fc receptors. The presence of high molecular weight species, or aggregates, disturbed measurements in these binding assays; up to 5% of aggregates in IgG samples changed the binding and kinetics to each of the Fc receptors. Overall, the screening assays described in this manuscript prove that rapid and multiplexed binding assays may be a valuable tool for lead optimization, process development, in-process controls, and biosimilarity assessment of IgGs during development and manufacturing of therapeutic IgGs.