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

Abstract

The interactions of therapeutic antibodies with fragment crystallizable γ (Fcγ) 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 Fcγ receptor classes (FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb) as well as FcRn 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 Fcγ receptors (FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb), while FcγRI and FcRn binding was not impacted. Deglycosylation completely inhibited binding to all Fcγ receptors, but showed no impact on binding to FcRn. On the other hand, afucosylation only impacted binding to FcγRIIIa and FcγRIIIb. 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.

Low pH Exposure During Immunoglobulin G Purification Methods Results in Aggregates That Avidly Bind Fcγ Receptors: Implications for Measuring Fc Dependent Antibody Functions

“…Similar results were also obtained by Dorion-Thibaudeau et al where even small percentages (2%) of IgG aggregates were found to affect Fc receptor binding (41). A previous study which investigated the effect of IgG aggregates on Fc receptor binding, also found aggregates bind more strongly to Fc receptors, with a much slower off-rate, significantly impacting affinity determinations (42). This is the result of an increased avidity effect, rather than a true difference in IgG affinity.…”

Assessment of susceptible chemical modification sites of trastuzumab and endogenous human immunoglobulins at physiological conditions

“…Several studies have evaluated Asn deamidation and Asp isomerization in recombinant antibodies using elevated temperature or other relevant stress conditions 25,27–30,45–49 . In the case of trastuzumab, LC-Asn-30 (CDR1) and HC-Asp-102 (CDR3) have been identified as the most vulnerable degradation sites.…”

Analytical Characterization of Monoclonal Antibodies with Novel Fc Receptor-Based Chromatography Technique

“…A recently published article reports that deamidated IgG samples caused aggregation or formation of higher molecular weight (HMW) species and thereby impacted the binding affinity. Asparagine deamidation led to reduced binding of IgG to the low affinity FcγRIIIa receptor [24]. IgGs may also be more prone to aggregation when glycans are absent, which in turn has an effect on Fc effector functions.…”

“…The interaction, if any, needs to be evaluated in case-by-case basis. Up to 5% of aggregates in IgG samples changed the binding and kinetics to each of the Fc receptors [24]. Methionine is easily oxidized to methionine sulfoxide which can also lead to light chain aggregation.…”