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

ABSTRACT: The interactions of therapeutic antibodies with fragment crystallizable ? (Fc?) 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?RI, Fc?RIIa, Fc?RIIb, Fc?RIIIa, and Fc?RIIIb) 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 Fc? receptors (Fc?RIIa, Fc?RIIb, Fc?RIIIa, and Fc?RIIIb), while Fc?RI and FcRn binding was not impacted. Deglycosylation completely inhibited binding to all Fc? receptors, but showed no impact on binding to FcRn. On the other hand, afucosylation only impacted binding to Fc?RIIIa and Fc?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.

SUBMITTER: Geuijen KPM

PROVIDER: S-EPMC5623700 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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

Geuijen Karin P M KPM Oppers-Tiemissen Cindy C Egging David F DF Simons Peter J PJ Boon Louis L Schasfoort Richard B M RBM Eppink Michel H M MHM

FEBS open bio 20170905 10

The interactions of therapeutic antibodies with fragment crystallizable γ (Fcγ) receptors and neonatal Fc receptors (FcRn) are measured <i>in vitro</i> 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 ...[more]

PMID: 28979843

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Project description:The neonatal Fc receptor (FcRn) is a key membrane protein that plays an integral role in serum immunoglobulin (IgG) recycling, which extends the half-life of antibody. In addition, FcRn is known to traffic antigen-bound immunoglobulins (Ag-IgGs), and to interact with immune complexes to facilitate the antigen cross-presentation of peptides derived from the immune complexes in antigen-presenting cells (APCs). Studies on the IgG-FcRn molecular interactions have primarily focused on the Fc region, and only recently have shown the potential impact of the antigen-binding fragment physiochemical properties on FcRn binding. However, the effect of the antigen physiochemical properties on IgG structure as it relates to Ag-IgG-FcRn binding is not well understood. Here we used an IgG-peptide antigen complex as a model system to investigate the structural effects of the antigen's physiochemical properties on the IgG structure, and the subsequent effects of Ag-IgG-FcRn interactions. We used hydroxyl radical footprinting-mass spectrometry to investigate the structural impact on an IgG upon antigen binding, and observed that the physicochemical properties of the antigen differentially induce conformational changes in the IgG FcRn binding region. The extent of these structural changes directly correlates to the magnitude of the affinity differences between the Ag-IgG complexes and FcRn. Moreover, the antigen's physicochemical properties differentially induce structural differences within the Ag-IgG-FcRn ternary complex. We also provide electron microscopy data that shows corroborating Fab-FcRn interactions, and confirms the hypothesis of potential 2:1 FcRn:IgG binding stoichiometry. These data demonstrate antigen-induced Fc structural rearrangements affect both the affinity toward FcRn and the trimeric antigen-IgG-FcRn complex, providing novel molecular insights in the first steps toward understanding interactions of FcRn-containing large(r)-sized immune complex.

Dataset Information

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

Publications

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

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