Qualification of a homogeneous cell-based neonatal Fc receptor (FcRn) binding assay and its application to studies on Fc functionality of IgG-based therapeutics.

The Fc region of IgG-based molecules plays an important role in determining their in vivo pharmacokinetic profile by its pH-dependent binding to the neonatal Fc receptor (FcRn) which is expressed on the endothelial cells lining blood vessels. By virtue of this pH-specific interaction with IgG-Fc, FcRn mediates IgG homeostasis in human adults by maintaining serum IgG levels, and also transfers maternal IgGs from mother to fetus via the placenta. The Fc-FcRn interaction is also critical for keeping IgG-based therapeutic molecules in circulation thereby enhancing their serum half life. A homogeneous cell-based flow cytometric FcRn binding assay was established to characterize the Fc-FcRn interaction of therapeutic IgG-based molecules. It is a competition-based assay, wherein the IgG-Fc containing test molecule competes with a fixed concentration of fluorescently-labeled IgG-Fc moiety in solution for binding to the cell-expressed FcRn. The cell-bound fluorescence is read on a flow cytometer. Response of the test sample is analyzed relative to the standard sample and the results are reported as % relative binding. The assay is robust and meets the qualification criteria for specificity, method linearity, accuracy and precision over the relative binding range of 60%-160%. This assay was shown to effectively characterize altered Fc-FcRn interactions for photo-stressed, heat-stressed, oxidized, and Fc mutant samples. It was observed that the relative binding of the IgG-Fc to the cell-surface-expressed FcRn in the assay varies across different molecules, even within the same IgG subclass. This indicates that the Fc-FcRn binding can be influenced by the antigen-binding region of the molecules in addition to the IgG subclass. Overall, this assay is reflective of the in vivo mechanism of immunoglobulin binding to membrane-bound FcRn, and can be used as an analytical tool for assessing lot-to-lot consistency and stability testing across different batches of the same molecule. Additionally, the assay can be used as an effective tool for elucidating the amino acids in the IgG-Fc domain that are critical for FcRn binding and also for comparing the binding of different IgG-Fc containing molecules to FcRn.