Methods to measure the binding of therapeutic monoclonal antibodies to the human Fc receptor FcγRIII (CD16) using real time kinetic analysis and flow cytometry

Two different methods have been developed to measure the binding of therapeutic antibodies to the low affinity human Fc receptor FcγRIII (CD16). The first measures binding of antibody to recombinant soluble receptor by surface plasmon resonance and the second uses flow cytometry to measure antibody binding to cells which express the receptor. Both methods have been formatted as parallel line assays and show high levels of accuracy, precision and linearity, making them suitable for comparability, potency and stability assays. They are both readily able to detect structural differences such as glycosylation, which affect Fc receptor binding. The same approaches can be used to measure the binding of any antibody to any Fc receptor. These assays show greater internal precision and long-term reproducibility than traditional cell-based assays such as antibody-dependent cell-mediated cytotoxicity. A combinational approach with a target binding might be appropriate for routine drug batch release as these assays are likely to be significantly more sensitive to small changes in drug structure or activity.

► New methods to measure IgG–FcR interactions by plasmon resonance and flow cytometry.
► Exemplified by the binding of alemtuzumab (Campath) to FcγRIII (CD16).
► Excellent precision and accuracy and very sensitive to changes in antibody glycoforms.
► Suitable for comparability studies, stability and lot release.
► Combined with an antigen binding assay this could replace complex functional assays.