Affinity of a therapeutic molecule binding to a target protein is one of the primary characteristics that delimits therapeutic potency in vivo and in vitro. It is critical to develop robust and reliable methods to study binding kinetics and equilibrium of therapeutic candidates to membrane protein targets given their prominence in drug discovery process. Unlike soluble proteins with well-defined protein concentration values, membrane protein expression is often heterogeneous and sensitive to culture conditions of the expressing cells. In addition to unknown membrane protein concentration, therapeutic protein binding to targets located on the cell surface can induce target internalization and/or signaling. Target mediated activities can concomitantly affect the concentration of antibody/antigen complex that is measured in cell binding assays for affinity determinations. To overcome some of these difficulties, we established an automated cell membrane equilibrium binding assay platform with integration of fluorescence-activated cell sorting (FACS) and kinetic exclusion assay (KinExA). Automation enabled sample preparation of four candidate therapeutic molecules for determination of their solution equilibrium affinity (KD) for the same target simultaneously. FACS analysis measures target protein density on the cell surface prior to membrane preparation, while KinExA quantifies free antibody concentration at equilibrium of antibody binding with cell membrane for KD determination. This presentation describes the development, application, and automation of solution based, label free binding analysis of membrane proteins, such as CD20, residing in their natural milieu.

Binding affinity determination of therapeutic antibodies to membrane protein targets

2019 Spring National Meeting

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