Surface plasmon resonance (SPR) biosensor analysis

A Biacore S51 SPR biosensor instrument (GE Healthcare) was used to characterize interactions between conjugates with known on- or off-target proteins. Human recombinant protein kinase ABL1 (ThermoFisher/Life Technologies) (20 μg/ml in 20 mM HEPES, pH 7.5) was immobilized directly on a CM5 biosensor chip surface by amine coupling, to a level of about 5000 RU. EGFR kinase domain L858R (ThermoFisher/Life Technologies) was immobilized directly on the biosensor chip surface by an amine-coupling procedure (32 μg/ml in 10 mM sodium acetate buffer, pH 4.0) resulting 11 000 RU. The running buffer contained: 50 mM Tris–HCl pH 7.4, 150 mM NaCl, 10 mM MgCl₂, 1 mM MnCl₂, 0.005% Tween-20. A 1:1 serial dilution of drug conjugates was injected over immobilized ABL1 at concentrations ranging from 0.06 to 2 μM. All experiments were performed at a flow rate of 30 μl/min and at 25°C and data was analyzed using Biacore S51 Evaluation software (GE Healthcare).