Hydrodynamic Radii of Ranibizumab, Aflibercept and Bevacizumab Measured by Time-Resolved Phosphorescence Anisotropy

To measure the hydrodynamic radii of intravitreal anti-VEGF drugs ranibizumab, aflibercept and bevacizumab with mu s time-resolved phosphorescence anisotropy. Ruthenium-based dye Ru(bpy)(2)(mcbpy - O - Su - ester)(PF6)(2), whose lifetime of several hundred nanoseconds is comparable to the rotational correlation time of these drugs in buffer, was used as a label. The hydrodynamic radii were calculated from the rotational correlation times of the Ru(bpy)(2)(mcbpy - O - Su - ester)(PF6)(2)-labelled drugs obtained with time-resolved phosphorescence anisotropy measurements in buffer/glycerol solutions of varying viscosity. The measured radii of 2.76 +/- 0.04 nm for ranibizumab, 3.70 +/- 0.03 nm for aflibercept and 4.58 +/- 0.01 nm for bevacizumab agree with calculations based on molecular weight and other experimental measurements. Time-resolved phosphorescence anisotropy is a relatively simple and straightforward method that allows experimental measurement of the hydrodynamic radius of individual proteins, and is superior to theoretical calculations which cannot give the required accuracy for a particular protein.