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

PURPOSE: To measure the hydrodynamic radii of intravitreal anti-VEGF drugs ranibizumab, aflibercept and bevacizumab with μs time-resolved phosphorescence anisotropy. METHODS: Ruthenium-based dye Ru(bpy)(2)(mcbpy − O − Su − ester)(PF(6))(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)(PF(6))(2)-labelled drugs obtained with time-resolved phosphorescence anisotropy measurements in buffer/glycerol solutions of varying viscosity. RESULTS: 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. CONCLUSIONS: 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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11095-016-1940-2) contains supplementary material, which is available to authorized users.