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

  • Pharm Res. 2016 Aug;33(8):2025-32. doi: 10.1007/s11095-016-1940-2.
Liisa M Hirvonen  1 ,  Gilbert O Fruhwirth  2 ,  Nishanthan Srikantha  3  4 ,  Matthew J Barber  1 ,  James E Neffendorf  4 ,  Klaus Suhling  5 ,  Timothy L Jackson  3  4
Affiliations
  • 1. Department of Physics, King's College London, Strand, London, WC2R 2LS, UK.
  • 2. Department of Imaging Chemistry and Biology, Division of Imaging Sciences and Biomedical Engineering, King's College London, St. Thomas' Hospital, Lambeth Wing, London, SE1 7EH, UK.
  • 3. School of Medicine, King's College London, London, SE5 9RS, UK.
  • 4. Department of Ophthalmology, King's College Hospital, London, SE5 9RS, UK.
  • 5. Department of Physics, King's College London, Strand, London, WC2R 2LS, UK. [email protected].
PMID: 27225494 DOI: 10.1007/s11095-016-1940-2
Abstract

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)(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.

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.

Keywords
fluorescence; hydrodynamic radius; phosphorescence; rotational diffusion; time-resolved anisotropy.
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