April 2014
Volume 55, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2014
The Relative Affinities of Ranibizumab and Aflibercept for Vascular Endothelial Growth Factor A (VEGF) - an Analytical Ultracentrifugation Study
Author Affiliations & Notes
  • Sandeep Yadav
    Genentech, Inc., South San Francisco, CA
  • Barthélemy Demeule
    Genentech, Inc., South San Francisco, CA
  • Jun Liu
    Genentech, Inc., South San Francisco, CA
  • Steven J Shire
    Genentech, Inc., South San Francisco, CA
  • Footnotes
    Commercial Relationships Sandeep Yadav, Genentech, Inc. (E); Barthélemy Demeule, Genentech, Inc. (E); Jun Liu, Genentech, Inc. (E); Steven Shire, Genentech, Inc. (E)
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 1942. doi:https://doi.org/
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    • Get Citation

      Sandeep Yadav, Barthélemy Demeule, Jun Liu, Steven J Shire; The Relative Affinities of Ranibizumab and Aflibercept for Vascular Endothelial Growth Factor A (VEGF) - an Analytical Ultracentrifugation Study. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1942. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract
 
Purpose
 

To evaluate the relative VEGF binding affinities of ranibizumab and aflibercept in free solution using sedimentation velocity analytical ultracentrifugation in a competitive binding mode.

 
Methods
 

Analytical ultracentrifugation experiments were performed at 20°C in phosphate-buffered saline (pH 7.2, 137 mM NaCl, 27 mM KCl, 8 mM Na2HPO4 and 1.5 mM KH2PO4). Sedimentation velocity was assessed in an Optima XL-I analytical ultracentrifuge equipped with absorbance optics, interference optics (Beckman Coulter, Fullerton, CA, USA), and fluorescence optics (Aviv Biomedical). Alexa Fluor 488 protein labeling kits were purchased from Molecular Probes (Eugene, OR, USA). First, the sedimentation coefficients of VEGF, ranibizumab, aflibercept and pre-formed ranibizumab-VEGF and aflibercept-VEGF complexes were obtained; second, competition experiments were conducted by challenging the pre-formed anti-VEGF/VEGF complexes with a different VEGF inhibitor.

 
Results
 

At equivalent molar ratio ranibizumab was able to displace aflibercept from preformed aflibercept-VEGF complexes in solution (Figure) whereas aflibercept was not able to displace ranibizumab from preformed ranibizumab-VEGF complexes.

 
Conclusions
 

These new data show that in solution ranibizumab is capable of displacing aflibercept from pre-formed complexes with VEGF indicating that it is very unlikely that aflibercept has 100-fold higher affinity for VEGF than ranibizumab as previously published by Papadopoulos et al. (Angiogenesis. 2012 Jun;15(2):171-85).

 
 
Figure. A pre-formed 1:1 aflibercept:VEGF complex (6.5S, dashed line) was challenged with one (dotted line) or two (solid line) molar equivalents of ranibizumab (3.7S). The appearance of a free aflibercept peak (5.3S) or a raised baseline between 5S and 6S, combined with evidence for the formation of a 2:1 ranibizumab:VEGF complex (6.2 S), suggests that ranibizumab was able to displace aflibercept from the complex with VEGF.
 
Figure. A pre-formed 1:1 aflibercept:VEGF complex (6.5S, dashed line) was challenged with one (dotted line) or two (solid line) molar equivalents of ranibizumab (3.7S). The appearance of a free aflibercept peak (5.3S) or a raised baseline between 5S and 6S, combined with evidence for the formation of a 2:1 ranibizumab:VEGF complex (6.2 S), suggests that ranibizumab was able to displace aflibercept from the complex with VEGF.
 
Keywords: 412 age-related macular degeneration • 609 neovascularization • 748 vascular endothelial growth factor  
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