Study of stability and biophysical characterization of ranibizumab and aflibercept.
The anti-vascular endothelial growth factor (VEGF) agents such as ranibizumab (Lucentis®) and aflibercept (EyLea®) are currently used as monthly or bimonthly intravitreal injections to treat potentially retinal diseases such as wet age-related macular degeneration (AMD) or diabetic macular edema (DME). Because of the complications associated with repeated intra-vitreal injections, there is considerable interest in developing a sustained delivery system. The purpose of this study was to examine the stability of both therapeutic proteins under physiological conditions as well as when incorporated into drug delivery systems (DDS). First, thermotropic properties in physiological conditions and at different pH values were evaluated by differential scanning calorimetry (DSC) to determine the protein denaturation temperature. Second, the effects of pH and incubation time on conformational changes and aggregation were evaluated by circular dichroism (CD), steady-state tryptophan fluorescence spectroscopy, and size-exclusion chromatography (SEC). Also, the ability of both proteins to bind to VEGF was tested in the aforementioned experimental conditions for up to 30 days. Finally, we investigated the stability of both proteins after a rapid screening method that simulates the first homogenizing step during the protein microencapsulation process. This method allowed the development of stable ranibizumab and aflibercept formulations that may be useful to entrap these proteins into microparticles selecting the most convenient organic solvent and protein stabilizers.
School of Material Science & Engineering, Nanyang Technological University, Singapore. Electronic address: mmoreno@ntu.edu.sg.
M R
MR
Moreno
School of Material Science & Engineering, Nanyang Technological University, Singapore.
T S
TS
Tabitha
School of Material Science & Engineering, Nanyang Technological University, Singapore.
J
J
Nirmal
School of Material Science & Engineering, Nanyang Technological University, Singapore.
K
K
Radhakrishnan
School of Material Science & Engineering, Nanyang Technological University, Singapore.
C H
CH
Yee
School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore.
S
S
Lim
School of Material Science & Engineering, Nanyang Technological University, Singapore.
S
S
Venkatraman
Tan Tock Seng Hospital, Singapore. Electronic address: rupesh_agrawal@ttsh.com.sg.
R
R
Agrawal
D002152:Calorimetry, Differential Scanning; D002845:Chromatography; D002942:Circular Dichroism; D004355:Drug Stability; D004655:Emulsions; D004797:Enzyme-Linked Immunosorbent Assay; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008269:Macular Edema / Q000188:drug therapy; D053758:Nanoparticles / Q000737:chemistry; D017433:Protein Structure, Secondary; D000069579:Ranibizumab / Q000737:chemistry*; D040262:Receptors, Vascular Endothelial Growth Factor / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D012997:Solvents / Q000737:chemistry; D013696:Temperature; D014466:Ultraviolet Rays; D042461:Vascular Endothelial Growth Factor A / Q000037:antagonists & inhibitors / Q000737:chemistry; D057135:Wet Macular Degeneration / Q000188:drug therapy
D004655:Emulsions; D011993:Recombinant Fusion Proteins; D012997:Solvents; D042461:Vascular Endothelial Growth Factor A; C533178:aflibercept; D040262:Receptors, Vascular Endothelial Growth Factor; D000069579:Ranibizumab
Aggregation state; Anti-vascular endothelial growth factor (VEGF) proteins; Biophysical characterization; Diabetic macular edema (DME); Emulsion; Excipients; Stability; Wet age related macular degeneration (AMD)