Abstract
Purpose :
Potency assays for biologicals are complex in nature, and reproducibility, response range, and accuracy depend on various factors such as nature of molecule, mechanism of action, target location, and concentration. Accurate and reproducible potency determination is vital for controlling product quality during manufacture and release. OCU200, a novel fusion biological consisting of human transferrin and tumstatin, is being developed for treatment of DME and wet age-related macular degeneration (wet-AMD). Transferrin facilitates targeting and delivery of OCU200 to the target choroidal endothelial cells through endo/transcytosis. To this end, we developed an enzyme-linked immunosorbent assay (ELISA)-based binding assay and biolayer interferometry (BLI) method to determine binding affinity as a measure of in vitro potency for OCU200.
Methods :
In the ELISA, biotinylated transferrin receptor was coated on a streptavidin-coated 96-well plate and varying concentrations of OCU200 were added. A detection antibody (anti-tumstatin-HRP) was applied to measure signal using a UV-visible spectrophotometer, and data was analyzed to determine the dissociation constant. Parallelly, a BLI method was developed to determine the molecular interaction between OCU200 and transferrin receptor. BLI analyzes the interference pattern of white light reflected from two surfaces: the layer of immobilized transferrin receptor on a fiber optic sensor and on the internal reflection surface. Interactions between OCU200 and immobilized transferrin receptor on the biosensor cause a shift in the refracted wavelengths. The shift in the refracted wavelength was measured in real-time, allowing determination of precise and accurate association and dissociation rates.
Results :
Dose dependent binding of OCU200 with transferrin receptor was observed in both ELISA and BLI assays. In the ELISA, the dissociation constant was found to be 3.7 ± 0.1 nM. Binding parameters in BLI, i.e. affinity constants (KD) found to be 0.38 ± 0.01 nM, the association rate (Kon) found to be 3.67E+05 (1/Ms), and dissociation rate (Koff) found to be 1.43E-04 (1/Ms).
Conclusions :
OCU200 binds to the transferrin receptor in a dose dependent manner, supporting our hypothesis that transferrin targets OCU200 to the choroid and retina mediated through the transferrin receptor. Binding affinity with receptor can be used a measure of potency for OCU200.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.