Breaking the Aggregation of the Monoclonal Antibody Bevacizumab (Avastin®) by Dexamethasone Phosphate: Insights from Molecular Modelling and Asymmetrical Flow Field-Flow Fractionation

ABSTRACT: Purpose: To investigate the mechanism behind the aggregation breaking properties of dexamethasone phosphate and related corticosteroids on the IgG1 antibody bevacizumab (Avastin®). Methods: An in silico 3D dimer model is developed to identify the bevacizumab-bevacizumab interface, and different corticosteroids are docked onto the model to distinguish preferred binding sites. In silico predictions are validated by in vitro stability studies, where the antibody is stressed in presence or absence of each corticosteroid and formed aggregates are quantified by asymmetrical flow field-flow fractionation. Results: The dimer model features one close crystal contact area: Lys445 on the Fc region interacts with one Fab arm of the second bevacizumab. Docking reveals an interaction between the phosphate group of dexamethasone phosphate and Lys445, while the rest of the molecule is hindering dimer formation. Predictions are confirmed in vitro, demonstrating that dexamethasone phosphate and betamethasone phosphate partly prevent antibody aggregation, whereas triamcinolone acetonide phosphate does not. Conclusions: Results suggest that bevacizumab monomers follow a specific mechanism to form dimers in which a protein-protein interaction hotspot can be distinguished. The dimer formation can be hindered by corticosteroids in a specific way. This approach allows a simple way to stabilize IgG1 antibodie

Intravitreal formulations targeting the retinal vasculature in posterior segment eye diseases

The objective of the thesis is the development of formulations for intravitreal injection that are targeting the retinal vasculature in posterior segment eye diseases. Due to the isolated structure of the eye, achieving therapeutically effective drug concentrations at the retinal tissues is challenging. The current work aims at improvement of the therapeutic efficacy of intravitreal injections. Two parts can be distinguished. The first part focuses on the prevention of angiogenesis, through the development of sustained release formulations for vasoactive compounds that are able to overcome retinal vascular occlusions. Injectable and biodegradable polymer systems, based on α-hydroxy acids, are investigated as carriers or prodrugs for this application. The second part aims at inhibiting angiogenesis, through the improvement of existing formulations that act against VEGF-A. Therapeutic antibodies are combined with corticosteroids or with small therapeutically inactive excipients. Goal of the former is a stable combo-formulation that might cause a synergistic effect, purpose of the second is to prevent aggregate formation in the antibody formulation