Preserved bevacizumab (Avastin®) eye drops for application in multidose containers - an in-vitro characterisation

Monoclonal antibodies have made an immense contribution to the treatment of various human diseases. We aimed at investigating an affordable treatment option for veterinary patients with corneal neovascularization by adding the preservative benzalkonium chloride (BAC) to bevacizumab (Avastin®) for usage in multidose containers. A comprehensive analytical similarity assessment of preserved and unpreserved bevacizumab after dilution and storage was carried out.

Diluted and preserved bevacizumab was analysed at different time points for a 4-week period and compared with unpreserved bevacizumab at the same concentrations at each time point. Native-PAGE, immunoblotting and HP-SEC were used to observe aggregation and degradation. DLS provided information about particle size and dispersity. Bevacizumab quantified by ELISA was conducted to determine its biological activity. Dose response curves and cell migration assays were performed to detect possible toxic effects and determine biological activity and efficacy of the drug using HUVECs.

Native-PAGE, immunoblotting and HP-SEC analysis did not show any changes or degradation products in the presence of BAC and after storage compared to unpreserved bevacizumab. The overlapping intensity-based particle size distribution obtained from DLS showed similarity in all tested groups and homogeneity was maintained. ELISA accurately detected bevacizumab at different concentrations. HUVECs incubated with preserved or unpreserved bevacizumab showed a comparable effect on cell migration. No decrease in cell viability was detected.

Equivalence tests demonstrated that bevacizumab is stable after dilution, storage and preservation with BAC. Our study shows that preserved bevacizumab applied in mutidose containers can be considered as a cost-effective alternative to the otherwise single-dose treatments.