Freeze Drying to Develop a Bevacizumab-based Tablet for Ocular Implantation

Abstract

 

Purpose

 

Bevacizumab has shown the potential to control scarring when administered into the subconjunctival space following glaucoma filtration surgery (GFS). To overcome the problem of rapid clearance, a solid dosage implantable tablet form of this antibody that provides prolonged release in the bleb is being developed. In an effort to optimise dose, we are examining each step used for tablet fabrication. As is often the case for any protein pharmaceutical presented in a solid reconstitutable form, a critical process step is lyophilisation, which can cause aggregation of the protein with loss of activity. The aim of this study is to characterise the lyophilisation of the bevacizumab formulation used to fabricate the subconjunctival implantable tablet. Determination of the glass transition temperature (Tg’) and collapse temperature (Tc) during lyophilisation assists in the development of a suitable freeze-drying cycle.

 

Methods

 

Differential Scanning Calorimetry (DSC) was used to measure the Tg’ of the excipients alone and in combination with the antibody. Freeze Drying Microscopy (FDM) was used to study the collapse temperature (Tc) for the excipients (trehalose and hyaluronic acid (HA)) and the formulation. The antibody formulation was characterised by size exclusion chromatography (SEC) and gel electrophoresis.

 

Results

 

DSC experiments indicate the sub-ambient glass transition temperature (Tg’) for bevacizumab to be -28.24°C (n=2) and that of trehalose to be -29.36°C (n=2). The melting endotherm of HA was -23.71°C (n=2) as no Tg’ could be observed. This can be attributed to the presence of buffer salts present in the HA formulation leading to a eutectic melt. FDM was used to monitor the progress of lyophilisation to determine the collapse temperature of the formulation (Fig. 1). The collapse temperature of the pharmaceutical formulation of bevacizumab was found to be -36°C (n=2).

 

Conclusions

 

Freeze-drying is a suitable technique to obtain a solid form of antibody that can be fabricated as a tablet. Characterisation of freeze-drying using FDM and DSC suggested that starting the primary drying below -36 °C may avoid collapse of the cake during scale up for freeze drying of the bevacizumab formulation.

 

 

Fig. 1. Progression of freeze drying of bevacizumab formulation for tablet fabrication using FDM: A) Sample frozen at -60°C B) Sample drying at -40°C C) Sample drying with collapse at -35°C

 

Keywords: 658 protein purification and characterization • 765 wound healing