Sir,

Intravitreal bevacizumab has been effective for vascular endothelial growth factor (VEGF)-mediated diseases of the retina and choroids.1, 2, 3 However, repeated injections may be required. An alternative mode of administration would be a biodegradable intravitreal implant4 of lyophilized bevacizumab, which has not been previously reported.

In an effort to assess the viability of a biodegradable intravitreal implant of lyophilized bevacizumab, we evaluated the effect of the lyophilization process of bevacizumab solution on the in vitro binding activity of bevacizumab to VEGF165.

The commercial solution of bevacizumab (Avastin 100 mg/4 ml; Genentech Inc., South San Francisco, CA, USA) was frozen at −40 °C for 24 h and then lyophilized (E-C Modulyo; E-C Apparatus Inc., New York, NY, USA). It was then stored in dry bottles in a low moist environment. The lyophilized bevacizumab was diluted in Tris-buffered saline at 1 mg/ml and used in western blot analysis.

The VEGF165 (Sigma-Aldrich, St Louis, MO, USA) was transferred to nitrocellulose membranes (Transblot 0.45 μM; Bio-Rad Laboratories, Richmond, CA, USA) after denaturation. The blot was blocked by 5% skim milk in Tris-buffered saline with 0.05% Tween (TTBS) for 1 h at room temperature and incubated overnight at 4 °C with primary antibody against VEGF or bevacizumab (Avastin lyophilized) at 1 : 2500 dilution. After washing with TTBS, we incubated blots with peroxidase-conjugated goat anti-human secondary antibody for 30 min at room temperature. After the final wash, we incubated the blot with chemiluminescence reagent (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and the signal was detected with Amersham film (Hyperfilm; GE Healthcare, Little Chalfont, Buckinghamshire, UK).

Gel electrophoresis confirmed the migration pattern of VEGF165 (Figure 1a). Subsequently, western blot analysis showed that bevacizumab could still bind to VEGF165 in vitro. A positive immunoreaction of bevacizumab with VEGF165 was revealed by a chemiluminescent reaction (Figure 1b).

Figure 1
figure 1

Western blot analysis of VEGF165. (a) Right lane: VEGF165 is shown on electrophoresis gel stain by Coomassie Brilliant Blue (Sigma-Aldrich) at 19 kDa. Left lane: Trypsinogen (24 kDa) and trypsin inhibitor (20 kDa) were used as controls. (b) Positive immunoreaction of bevacizumab with VEGF165 transferred to nitrocellulose membranes was revealed by a chemiluminescent reaction from peroxidase-conjugated goat anti-human secondary antibody (arrow). The corresponding positions of the controls were marked on the left side of the membrane.

Full size image

This study shows that bevacizumab is still active after freezing and lyophilization, which are essential steps in the development of a biodegradable intravitreal implant. Even deep freezing did not alter the binding activity of bevacizumab to VEGF165, which was a concern.5 Upcoming in vivo studies on chick embryos will allow us to definitely test the bioactivity of bevacizumab biodegradable intravitreal implants.