Preliminary study of the impact of hydrogen peroxide on three peptides.

Introduction:
In pharmacy units, surface bio-decontamination of drugs and medical devices (MD) is usually carried out using oxidizing agents, such as hydrogen peroxide (H2O2). The permeation of H2O2 into bags without overwrapping following a bio-decontamination has been demonstrated. The aim of this study is to investigate the impact of H2O2 on three peptides in order to determine whether further study should be considered when preparing peptide drugs in the presence of residual H2O2.

Materials and Methods:
The peptides tested were ziconotide (25 amino acids (AA)), glucagon (29 AA) and octreotide (8 AA), at a concentration of 10 µg/mL. Glucagon and octreotide were used as controls. Increasing concentrations of H2O2 (0.01 ppm, 0.05 ppm and 0.1 ppm) were added to the previous solutions. The analysis was carried out over 48 hours after adding the H2O2 to the vials. The assay was carried out using ultra-high performance liquid chromatography (Waters) coupled with a UV detector at 210 nm, on a BEH C18 1.7 μm, 2.1 mm × 50 mm column (Waters) heated to 50° C. The mobile phase was a water / acetonitrile + 0.1 % trifluoroacetic acid gradient. The samples were stored at 6° C in the autosampler. To assess stability over time, the pure peptide solutions were first injected, then, the peak areas were compared before and after the addition of H2O2.

Results:
Without H2O2, the three peptides seemed stable (84-107%) over time. With H2O2, the degradation rate (DR) of ziconotide was higher than 10% after 2 hours of contact at 0.05 and 0.1 ppm. The degradation increased and reached 95% after 48 hours at 0.1 ppm. The DR of glucagon was higher than 10% after 2 hours of contact and reached 42% after 48 hours at 0.1 ppm. Octreotide did not appear to be degraded by H2O2. The variations observed over time were similar for all the H2O2 concentrations and seemed to be related to the analytical method inaccuracies.

Discussion-conclusion:
This preliminary study showed that the effect of H2O2 varies depending on whether or not the peptides contain a methionine (Met), which is an AA sensitive to the experimental conditions of the study. The Met-12 on the surface of the ziconotide is highly exposed to oxidation, as the Met-27 on the surface of the glucagon. The difference in DR could be explained by the difference in the sequence between the two peptides. The octreotide, does not contain a Met, so, it remains stable in the presence of H2O2. Residual levels of H2O2 in isolators or MDs might cause significant degradation of ziconotide over time. To improve the study, a validation of the analytical method will be made in order to compare the preparation of ziconotide in an atmosphere containing, or not, residual H2O2. Likewise, it would be interesting to study whether the oxidation of ziconotide alters its analgesic action.