OXIDATION
Oxidation of drug substances is a common pathway for drug degradation both in liquid and solid formulation, particularly during
storage. Factors promoting the oxidation rate of proteins during storage include oxygen (from head space), light, peroxides,
and heavy metal ions. Modifications to protein therapeutics through oxidation can lead to a range of functional consequences,
such as inactivation or activation of the drug product, altered binding activities, increased susceptibility to aggregation
and proteolysis, and altered immunogenicity. Methionine residues on the surface of the protein are the most susceptible to
oxidation, and formulation excipients are often used to protect the protein from the oxidation process. Moreover, FDA guidelines
suggest that oxidation must be controlled in the product formulation of therapeutic proteins (4).
To examine the ability of rAlbumin to act as an antioxidant, pharmaceutically relevant concentrations of Insulin-like growth
factor-I (IGF-I) were exposed to trace amounts of the oxidizing agent hydrogen peroxide (H2O2). IGF-I, an important anabolic growth factor, has been shown to be susceptible to oxidation, particularly during storage,
and was therefore chosen as an appropriate model to investigate the ability of rAlbumin as an excipient to inhibit oxidation
(5).
Figure 1
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rAlbumin and L-methionine, a commonly used antioxidant, were tested at a range of concentrations for their ability to prevent
oxidation of IGF-I. Either rAlbumin or L-Methionine was dissolved in a buffer solution. The IGF-I (20 μg/ml) protein was then
added to all samples followed by the H2O2 to a final concentration of 0.0005%. The reaction was terminated with catalase and the degree of oxidation analyzed by reverse-phase
high performance liquid chromatography (HPLC). Percent oxidation of IGF-I was calculated against the main IGF-I peak for all
samples.
Figure 2
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Results displayed in Figure 1 demonstrate rAlbumin to be an effective antioxidant. Oxidation of IGF-I was significantly reduced
in the solutions containing increasing concentrations of rAlbumin, with the highest concentration (20 mg/ml) reducing IGF-I
oxidation by 93%. Due to the susceptibility of IGF-I to oxidation during storage, it is important to note that the initial
IGF-I sample already contained 11.6% of the oxidized form. When compared with the commonly used antioxidant L-Methionine,
rAlbumin protected IGF-I at molar concentrations approximately 13-fold less than that of L-methionine (see Figure 2).
Human serum albumin (HSA) functions as a potent antioxidant primarily due to a single free-thiol at position Cys 34, with
HSA-SH acting as a potential scavenger for reactive oxygen species (ROS). The albumin molecule has six methionine residues
available as an ROS scavenging system and, in addition, has the ability to bind heavy metal ions, such as Cu2+ and Fe3+, which reduces the availability of these ions to cause oxidation.
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