Stabilization of Interferon alpha-2b in a Topical Cream - Pharmaceutical Technology

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Stabilization of Interferon alpha-2b in a Topical Cream
The authors describe a proprietary process for producing a stable, topical interferon alpha-2b formulation that can deliver large drug molecules into the skin or mucosa.

Pharmaceutical Technology
Volume 33, Issue 7, pp. 80-86

Identification of Component A in the formulation

The prevention of IFNα-2b oxidation was the most significant challenge in developing a topical IFNα-2b formulation. IFNα-2b contains five methionine residues (Met16, Met21, Met59, Met111, and Met148), which are susceptible to oxidation in solution (6). Giltlin et al. demonstrated that the oxidation of the Met111 residue produced a methioninesulfoxide derivative that has similar biological activity to the native IFNα-2b (7). This derivative is potentially a major oxidative product in Interferon alpha-2b Cream and is generally known as Component A. In the authors' research, activity of IFNα-2b was measured using a cell-based antiviral assay (AVA) that was shown to be nonspecific for the active pharmaceutical ingredient (API) versus Component A. A sensitive reversed-phase high-performance liquid chromatography (RP-HPLC) assay using fluorescence detection was developed to monitor the formation of the oxidative degradation product (Component A) and to quantify the amount of IFNα-2b.

Figure 1: Reverse-phase high-performance liquid chromatography assays using fluorescence detection as follows: (a) unoxidized interferon alpha-2b (IFNα-2b) in solution 80 g/g; (b) oxidized IFNα-2b in solution 80 g/g after 5 h; (c) spiked-placebo formulation with stressed active pharmaceutical ingredient and recovery of Component A; (d) active formulation after 5 months at real-time storage; (e) forced degradation of active formulation after 5-h exposure to 0.25% hydrogen peroxide (H2O2); and (f) forced degradation of active formulation after 5-h exposure to 3% H2O2. EU is emission unit; IFN = IFNα-2b.
When analyzing concentrated solutions containing 80g/g IFNα-2b, Component A was identified as eluting before the principal IFNα-2b peak at approximately 1% of the principal peak area (see Figure 1a). To confirm the formation of Component A, the forced degradation of IFNα-2b in solution showed that while the principal IFNα-2b peak decreased by 16%, a corresponding increase was observed in the Component A peak. The forced degradation of IFNα-2b in solution was conducted in the presence of an oxidizing agent (0.25% hydrogen peroxide [H2O2]) for 5 h, and the reaction was stopped by the addition of methionine (see Figure 1b) (6).

The AVA and the enzyme-linked immunosorbent assay detected no change in biological activity and content, respectively, as a result of oxidative stress. Formation of aggregates was not observed upon oxidative stress of IFNα-2b, as suggested by ultraviolet–visible spectroscopic data and sodium dodecyl sulfate polyacrylamide gel electrophoresis studies (Data not shown).

In the chromatogram of stressed IFNα-2b in solution (see Figure 1b), Component A was positively identified as eluting prior to the principal IFNα-2b peak, and the peak area was used to calculate its theoretical concentration (assuming Component A was equivalent to IFN-a2b). The stressed API solution was used to spike placebo formulation at a target concentration of 125 ng/g Component A (see Figure 1c). The percent recovery of Component A from spiked placebo formulation (see Table II) confirmed that if IFNα-2b had been oxidized in the formulation, the main oxidative product would have been extracted and detected using the RP-HPLC method.


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