Assessment of the Degradation Pattern and Extent of PEG Interferon α-2b Using a Stability-Indicating SE-HPLC Assay

Abstract

Covalently attaching polyethylene glycol (PEGylation) to therapeutic proteins is an increasingly important tool for improving stability, pharmacokinetic and pharmacodynamic properties. In this work, degradation of pegylated interferon α-2b (mono-PEG-IFN) was induced using various physicochemical stress conditions (mechanical agitation, pH, temperature, and repeated freeze-thaw). Stability-indicating SE-HPLC assay was validated and employed for monitoring mono-PEG-IFN in the presence of all degradation products. Results were expressed in terms of percentage decrease in mono-PEG-IFN concentration (%Degradation) and peak area normalization method (%Purity). Separation was carried out using a mobile phase of phosphate buffer (100 mM, pH 6.8):1-propanol (80:20 v/v) at 1.0 mL/min and 214 nm. Incubation at pH 4.0–10.0, 37 °C for up to 4 weeks resulted in the formation of aggregates, small molecular weight peptide fragments and mostly depegylated interferon. Similar degradation pattern but to lower extent was noted under short-term storage conditions (24 h at 2–8 and 37 °C). No degradation was noted when the lyophilized powder was stored for 30 months at 2–8 °C, under real-time stability conditions. It should be noted that expression of the results using %Purity, currently employed for batch release was not a reliable approach. Alternatively, the stability of mono-PEG-IFN should be expressed as %Degradation that was shown to reveal minor changes in product stability. Results raised a concern about the efficacy and safety of reconstituted multi-dose vials of pegylated therapeutics that are stored refrigerated. The need for in-house validated testing protocols developed by local regulatory authorities to prevent access of substandard biotherapeutics to local markets is discussed.