Study of aggregation, denaturation and reduction of interferon alpha-2 products by size-exclusion high-performance liquid chromatography with fluorescence detection and biological assays.

Interferon alpha-2 (IFN alpha-2) products have been widely used as antivirals for the treatment of serious diseases such as hepatitis B and C. However, reports of adverse reactions following treatment have prompted investigations into the cause of these undesirable events. In this study size-exclusion HPLC (SE-HPLC) methods coupled with intrinsic fluorescence detection were developed for evaluating the stability and degradation profiles of IFN alpha-2 drug substances and drug products. The method allowed baseline resolution of the active ingredient from the excipients present in the finished products that included large amounts of albumin. Limits of detection (S/N>or=3) for IFN alpha-2a and IFN alpha-2b were 32 ng/mL and 28 ng/mL, respectively and good repeatability of chromatographic profiles (%RSD<2.1) was obtained. High molecular weight (HMW) aggregates with apparent molecular weight of approximately 650 kDa as well as dimers, denatured and reduced variants were successfully identified and separated from native IFN alpha-2 proteins. This chromatographic method, which quantitatively measures physical and chemical changes taking place in solution formulations, was found to be capable of monitoring IFN alpha-2a and IFN alpha-2b stability. Potency assay results revealed up to 87% decrease in biological activity of the physically and chemically altered variants compared to the original IFNs.