Thermal stability of the WHO international standard of interferon alpha 2b (IFN-α2b): Application of new reporter gene assay for IFN-α2b potency determinations

Abstract

A World Health Organization requirement for biological standards is that they should exhibit long-term stability at their recommended storage temperature. Thermal stability is usually predicted in accelerated thermal degradation studies, where ampoules of the lyophilized standard are stored at elevated temperatures for relatively short times before testing. To confirm the predicted thermal stability of the 2nd international standard of human interferon alpha 2b (IFN-α2b; 95/566), we tested the potency of the ampouled contents of this standard after 9 years storage at the customary storage temperature of − 20 °C in comparison with ampoules of the IS which had been stored continuously at temperatures ranging from − 150 °C to 56 °C. Since IFN-α2b potency estimates derived from the results of antiviral assays (AVA) showed high within-assay variability, we investigated a novel reporter gene assay (RGA) based on induction of secreted alkaline phosphatase (SEAP) for comparability and precision of such estimations. We show that this RGA generated comparable estimates with overall lower variation. Additionally, the SEAP conversion of p-nitrophenyl phosphate to yellow product could be followed kinetically. Absorbance readings were shown to increase with time in proportion with increasing concentration of IFN-α2b. When the time-dependent increments of absorbance were plotted graphically, the slopes of lines corresponded to concentration. This approach enabled single dilutions of IFN samples, identical in molecular structure to an IFN-α2b standard, to be used for potency estimates by interpolation of slope value against those of the standard at fixed concentrations. It appears attractive for high through-put potency testing of various R&D IFN-α2b samples.