Acta Pharm. 50 (2000) 209-218
The SDS-PAGE technique for determination of the commercially available therapeutic proteins interferon alpha 2a and 2b was studied. The aim of the study was to estimate whether the requirements of the European Pharmacopoeia for IFN-a2 concentrated solution could also be applied to finished products. Different staining procedures and gel concentrations were tested for maximum sensitivity and protein separation. A 14% acrylamide/bisacrylamide gel concentration was found to be optimal. The silver staining procedure according to Blum et al. (13) provided high sensitivity down to 1 ng on a colourless, transparent background. Detection with Coomassie Brilliant Blue staining achieved a sensitivity of approximately 17 ng, making the identification of IFN-a2 in lyophilised products still possible. The molecular mass was determined by densitometry using a four-parameter regression analysis and compared to the pharmacopoeial standard. Solutions of IFN-a2 contain no human serum albumin which interferes with the detection of IFN-a2 impurities and can therefore be examined for purity as well as identity. However, strict pharmacopoeial requirements could only be followed when analysing solutions of 15 x 106 IU mL -1 or higher concentration, using sensitive silver staining. Impurities of molecular mass higher than that of IFN-a2 were detected in the samples under non-reducing conditions, impurities of molecular mass lower than that of IFN-a2 were observed in almost all of the samples under reducing conditions. The findings of this study have been adopted for routine analysis of IFN-a2 products.
Keywords: IFN-alfa2a, IFN-alfa2b, SDS-PAGE, silver staining, Coomassie blue staining, purity