Method Development and Validation of SDS-PAGE for Quality Control Testing of Pegylated Interferon Alpha-2a

Hepatitis is one of the most prevailing diseases in Pakistan. Free IFN & Pegylated IFN α-2a is being used to cure chronic hepatitis diseases. A method of impurities determination and molecular weight estimation of PEG-IFN α-2a and Free Interferon α-2a using SDS-PAGE is developed for the purpose of lot release during Quality Control testing. It is the most popular cost-effective method to estimate the molecular weights (MWs) of protein subunits with considerable accuracy. In our study method for the identification and estimation of MWs of PEG & Free IFN were validated first before determining MW’s and impurities of IFNs from different sources. The Rf values showed consistency in precision. The Rf values were also consistent and comparable in 6 independent samples run by second analyst with CV% less than 0.5%. Beside repeatability and reproducibility, limit of quantification was also determined by two different staining techniques i.e. Coomassie and silver staining. Staining technique using silver ions found more sensitive than Coomassie as it detected up to 0.002μg of protein as compared to 0.05μg detected by Coomassie stain, therefore sliver staining technique is considered more sensitive and preferred technique to estimate impurities. The method has also proved to have resolution power to differentiate between free IFN and PEG-IFN. No affect on method was observed by small variations between gel compositions from 14% to 15%, variation in voltage from 105 to 110V and running buffer pH from 8.6 to 8.8. The analytical method validation showed no interference and thus specific for identification and estimation of molecular weights of free Interferon and Pegylated Interferon. Thus, the suggested method could be used for routine analysis of free IFN and PEG-IFN for quality control, stability studies and for further studies like western blotting etc.