Determination of recombinant Interferon-alpha 2 in E. coil periplasmic extracts by reversed-phase high-performance liquid chromatography

Reversed-phase high-performance liquid chromatography (RP-HPLC) has been used to analyze Interferon alpha-2 (IFN-alpha 2) as a pure protein or as a pharmaceutical preparation: a method for analyzing periplasmic IFN-a2 directly in osmotic shock extract has, however, never been reported. This work describes an RP-HPLC methodology for the qualitative and quantitative analysis of human IFN-alpha 2a and IFN-alpha 2b directly in bacterial periplasmic extracts or in purified preparations. The analytical method has been set up and validated for accuracy, precision, linearity, sensitivity and specificity. A recovery test indicated an average bias of similar to 1%, intra-day and inter-day quantitative determinations presented relative standard deviations always <= 5%, while the working sensitivity was of similar to 0.3 mu g of IFN-alpha 2 (RSD = 5%). The method proved to be suitable for detecting and quantifying also glycosylated and oxidized forms and N-methionylated IFN-alpha 2 molecules, it was, however, not able to distinguish between IFN-alpha 2a and IFN-alpha 2b. This rapid methodology allows the application of RP-HPLC as a powerful tool to monitor the production yield and quality of IFN-alpha 2 in osmotic shock fluids, right after, or even during the fermentation process. (AU)