Development and Validation of a Stability-Indicating Size-Indicating Size-Exclusion LC Method for the Determination of rhIFN-alpha 2a in Pharmaceutical Formulations

摘要

A size-exclusion LC method was validated for the determination of interferon-alpha 2a (rhIFN-alpha 2a) in pharmaceutical formulations without interference from human serum albumin. Chromatographic separation was performed on a BioSep-SEC-S 2000 column (300 x 7.8 mm id). The mobile phase consisted of 0.001 M monobasic potassium phosphate, 0.008 M sodium phosphate dibasic; 0.2 M sodium chloride buffer, pH 7.4, run at a gradient flow rate and using photodiode array detection at 214 nm, was used. Chromatographic separation was achieved with a retention time of 17.2 min, and the analysis was linear over the concentration range of 1.98 to 198 mu g/mL (r(2)=0.9996). The accuracy was 101.39%, with bias lower than 1.67%. The LOD and LOQ were 0.87 and 1.98 mu g/mL, respectively. Moreover, method validation demonstrated acceptable results for precision and robustness. The method was applied to the assessment of rhIFN-alpha 2a and related proteins in biopharmaceutical dosage forms, and the content/potencies were correlated to those given by a validated RP-LC method and an in vitro bioassay. It was concluded that use of the methods in conjunction allows a great improvement in monitoring stability and QC, thereby ensuring the therapeutic efficacy of the biotechnology-derived medicine.