title: Development and validation of a stability-indicating size-indicating size-exclusion LC method for the determination of rhIFN-alpha2a in pharmaceutical formulations.
abstract: A size-exclusion LC method was validated for the determination of interferon-a2a (rhlFN-alpha2a) 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 microg/mL (r2 = 0.9996). The accuracy was 101.39%, with bias lower than 1.67%. The LOD and LOQ were 0.87 and 1.98 microg/mL, respectively. Moreover, method validation demonstrated acceptable results for precision and robustness. The method was applied to the assessment of rhlFN-alpha2a 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.
authors.affiliation: Federal University of Santa Maria, Postgraduate Program in Pharmaceutical Sciences, 97105-900 Santa Maria-RS, Brazil.
authors.forename: Estevan Sonego
authors.initials: ES
authors.lastname: Zimmermann
authors.forename: Lucélia Magalhães
authors.initials: LM
authors.lastname: da Silva
authors.forename: Guilherme Zanini
authors.initials: GZ
authors.lastname: Calegari
authors.forename: Fernanda Pavani
authors.initials: FP
authors.lastname: Stamm
authors.forename: Ricardo Bizogne
authors.initials: RB
authors.lastname: Souto
authors.forename: Sérgio Luiz
authors.initials: SL
authors.lastname: Dalmora
mesh_terms: D045744:Cell Line, Tumor; D002853:Chromatography, Liquid / Q000379:methods*; D004355:Drug Stability; D006801:Humans; D016898:Interferon-alpha / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D016898:Interferon-alpha