Evaluation of recombinant human interferon beta 1b by liquid chromatography methods and bioassay

Resumo

Recombinant human interferon beta 1b (rhIFNβ−1b) is clinically used to treat multiple sclerosis. A reversed−phase liquid chromatography (RP−LC) method was carried out on a Jupiter C4 column (250 mm × 4.6 mm i.d.). The mobile phase A consisted of 0.1% trifluoroacetic acid (TFA) in water, and the mobile phase B was acetonitrile with 0.1% TFA run at a flow rate of 1.0 mL/min. A size exclusion liquid chromatography (SE−LC) method was carried out on a BioSep−SEC−S 2000 column (300 mm × 7.8 mm i.d.). The mobile phase consisted of 1 mM monobasic potassium phosphate, 8 mM sodium phosphate dibasic and 200 mM sodium chloride buffer pH 7.4, run isocratically at a flow rate of 0.8 mL/min. Retention times were 31.87 and 17.78 min, and calibration curves were linear over the concentration range of 1−200 µg/mL (r2 = 0.9998) and 0.50−200 µg/mL (r2 = 0.9999), respectively, for RP−LC and SE−LC, with detection at 214 nm. Liquid chromatography (LC) methods were validated and employed in conjunction with the in vitro bioassay to assess the content/potency of rhIFNβ-1b, contributing to improve the quality control and to ensure the efficacy of the biotherapeutic.