RP-HPLC determination of recombinant human interferon omega in the Pichia pastoris fermentation broth

Abstract

A rapid and valid reversed-phase high performance liquid chromatography (RP-HPLC) method for determination of recombinant human interferon omega (rhIFNω) in the yeast Pichia pastoris fermentation broth was developed. The method is based on the hydrophobicity of rhIFNω followed by RP-HPLC separation with UV detection. The chromatography analysis was performed on EC 250/4 NUCLEOSIL 300-5 C18 (250 mm × 4 mm i.d., 300 Å, with a particle size of 5 μm) column. The compositions of the mobile phase A and B were 999:1 (v/v) water/TFA and 999:1 (v/v) acetonitrile/TFA at a flow rate of 1.0 ml min −1. Detection was done by spectrophotometry at 280 nm and the column temperature was 30 ± 1 °C. Calibration curve was linear ( r = 0.9986, n = 7) in the range of 0.074–0.555 mg ml −1 for rhIFNω and the regression equation was y = 2.02 × 10 6 x − 1.27 × 10 5. Limit of detection for rhIFNω was 0.053 mg ml −1. The values of R.S.D. (%) of intra-day and inter-day precision were <5.65 and <5.68 ( n = 6), respectively. The R.S.D. (%) values and the average recovery rate of recovery experiment were <1.23 ( n = 3) and 97.97%.