RP-HPLC determination of recombinant human interferon omega in the Pichia pastoris fermentation broth.
A rapid and valid reversed-phase high performance liquid chromatography (RP-HPLC) method for determination of recombinant human interferon omega (rhIFNomega) in the yeast Pichia pastoris fermentation broth was developed. The method is based on the hydrophobicity of rhIFNomega followed by RP-HPLC separation with UV detection. The chromatography analysis was performed on EC 250/4 NUCLEOSIL 300-5 C18 (250 mm x 4 mm i.d., 300 A, with a particle size of 5 microm) 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 degrees C. Calibration curve was linear (r=0.9986, n=7) in the range of 0.074-0.555 mg ml(-1) for rhIFNomega and the regression equation was y=2.02 x 10(6)x-1.27 x 10(5). Limit of detection for rhIFNomega 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%.
Liu
Hong
H
College of Bioengineering, Key Laboratory of Biomechanics and Tissue Engineering under the State Ministry of Education, Chongqing University, 174 Shapingba Street, Chongqing 400044, PR China. lhphch@126.com
Pan
Hong-Chun
HC
Peng
Li
L
Cai
Shao-Xi
SX
D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D003470:Culture Media; D005285:Fermentation; D007202:Indicators and Reagents; D007370:Interferon Type I / Q000032:analysis* / Q000096:biosynthesis; D010843:Pichia / Q000737:chemistry*; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis; D012015:Reference Standards; D015203:Reproducibility of Results
D003470:Culture Media; D007202:Indicators and Reagents; D007370:Interferon Type I; D011994:Recombinant Proteins; C063743:interferon omega 1