The urea denatured recombinant human granulocyte colony-stimulating factor (rhG- CSF) which was expressed in Escheriachia coli (E. coli) was refolded with simultaneous purification by strong anion exchange chromatogra...The urea denatured recombinant human granulocyte colony-stimulating factor (rhG- CSF) which was expressed in Escheriachia coli (E. coli) was refolded with simultaneous purification by strong anion exchange chromatography (SAX) in the presence of low concentration- of urea. The effect of urea concentration on this refolding process was investigated. The obtained refolded rhG-CSF has a high specific activity of 2.3×108 U/mg, demonstrating that the proteins were completely refolded during the chromatographic process. With only one step by SAX in 40 min, purity and mass recovery of the refolded and purified rhG-CSF were 97% and 43%, respectively.展开更多
An on-line UV spectrometric method for the quantitative determination ofmethanol increment of methanol-water in the mobile phase (i.e., of greater concentration than thatof the mobile phase) by frontal analysis (FA) o...An on-line UV spectrometric method for the quantitative determination ofmethanol increment of methanol-water in the mobile phase (i.e., of greater concentration than thatof the mobile phase) by frontal analysis (FA) of insulin in reversed phase liquid chromatography(RPLC) was presented. When the methanol increment concentration ranged from 0.05% to 0.50%,V(CH_3OH)/V(H_2O), a set of elution curves could be obtained at 198 nm by a diode-array detector inthe presence of 47% methanol, V(CH_3OH)/V(H_2O) containing 0.03% hydrochloric acid,V(CH_3OH-H_2O)/V(HCl) in the mobile phase. The plateau height of the elution curves of the methanolincrement was found to be proportional to the methanol increment in the mobile phase. The methanolincrement could be determined on a quantitative basis. When the method was used to investigate theelution curve of insulin by FA in RPLC, a small plateau, being the methanol increment, was detectedbefore the usual insulin plateau of each elution curve. In this case the methanol increment wasfound to vary with insulin concentration in the mobile phase. When that concentration was between0.025 mg/mL and 0.30 mg/mL, the methanol increment could be determined in the range from 0.03% to0.19% with a deviation of ±10%. A nuclear magnetic resonance spectrometer (NMR) was also employedto confirm the obtained result. A methodology with a very rigorous experimental procedure forobtaining results of such accuracy is also included. The presented result may be used to prove thata displacement process definitely occurs as insulin is adsorbed by the RPLC stationary phase inFA.展开更多
基金This work is supported by the National Natural Science Foundation of China(No．20175016)
文摘The urea denatured recombinant human granulocyte colony-stimulating factor (rhG- CSF) which was expressed in Escheriachia coli (E. coli) was refolded with simultaneous purification by strong anion exchange chromatography (SAX) in the presence of low concentration- of urea. The effect of urea concentration on this refolding process was investigated. The obtained refolded rhG-CSF has a high specific activity of 2.3×108 U/mg, demonstrating that the proteins were completely refolded during the chromatographic process. With only one step by SAX in 40 min, purity and mass recovery of the refolded and purified rhG-CSF were 97% and 43%, respectively.
文摘An on-line UV spectrometric method for the quantitative determination ofmethanol increment of methanol-water in the mobile phase (i.e., of greater concentration than thatof the mobile phase) by frontal analysis (FA) of insulin in reversed phase liquid chromatography(RPLC) was presented. When the methanol increment concentration ranged from 0.05% to 0.50%,V(CH_3OH)/V(H_2O), a set of elution curves could be obtained at 198 nm by a diode-array detector inthe presence of 47% methanol, V(CH_3OH)/V(H_2O) containing 0.03% hydrochloric acid,V(CH_3OH-H_2O)/V(HCl) in the mobile phase. The plateau height of the elution curves of the methanolincrement was found to be proportional to the methanol increment in the mobile phase. The methanolincrement could be determined on a quantitative basis. When the method was used to investigate theelution curve of insulin by FA in RPLC, a small plateau, being the methanol increment, was detectedbefore the usual insulin plateau of each elution curve. In this case the methanol increment wasfound to vary with insulin concentration in the mobile phase. When that concentration was between0.025 mg/mL and 0.30 mg/mL, the methanol increment could be determined in the range from 0.03% to0.19% with a deviation of ±10%. A nuclear magnetic resonance spectrometer (NMR) was also employedto confirm the obtained result. A methodology with a very rigorous experimental procedure forobtaining results of such accuracy is also included. The presented result may be used to prove thata displacement process definitely occurs as insulin is adsorbed by the RPLC stationary phase inFA.
