Protein Folding Liquid Chromatography Of Recombinant Human Interferon-γ

Based on the mechanism of protein folding by liquid chromatography, the efficiency of refolding with simultaneous purification of recombinant human interferon-γ(rhINF-γ) produced by E.coli was investigated. It mainly focused on the methods for increasing in the refolded efficiency and the mass recovery of rhINF-γby protein folding liquid chromatography (PFLC).The thesis contains six parts.1. Review: The survey of recombinant protein drugs made in our country and the significance of protein folding were introduced briefly. The methods of protein folding including the recent development of recombinant proteins and rhINF-γwerealso introduced and reviewed. It contained 137 references.2. The theoretical basis of protein folding, especially, the mechanism of proteinrefolding by hydrophobic interaction chromatography (HIC) was emphasized. It is as the fundamendal of this thesis.3. Using seven types of' stationary phase of hydrophobic interaction chromatography (STHIC) with different end groups of PEG-200, PEG-400, PEG-600, PEG-1000, furfural, oxethyl and phenyl, the sample size was investigated to find out the reasons of mass loss of rhIFN-γduring its refolding with simultaneous purification by PFLC. When the breakthrough volume or mass of 7.0 mol·L^-1 guanidine hydrochloride (GuHCl) solution and impure proteins didn't influence on all rhIFN-γinjected to be retained on the column, the optimum loading volume was investigated. Comparing the purity, specific bioactivity and mass recoveries of rhIFN-γon STHICs with different end groups, we found that PEG-200 STHIC was the most excellent STHIC. So, the PEG-200 STHIC was preferred stationary phase. The efficiency of purification of recombinant human stem cell factor (rhSCF) was also studied on STHICs with different end groups of PEG-200 and PEG-600. The results of refolding and purification prove that a weaker hydrophobic STHIC is advantageous. 4. Effects of mobile phase composition, gradient mode, flow rate and pH on the purity, specific bioactivity and mass recoveries of rhIFN-γfor renaturation with simultaneous purification were investigated on the PEG-200 STHIC. The conditions of refolding with simultaneous purification of rhIFN-γwere optimized. The base of renaturation with simultaneous purification of rhIFN-γwas established on large scale.5. Under the selected and optimized chromatographic conditions, the extract of rhIFN-γwas renatured with simultaneously purified by using the PEG-200 STHIC only with one-step. The fraction of the renatured and purified rhIFN-γfrom the PEG-200 STHIC was desalted by the size exclusion chromatography, subsequently freeze-dried to powder. Then, the powder was identifed by MALDI-TOF-MS and reverse phase liquid chromatography (RPLC). The obtained mass recovery, purity, specific bioactivity of the renaturation with simultaneous purification of rhIFN-γby PEG-200 STHIC were 93.7%, >95%, and 4.3×10⁷I.U./mg, respectively. The mass recovery, purity of the desalted by Sephadex G-25 were 92.0% and >95%, respectively. The purity of the freeze-dried monomeric and dimmer rhIFN-γwere >95% and <4.5%, respectively, and the specific bioactivity was 9.5×10⁸ I.U./mg. Comparing the purification technology with other thesis, the results showed that the preparation of rhIFN-γin this purification technology is a kind of simple and efficient one.6. The separation efficiency and its influent fators were studied on chromatographic cake. The results proved that chromatographic cake had the characters including higher speed separation, large volumn and high viscity sample injection. The good results of renaturation with simultaneous purification of rhIFN-γand rhSCF on a weak hydrophobic STHIC with end groups of PEG-200 by chromatographic cake were gained.