Refolding And Simultaneous Purification Of RhG-CSF And RhTNF-? Using Dual-function Chromatographic Stationary Phase

In the field of bio-pharmaceutical technology,as the overexpression of recombinant protein drugs in Escherichia coli(E.coli)often results in the accumulation of the protein product to form inactive and insoluble deposits inside the cells,called the inclusion bodies.The inclusion body is usually insoluble in water.The solubilization is possible through the introduction of high concentration of denaturing,such as 7.0 mol/L guanidine hydrochloride(GuHCl)or 8.0 mol/L urea.In order to obtain the target protein with biological activity,the insoluble inclusion body proteins were refolded,i.e.,protein refolding(renaturation).The methods frequently used for the renaturation of inclusion body include dilution and dialysis.Though these methods are simple,the recoveries of the mass and bioactivity of target protein obtained are very low,typically only 5%to 20%.Furthermore,the folded protein can not easily separated from misfolded forms,which affects the cost of therapeutic protein.Therefore,the refolding of recombinant protein drugs meets the bottleneck restricted the industrialization of bioengineering technology.It is of great significance to improve the yiqld of refolding as well as to develop an efficient way to produce recombinant proteins with refolding at a large scale.In recent years,liquid chromatography has been widely used in the refolding and purification of recombinant protein drugs,and obtained the higher mass recovery and biological activity.Therefore,it is regarded as an efficient,and close to ideal refolding method named as protein folding liquid chromatography(PFLC).We firstly synthesized a novel dual-function chromatographic stationary phase which simultaneously containing ion-exchange and hydrophobic group.The dual-function column can efficiently separate proteins in IEC and HIC mode,its ability to separate protein can be comparable to a corresponding single mode column.Therefore,a dual-function column can replace two conventional ion exchange and hydrophobic column to achieve "a column with two" function,which is vividly called "2D column".Based on this dual-function column,2DLC can be established using only a single column.Compared with conventional two-dimensional chromatography,the dual-function column can complete the HIC-IEC or IEC-HIC dimensional chromatography process.In this paper,the recombinant protein drugs rhG-CSF and rhTNF-a is refolded and simultaneously purified using the synthetic dual-function column and building single column two-dimensional liquid chromatography.This thesis is composed of four parts as follows.1.ReviewThis chapter gives a comprehensive review on new refolding technology of denatured protein in vitro and the advantages of refolding protein by liquid chromatography.We also prospected the application of mixed-mode chromatography and two-dimensional liquid chromatography using a single column(2DLC-1C)in separation and purification protein.A total of 75 reference were cited in this chapter.2.Refolding and simultaneous purification of recombinant human granulocyte colony stimulating factor(rhG-CSF)using dual-function chromatographic stationary phaseRenaturation and simultaneous purification of rhG-CSF by WAX/HIC column in IEC and HIC mode was studied.We examine the impact of composition of the mobile phase containing the urea concentration,pH,the ratio of GSH/GSSG,the concentration of ammonium sulfate and loading volume on refolding and purification of rhG-CSF.The result shows the WAX/HIC column can refold and simultaneously purify rhG-CSF in the two modes.The purity of 96.23%and mass recovery of 39.93%of refolded rhG-CSF can be achieved in IEC mode.The mass recovery and purity are 39.74%and 96.03%in HIC mode.3.Refolding and simultaneous purification of recombinant human tumor necrosis factor(rhTNF-?)using dual-function chromatographic stationary phase in IEC modeWAX/HIC dual-function column was applied to the refolding and simultaneous purification of rhTNF-a in IEC mode.Several chromatographic parameters affecting the refolding yield of the denatured/reduced rhTNF-a,such as the urea concentration,pH value and concentration and ratio of GSH/GSSG in the mobile phase were investigated in detail.At the optimal conditions,rhTNF-a can be renatured and purified with 30 min by one step.The purity of 96.72%and mass recovery of 63.25%of refolded rhTNF-a can be achieved.The result shows WAX/HIC column can achieve the refolding and simultaneous purification of rhTNF-a in IEC mode.4.Refolding and simultaneous purification of recombinant human tumor necrosis factor(rhTNF-?)using dual-function chromatographic stationary phase in HIC modeFirstly,we seperate and purify rhTNF-a using WAX/HIC dual-function column in HIC mode.The strong hydrophobility of rhTNF-?,together with the strong hygrophobic interaction between rhTNF-? and statinary phase in the HIC mode,leads to the rhTNF-?difficult to be eluted.Therefore we examined the effect of urea concentration in mobile phase on the elution of rhTNF-?.The results revealed that only when the urea concentration in mobile phase is 8.0 mol/L,the rhTNF-? can be eluted from stationary phase.Furthermore,the effects of loading volume on the purification of rhTNF-? was also investigated.When the 8.0mol/L urea was added in elution buffer B and sample loading volume was 0.3ml,the final mass recovery and purity of rhTNF-a after a linear gradient elution were 70.27%and 90.45%,respectively.Secondly,off-line 2DLC-1C was set up and can successfully employ to renaturate rhTNF-a.The rhTNF-a solution extracted by 8.0 mol/L urea from inclusion bodies were isolated and purified in HIC mode.After the chromatographic fractions of target protein were collected,the collected chromatographic fractions were refolded and simultaneously purified in IEC mode.The mass recovery of rhTNF-a is 46.78%.The SDS-PAGE indicates the purity is 98.32%by off-line 2DLC-1C.The results further indicate 2DLC-1C can refold and simultaneous purity recombinant protein drugs in IEC and HIC mode.2DLC-1C can also develop new technology,namely single column two-dimensional protein folding liquid chromatography.The new technology can replace two columns with a column,which can greatly simplify the production process,reduce costs and has important application value for the development of biotechnology.