Study On The Synthesis And Application Of High Performance Liquid Chromatographic Stationary Phase Modified With Tryptophan

At present,the development of genetic engineering technology enables a variety of recombinant protein drugs to successfully apply in clinical which produces enormous economic and social benefits.Thousands of recombinant proteins expressed in E.coli exist in the inclusion bodies,accounting for 70-80%.The refolding of these inclusion bodies is a "bottleneck" for obtaining high activity and quality proteins during the preparation of recombinant protein drugs.As we all know,high performance liquid chromatography(HPLC)has many advantages such as high separation efficiency,fast purification speed,practicality and easy to scale up,which plays an important role in the renaturation with purification of inclusion bodies.However,unresolved questions remain about low refolding efficiency on-column refolding with HPLC,especially application in large-scale prep-aration.Therefore,HPLC stationary phase based on silica gel functionalized with tryptophan as the ligand was prepared in this work;it had typical characters of high performance hydrophobic interaction chromatography(HPHIC)and characteristic of weak cation exchange chromatography(WCX).Therefore,their separation performance for standard proteins in both WCX and HIC modes,applied to separate egg white and to refold with simultaneous purification of RGD-tagged recombinant human Notch ligand Delta-like 1 were studied.This article includes four sections in following:1.Literature reviewIn recent years,with the research and development of HPLC enables HPLC to widely employ in the separation of biological macromolecules,renaturation with purification of denatured proteins,including HPHIC,HPIEC,MMC and their application in the large scale.Here the above application development will be reviewed.2.The synthesis and separation performance of HPLC stationary phase modified with tryptophanThe chromatography stationary phase based on silica gel was prepared using tryptophan as the ligand with hydrophobic group and charged group by epoxidation,which has HIC and WCX modes.It was characterized by solid fluorescence and elemental analysis,separation performance for standard proteins in both WCX and HIC modes,and applied to further separate egg white were studied.As a result,five kinds of standard proteins(Cyt-C,Rnase-A,Lys,a-Amy,Ins)could be baseline separated in HPHIC mode;Two kinds of standard proteins(RNase A,Lys)could be separated in WCX mode.Moreover,Lys could be separated from egg white one-step in HPHIC mode,the mass recovery and purity were 97.8%and 100%respectively.3.Refolding with simultaneous purification of RGD-rhDlll by tryptophan bonding HPLC stationary phaseIn this study,we first screened RGD-rhDll1 engineering bacteria expressed in shake flask,after ultrasonication,washing and centrifugation,the inclusion body was dissolved in 8.0 moL/L urea obtaining denaturing solution;Then the denaturing solution was refolded with simultaneous purified by tryptophan bonding HPHIC stationary phase.The effects of different gradient elution and pH on refold with simultaneous purification of RGD-rhDll1 were investigated in WCX mode.As a result,when the pH4.0 of equilibrium liquid,using isocratic elution,RGD-rhDll1 is retained,this suggests that the stationary phase has a certain electrostatic interactions.In addition,the effects of different gradient elution,pH and different stationary phas on refolding with simultaneous purification of RGD-rhDlll were investigated in HPHIC mode.The results indicated that when pH7.5 and the flow rate was 1.0 mL/min,a mass recovery of 68.6%could be obtained and the purity was 95.8%.4.Refolding with simultaneous purification of RGD-rhDll1 by tryptophan bonding HPHIC chromatographic cakeIn this part,refolding with simultaneous purification of RGD-rhDll1 by chromatographic cake was studied,which was synthesized independently by our group.The effects of different concentrations urea in mobile phase,the loading volume and the flow rate on refolding with simultaneous purification of RGD-rhDlll were investigated.The results showed that when the 3.0 mol/L urea was added in mobile phase,the flow rate was 2.0 mL/min and sample loading volume was 0.2 mL,the mass recovery and purity of RGD-rhDll1 were 67.0%and 98.2%,respectively.