Preparation Of Cellulose-based MONOLITH Material And Its Application In The Purification Of Protein Drugs

In recent years,protein drugs obtained by recombinant DNA technology have overcome the critical problem of their limited source and have been rapidly developed.Due to their characteristics such as high activity,specific action sites,low toxicity and side effects,and clear biological functions,recombinant proteins drugs are commonly used in the treatment of cancer,infectious diseases,and autoimmune diseases.However,in the preparation of recombinant protein drugs,the separation and purification process in the downstream technology has become a bottleneck due to the problems of low separation efficiency,complicated process,and large cost.Therefore,the research content of this dissertation is to construct a new type of stationary phase material-functional cellulose MONOLITH?Ce MONOLITH?for immobilized metal ion chelate affinity chromatography?IMAC?to achieve efficient and rapid separation and purification of recombinant protein drugs.The main research content is as follow:1.Preparation of Ce MONOLITH material:Ce MONOLITH materials was fabricated through a TIPS method of cellulose acetate?CA?used as the starting material,followed by its alkaline hydrolysis reaction,the obtained materials were characterized by FT-IR,SEM and N₂ adsorption/desorption.In the preparation of CA MONOLITH material,the influence of the TIPS preparation conditions on the internal porous morphology of CA MONOLITH was investigated.Under the optimal preparation conditions?the molecular weight of CA:5.0×10⁴,the concentration of CA:200 mg/ml,the mixing ratio of 1-hexanol/DMF:1.5/1?v/v??,CA MONOLITH material with a hierarchically porous structure?macropore:3.6?m,mesopore:7.8 nm?was obtained.In the preparation of Ce MONOLITH,the hydrolysis time of CA MONOLITH was investigated.The results show that Ce MONOLOTH obtained after3 h is the most fully hydrolyzed and the original hierarchically porous structure is maintained.2.Surface modification and functionalization of Ce MONOLITH materials:The functionalized Ce MONOLITH(Mⁿ⁺-EDTA/NTA-Ce MONOLITH)was obtained through the esterification reaction of the hydroxyl groups on the surface of Ce MONOLITH with the anhydride compounds EDTAD and NTAA,followed by chelating and adsorbing the metal ion Mⁿ⁺.The effects of esterification temperature and time on the internal morphology of Ce MONOLITH and the amount of the introduced ligand were investigated by FT-IR,SEM and elemental analysis.Under the optimal reaction temperature and time were 50°C and 24 h,48.94 and 78.57 mmol/g of EDTA and NTA were introduced into the MONOLITH materials.Then the chelating ability for different Mⁿ⁺of EDTA-Ce MONOLITH and its separation and purification performance after chelating Mⁿ⁺for recombinant protein His-tag TPL in E.coli lysate were investigated by AAS and SDS-PAGE,respectively.The results showed that the optimal chelated metal ion was Ni²⁺and EDTA-Ce MONOLITH chelated with Ni²⁺has a specific and efficient separation effect on the His-tag TPL.3.Separation and purification of His-tag protein drugs by Functionalized Ce MONOLITH:First,the His-tag HPL was isolated and purified using the specific affinity between His-tag and Ni²⁺chelated in the EDTA/NTA-Ce MONOLITH.the maximum affinity of MONOLITH as well as the purity,conformation,and activity of the His-tag TPL protein after isolation were investigated through a series of analytical methods including SDS-PAGE,HPLC,CD,TPL,and UV.The experimental results showed that the maximum affinity of Ni²⁺-EDTA/NTA-Ce MONOLITH of His-tag TPL was 76.92 and 64.57 mg/g,respectively,and the secondary conformation and the activity of His-tag TPL did not change.Then,the separation and purification performance of His-tag TPL,His-tag BAFF and His-tag IL-lra expressed in E.coli lysates by Ni²⁺-EDTA/NTA-Ce MONOLITH were further investigated.The results of SDS-PAGE and UV showed that Ni²⁺-EDTA-Ce MONOLITH had a relatively higher specific and efficient affinity adsorption efficiency,and the adsorption capacities for the three proteins were 74.51,64.92,and 24.17 mg/g,respectively.Finally,the reusability of MONOLITH materials was investigated.The results showed that the affinity of Ni²⁺-EDTA/NTA-Ce MONOLITH after 5 times of repeated use was94.37%and 86.35%of the original adsorption amount,which remained high protein separation and purification capacity,with excellent recycling performance.