Preparation Of Bifunctional MONOLITH Material And Its Application In Separation,Purification And Site-Specific Modification Of Recombinant Protein Drugs

Protein drugs are a class of effective drugs that can be used to treat cancer,infectious diseases,cardiovascular diseases and neurological diseases and other serious diseases that endanger human life.They have characteristics including high activity,small toxic and side effects,specific action sites and clear biological functions,therefore protein drugs play an increasingly important role in clinical practice.In recent years,with the rapid development of recombinant DNA technology,protein drugs can be obtained in large quantities through gene technology,and soon become a hot spot in biomedical industry.However,recombinant protein drugs currently face a lot of problems.Firstly,the separation and purification steps of the preparation process are complicated and costly with low efficiency.In addition,the molecular weight of most proteins is relatively small and they are easily hydrolyzed by enzymes and eliminated by the kidneys,resulting in frequent administration to maintain an effective blood concentration.Therefore,this thesis constructed a bifunctional cellulose-based MONOLITH to achieve the efficient purification and the site-specific modification of recombinant interferon?-2b.The main contents are as follows:1.Preparation of cellulose acetate-based and cellulose-based MONOLITH.Cellulose acetate-based MONOLITH(CA MONOLITH)was prepared by thermally induced phase separation(TIPS),and then the cellulose-based MONOLITH(CE MONOLITH)with hierarchically porous structure was obtained by alkaline hydrolysis.The CA and CE MONOLITH were characterized by FT-IR,SEM,and nitrogen adsorption/desorption.The internal morphology of CA MONOLITH was adjusted by changing the preparation parameters of TIPS,such as the concentration of CA solution and the ratio of mixed solution.The optimum preparation conditions were selected as the concentration of CA was 220 mg/m L and the volume ratio of 1-hexanol/DMF was 1.4/1.And the resultant CA and CE MONOLITH with specific surface areas of 32.28 m²/g and 30.87 m²/g were obtained.During the preparation of CE MONOLITH,the best hydrolysis conditions were achieved by investigating the temperature and time of alkaline hydrolysis.The best alkaline hydrolysis conditions were:hydrolysis temperature was 30°C and hydrolysis time was 3 h.The SEM images before and after hydrolysis showed that the internal morphology of CE MONOLITH after hydrolysis retained the original hierarchically porous structure.2.Preparation of functional cellulose-based MONOLITH for the separation and purification of recombinant interferon?-2b.First,the hydroxyl groups of CE MONOLITH were esterified with ethylenediamine tetraacetic dianhydride(EDTAD)and the EDTA ligands were introduced.After chelating with Ni²⁺,the obtained functional cellulose-based MONOLITH(Ni²⁺-EDTA-CE MONOLITH)was used to separate and purify the his-tagged interferon?-2b.The Ni²⁺-EDTA-CE MONOLITH and the purified his-tagged interferon?-2b were characterized by FT-IR,SEM,AAS and SDS-PAGE.The FT-IR results confirmed that the EDTA ligands were successfully introduced into CE MONOLITH;SEM showed that EDTA-CE MONOLITH retained its original internal morphology after introducing EDTA;by changing the initial concentration of Ni²⁺solution,it was calculated that the maximum Ni²⁺adsorption capacity of EDTA-CE MONOLITH was132.86 mg/g;it can be known by SDS-PAGE results that EDTA-CE MONOLITH had high purification ability for his-tagged IFN?-2b.The maximum adsorption capacity of Ni²⁺-EDTA-CE MONOLITH calculated by Bradford method was 29.17 mg/g.Finally,the reusability of Ni²⁺-EDTA-CE MONOLITH was investigated.The results showed that after 5consecutive cycles,it can still maintain adsorption capacity of 90.6%.3.Preparation of functional cellulose-based MONOLITH for site-specific modification of interferon?-2b.First,the hydroxyl groups of CE MONOLITH were esterified with methacrylic anhydride(MAA)to introduce C=C bonds.Then the NHS-CE MONOLITH was obtained by introducing highly reactive succinimide ester(NHS)groups to MAA-MONOLITH by click chemistry method.After that,the efficient immobilization of transglutaminase was realized by fixed-bed flowing to carry out the site-specific modification of IFN?-2b to obtain another functional cellulose-based MONOLITH(TGase-CE MONOLITH).The fabricated MAA and NHS-CE MONOLITH were characterized by FT-IR,elemental analysis,circular dichroism spectroscopy,and MALDI-TOF mass spectrometry.FT-IR spectra confirmed that MAA and NHS-CE MONOLITH were successfully fabricated.Through elemental analysis and the quantitative determination of NHS groups,it was confirmed that NHS groups havd been successfully introduced into MAA-CE MONOLITH and the introduction amount of NHS was 187.94?mol/g.Through Bradford method,it can be calculated that the immobilization capacity of NHS-CE MONOLITH for TGase was 38.6 mg/g.Through enzyme activity test,it can be calculated that the enzyme activity immobilized on TGase-CE MONOLITH remained 16.27%.Finally,the site-specific single-modified PEG_5k-IFN?-2b was obtained through the catalysis of TGase-CE MONOLITH,which was confirmed through SDS-PAGE and MALDI-TOF mass spectrometry.