Specific Small Peptides Functionalized Affinity Monolith For Enrichment And Purification Of Monoclonal Antibody Drugs

With the rapid development of monoclonal antibody drug in therapeutics and diagnostics,the techniques of monitoring concentration and purification become very important.Currently,protein A affinity ligands that is the most widely used in agarose affinity chromatography has strong specificity,but its high cost,poor stability,easy degradation,pH-sensitive,short lifespan,and non-specific adsorption by agarose matrix also bring great difficulties to the purification and analysis.Due to cheap,stable chemical properties,good biocompatibility,mild elution conditions and long service life,small peptide ligand has been one of the best candidates to replace protein A.On the other hand,Organic polymeric monolith column obtained by in situ polymerization exhibits high porosity,large specific surface area,easy modification and excellent acid-alkali tolerance.What's more,hydrophilic crosslinking agent can significantly reduce nonspecific adsorption on the surface of the material.In this work,to make full use of those advantages,small peptide was immobilized on the monolithic matrix by different preparation plan for the enrichment and purification of monoclonal antibody drug.Combined with the present research of the small peptide ligands,the prospect is proposed on the application and development of the small peptide functionalized monoliths.In the first chapter,the category and the development of protein purification technology and the research of small peptide ligands affinity purification strategy were systematically introduced.The effect of preparation methodology of the monoliths on separation and purification results and their respective advantages and disadvantages were also summarized.On this basis,the innovative points and significances of the current researches were finally highlighted.In the second chapter,the small peptide DAAG containing histidine-tag?His-tag-DAAG?was designed as affinity ligand.A DAAG functionalized monolith was fabricated through a metal ion chelation-based multi-step approach.Firstly,the polymerization mixture containing the functional monomers?GMA,EDMA?,AIBN?the initiator?and a ternary porogenic solvent?water,1,4-butanediol and n-propanol?was employed to prepare poly?GMA-co-EDMA?monolith;Thereafter,ANTA solution was continuously pumped through the capillary/pipette and reacted with the epoxy groups on the poly?GMA-co-EDMA?monolith;Then the capillary or pipette was then rinsed with NiSO₄ solution for completing the chelating reaction between Ni²⁺and the NTA carboxylate groups.Finally,the Ni²⁺based IMAC monolith was rinsed His-tag-DAAG solution in order to immobilize the peptide through chelating reaction between the imidazole group of His and Ni²⁺.Systemically characterized by scanning electron microscopy?SEM?,energy-dispersive X-ray spectrometry?EDS?,,elemental analysis,thermogravimetric analysis?TGA?and Fourier Transform Infrared Spectrometry?FT-IR?mercury injection method,Brunauer-Emmett-Teller?BET?method and micro-LC were performed.In the third chapter,the specific binding ability of the DAAG functionalized monolith was evaluated and compared to the poly?GMA-co-EDMA?,poly?GMA-co-EDMA?-NTA and Ni²⁺based IMAC monolith using a series of proteins as probes including hIgG,trastuzumab,human serum albumin?HSA?,bovine serum albumin?BSA?,?-lactoglobulin,myoglobin and trypsin.The dynamic binding capacity?DBC?and reusability of the DAAG functionalized monolith were also tested.Finally,the purification of trastuzumab or hIgG from biological samples was carried out using the DAAG functionalized monolith and the collected fractions were confirmed by SDS-PAGE and MALDI-TOF MS.In the fourth chapter,as the metal ion chelation-based multi-step approach is relatively complicated,the one-step or simpler multi-step approach was explored.The monolith is prepared by the one-step and polymerization mixture containing modified EDPW as monomer,MeOH and THF as porogens,cross-linker MBA and initiator AIBN.After a systematical optimization of the polymerization conditions,the resulting monolith showed good permeability and stability.However,the results of specific test indicated that affinity ligands might be covered and failed to interact with target.For simplified multi-step,poly?VIM-co-MBA?monolithic column was prepared with VIM as monomers,MBA as crosslinking agent,methanol and tetrahydrofuran as porogenic solvent and polymerization conditions were systematically optimized.As before,Ni²⁺and His-tag-DAAG were immobilized and DAAG functionalized monolith was obtained.The results of specific tests showed the peptide immobilization by this simpler multi-step approach is a feasible scheme.In the fifth chapter,the conclusions and prospects of small peptide functionalized monoliths,including the preparation methods and its applications in enrichment and purification of mAbs were further described.