Research On Preparation And Antibody Adsorption Properties Of Tetrapeptide Biomimetic Chromatographic Resins

Today,Protein A-based chromatography is commonly applied in the capture step for antibody purification.However,the use of protein A chromatography is less attractive because of toxic ligand leakage as well as high cost,etc.As a potential alternative to protein A chromatography,peptide biomimetic chromatography has the advantages of cheaper to produce,milder elution,and resistance to enzymatic degradation.With the help of experiment and molecular simulation,this work tried to understand the interaction between tetrapeptide biomimetic resins and antibody or impure protein,design and screen novel tetrapeptide ligand,and explore a new process for antibody purification by tetrapeptide biomimetic chromatography.Firstly,five tetrapeptide biomimetic ligands(Ac-HYRF,Ac-HWRH,Ac-YHRI,Ac-FHRA,Ac-FYRH)selected in previous study were coupled on the agarose gel with help of spacer to investigate their adsorption and separation performance.Static adsorption results showed that five resins have good binding ability for both human immunoglobulin G(hIgG)and bovine serum albumin(BSA).The difference is that the adsorption of hIgG relied on the combined effect of hydrophobic and electrostatic interaction but adsorption of BSA almost entirely dependent on electrostatic interactions.However,further improvement for tetrapeptide ligand was needed due to the poor performance in separating hIgG from protein mixture by Ac-FYRH-4FF resin.To improve the adsorption selectivity of tetrapeptide ligands,Cysteine,Tryptophan and Glutamate were introduced as the replacement of arginine and three new tetrapeptide ligands(Ac-FYCH,Ac-FYWH and Ac-FYHE)were designed.Then new tetrapeptide ligands were evaluated by molecular docking,static/dynamic adsorption and antibody separation.It was found that ligand Ac-FYHE has high binding capacity and selectivity for hIgG.At pH 7,dynamic binding capacity(Q10%)of Ac-FYHE-4FF resin was 24.1 mg/mL for hIgG but just 2.1 mg/mL for BSA.when Ac-FYHE-4FF resin was used to separate hIgG from protein mixture,the purity and recovery reached 93.4%and 92.1%,respectively.Finally,the adsorption and separation properties of the potential resins Ac-FYHE-4FF were further investigated.As a result,when the ligand density increased,the adsorption capacity of antibody hIgG(Qm)increased while the adsorption efficiency of the ligand decreased.With the addition of 0.5 M NaCl,Qm decreased by less than 20%but Qm decreased by 70%with the addition of 50%ethylene glycol,which indicated that hydrophobic interaction would be the driving force for the binding between Ac-FYHE-4FF resin and hIgG Besides,pH 7.5 and 3.5-5.0 could be optimal loading and elution conditon for hIgG respectively.And the Ac-FYHE-4FF resin was applied to separate mAb or hIgG from CHO cell culture supernatant and human serum,the purity and recovery were both more than 90%with only one-step separation,which indicated that the Ac-FYHE-4FF resin developed in this work would be promising for antibody purification.In this work,a novel tetrapeptide ligand,Ac-FYHE,with high adsorption capacity and selectivity for antibody has been obtained and successfully applied to the antibody purification,which would contribute to the promotion and application of peptide biomimetic chromatography.