Establishing A Methodology For Affinity Ligands Screening Of Monoclonal Antibody With STD-NMR

Biological medicine attentions greatly stimulate the development of downstream purification technology of monoclonal antibodies.Protein A,the current affinity ligand for antibody purification,still has several inherent shortcomings,such as high cost,harsh elution conditions and low stability,which lead to the secondary contamination and biological toxicity in purification.Discovery of the novel high-quality affinity ligands has become a bottleneck for antibody purification.In recent years,a group of peptide ligands have become the research hotspot.However,the designs of them were mostly based on the virtual screening approaches and still suffered from drawbacks on false positive result and low hit rate.Therefore,establishment of a direct screening protocol aiming to the actual binding environment is greatly needed.STD-NMR has advantages in that the size of protein is not limited and most importantly it can screen the ligand in actual binding environment.Therefore,here we established a novel STD-NMR approach for the selective ligand screening,and meanwhile we evaluate this approach by isothermal titration thermography(ITC)and quartz crystal microbalance(QCM).Then the screened biomimetic peptide was used for the antibody affinity media preparation and antibodies purification.The following results were obtained:1.Firstly,a none-competitive titration approach was established to distinguish the specificity and nonspecific ligand.We evaluated a series of approaches from multiple scales,such as,STD-NMR,ITC,QCM,and investigated their possibilities in mining the difference between two selective interaction modes.The results showed that the specific ligand tended to a linear STD signal intensity,with relatively higher ΔH,lowerΔS and lower adsorption amount,while the nonspecific ligand was completely the opposite.Then,the STD none-competitive titration experiment was used to screen two biomimetic ligands,RGWLC and FYEILHC,previously designed by our group.The results showed that FYEILHC had better specificity than RGWLC and was more suitable for the affinity media design.2.Finally,we utilized this biomimetic peptide as ligand by grafting it on the supermacroporous dextran-poly(glycidyl methacrylate)microspheres(dextran-PGMA)and developed it into an efficient IgG chromatographic media.The scan electronic microscopy(SEM)demonstrated that the macroporous structure of microspheres can be mostly maintained after coupling with the biomimetic peptide.AKTA protein purification system was used to evaluate the column with macroporous affinity media.The results showed that the biomimetic peptide ligand grafted dextran-PGMA microsphere had low cost,high-throughput separation,chemical stability and great potential to purify IgG from the plasma.To sum up,we established a method for screening specific and non-specific ligands for the first time,and prepared an affinity media by screening the biomimetic peptide ligand and grafting them on gigaporous dextran-PGMA microsphere.The study provides a new preparation method for the design of antibody purification media.