Synthsis Of Affinity Adsorbent With Biomimetric Peptide Ligand And The Performance In Antibody Purification

In recent years,monoclonal antibodies have been widely applied in the diagnosis and treatment of diseases because of their high specificity and low immunogenicity.At present,protein A chromatography has become the "gold standard" in the process of antibody separation and purification because of its high specificity.However,it also has faced a series of problems,such as high prices,poor enzyme stability,easy to ligand leakage and harsh elution conditions.The biomimetic affinity peptides obtained by rational design based on molecular simulation can not only overcome the problems of protein A,but also have high selectivity and appropriate affinity similar to protein A,which can be regarded as an alternative ligand for protein A.In order to further improve the performance of biomimetic peptide affinity medium,we selected the biomimetic octapeptide FYWHCLDE which was selected in our previous work,and the amino acids residues in polypeptide were replaced by its enantiomer amino acids.First,the stability of L-FYWHCLDE and D-FYWHCLDE affinity ligands againstα-chymotrypsin was investigated,and then they were coupled to the matrix Sepharose6 fast flow to investigate the static adsorption performance of the medium for h Ig G at different p Hs.The results of enzyme stability test showed that more than 50% of the peptides were degraded in L-FYWHCLDE peptide affinity medium after incubation with the enzyme for 8 h,while only about 8% was degraded in D-FYWHCLDE peptide affinity medium,indicating that D-type peptide affinity medium had higher enzyme stability.The results of static adsorption experiments showed that changing the configuration of amino acids did not affect the specificity and affinity of the peptide for h Ig G.The maximum static adsorption capacity of the two peptide affinity media were obtained to be 111.32 mg / ml for L-type peptide affinity medium and 129.81 mg / ml for D-type pepide affinity medium at p H 6.0.Then D-type peptide affinity medium was used to isolate and purify monoclonal antibodies from cell culture supernatant.The influence of salt concentration in the elution buffer was investigated.It was found that the highest recovery(94.97%)and purity(more than 98%)of monoclonal antibody could be obtained when 0.5 mol/L Na Cl was added into the equilibrium buffer.Under this condition,meanwhile,the purity(more than 90%)and recovery(more than 85%)of Ig G isolated from human serum decreased slightly.This could be attributed to the fact that,there were many kinds of immunoglobulins in serum,and this peptide ligand had affinity for all of them.After 40 cycles of purification of Ig G from serum by two kinds of affinity media and 12 hours of in situ CIP with 0.1 mol/L Na OH,DBC decreased differently in two peptide affinity chromatography.It was found that the reusability and alkali resistance of D-type peptide affinity medium were higher than that of L-type peptide affinity medium.These results indicated that the peptide affinity ligands composed of unnatural amino acids were more stable,which is helpful to improve the service life of the medium,and exhibited great potential for the separation and purification of antibodies.