Identification Of Antibody Ligands And Protein Adsorption Onto Dextran-grafted Matrices

Antibodies are of great application value in the biopharmaceutical field,and immunoglobulin G(IgG)is a typical type of antibodies.In this work,affinity peptide ligands of hIgG were identified.Dextran-grafted Sepharose FF resins and capillaries which mimicked pores of Sepharose gel particles were fabricated,and then the affinity peptide ligand of hIgG or ionic group was immobilized to investigate protein adsorption-transport.The details of this work are summarized as follows.Firstly,the untested 11 peptides from the 15 potential peptide ligands of hIgG previously obtained by a biomimetic design strategy were identified.Two octapeptides(FYCHWQDE and FYCHNQDE)were found to show high affinity for hIgG.Both the peptides specifically bound to Fc fragment of hIgG mainly by electrostatic interactions,and hIgG could be efficiently purified from human serum with the two peptide affinity columns.Thus,a total of five among the 15 peptides were identified as high-affinity ligands of hIgG.Furthermore,the five peptide ligands were all derived from the same peptide model FYxHxxxE(where x denotes any amino acid),containing four common hot spots F132,Y133,H137 and E143 of the affinity motif of Protein A.Then,the octapeptide ligand FYCHWQDE of hIgG was immobilized on Sepharose FF resins to study the effect of ligand density on hIgG adsorption.The results indicated that the adsorption capacity of hIgG increased with ligand density and the uptake rate of hIgG mildly decreased with increasing ligand density.Moreover,the dynamic binding capacity of hIgG were the maximum on the affinity column with ligand density of 45.3 ?mol/mL.By using this affinity column,hIgG was efficiently purified from human serum.To investigate the role of dextran grafting in hIgG adsorption,the octapeptide ligand FYCHWQDE of hIgG was immobilized on dextran-grafted Sepharose FF resins.The results showed that the uptake rate of hIgG was obviously enhanced via chain delivery effect on the dextran-grafted ion exchangers,but not accelerated by dextran grafting on the dextran-grafted FYCHWQDE resins.Analyses of different roles of grafted polymer in protein adsorption in different adsorbents revealed three necessary elements for the happening of chain delivery in polymer-grafted adsorbents,namely long and flexible polymer chains for ligand coupling,low hydrophobic ligands and multivalent proteins with more than one binding sites for the ligands.In order to observe protein adsorption behaviors on ionic surfaces,a microscopic method was developed to directly visualize protein adsorption and transport in bare capillaries modified with ion exchange groups.Silica capillaries were coated with poly(vinyl alcohol)to eliminate nonspecific protein absorption and then functionalized with DEAE or DEAE-dextran to prepare anion-exchange capillaries.Protein concentration profiles were acquired from microscopic images and analyzed with a one-dimensional diffusion-adsorption model to determine the effective diffusivities of protein.The results indicated that surface diffusion of bound protein was negligible on the surface of DEAE modified capillaries,but significantly contributed to protein transfer in the DEAE-dextran-grafted capillaries via chain delivery effect.The chain delivery was little affected by protein concentration,but markedly increased with ionic capacity.This work found new affinity peptide ligands of hIgG and revealed characteristics of protein adsorption and transport in dextran-grafted adsorbents.The results will benefit the development and application of polymer-grafted chromatographic resins.