Protein Retention In Polymer-grafted Ion Exchange Chromatographic Resin And Its Thermodynamic Analysis

In recent years,polymer-grafted ion-exchange chromatography has received extensive attention in the research and process development of biopharmaceuticals and related fields,and has shown its great advantages in adsorption capacity and mass transfer performance.Due to lack of systematic investigation to protein retention in polymer-grafted ion exchange chromatography,so far,researchers still lack a deep understanding and comprehensive description about its retention mechanism of proteins.In this work,two commercial dextran-grafted cation exchange adsorbents,SP Sepharose XL and Capto S,were used to investigate the effects of liquid phase on protein retention and thermodynamic behavior,using bovine serum albumin,ovalbumin,β-lactoglobulin B,myoglobin and cytochrome C as the model proteins.The result was also compared with SP Sepharose FF,a non-grafted cation-exchange adsorbent.Linear gradient elution showed that with an increase of buffer pHs,the retention factors of all the proteins in cation exchange chromatography decreased,demonstrating the dominant role of electrostatic interaction for protein binding on cation exchange adsorbents.The evidences further revealed that the scattered positive charges on the surface of protein molecules,rather than net charge of protein,determined protein retention in cation exchange chromatography.Likely,four counterions showed different preferences on the cation exchange adsorbents,which significantly affected the protein retention.Compared with SP Sepharose FF,the pore structure characteristics of dextran-grafted cation exchange adsorbents led to distinct distribution of cation exchange ligand in pore space,thus brough about a longer retention of the protein in the column filled with dextran-grafted cation exchange adsorbents.The isothermal titration calorimetry showed that,all of the four protein binding to cation exchange adsorbents was exothermal enthalpy-driven process,andΔH_ads values increased with increasing protein surface coverage on the cation exchange adsorbents.During protein binding,released heat decreased with an increase of buffer pH.The presence of counterions aggravated the chaos of the system,causingΔH_ads andΔS_adsincreased.TheΔS_adsvalues andΔH_ads values of bovine serum albumin on SP Sepharose XL were smaller than their corresponding values on SP Sepharose FF,which could be attributed to the structure characteristics of dextran-grafted cation exchange adsorbents.These results in this research help us well-understand adsorption mechanism of protein in polymer-grafted adsorbents and give scientific guidance for the development of chromatographic materials.