Chromatography is the main purification unit operation for biopharmaceuticals and other biological products. In order to optimize or perform stable chromatographic separation processes many variables or parameters such as flow-rate, mobile phase and column dimension must be carefully determined. One of the important parameters is the temperature of the mobile and stationary phases. The temperature affects mass transfer as well as adsorption/retention.In this study, we carried out linear gradient elution (LGE) experiments of proteins with ion-exchange chromatography (IEC) media over a wide range of temperature (288-308K). From the peak salt concentration IR as a function of normalized gradient slope GH the binding site B, the equilibrium coefficient Ke and IR at a fixed GH value were determined. The B value did not change appreciably with temperature. The temperature dependence of Ke was different from protein to protein. Consequently, there were three different retention behaviors, shifted to larger elution volumes, shift to smaller elution volumes and constant elution volume.The peak width was analyzed based on our method for determining HETP from LGE curves. The results were well correlated with the dimensionless velocity, ?=udp/Dm where dp=particle diameter, u=mobile phase velocity and Dm=molecular diffusion coefficient(determined by the Taylor dispersion method).The dynamic binding capacity DBC and static binding capacity SBC values of proteins onto IEC were also measured as a function of temperature. The DBC/SBC values were well correlated with the dimensionless variable udp2/(DsZ)  where Z= column bed height and Ds =stationary phase (pore) diffusion coefficient (determined from pulse response curves at non-binding conditions).These results are useful for optimizing the separation process and for predicting the variations of DBC or retention volume with temperature for the production process.

Temperature effects on chromatography performance of proteins and other biological products

247th National Meeting (2014)

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