Microcalorimetric study of adsorption of human monoclonal antibodies on cation exchange chromatographic materials

Adsorption of two human monoclonal antibodies on two different strong cation exchange resins is studied by isothermal titration microcalorimetry and independent adsorption isotherm measurements. The pH value is varied between 4.5 and 7.0, using different buffer systems, the temperature is always 25 °C. The adsorption isotherm data is fitted using two different Langmuir type models. Combining the calorimetric and the adsorption data, the specific enthalpy of adsorption of the protein Δ h p ads is determined. At pH values near 7.0, where the antibodies are only weakly charged, the adsorption is exothermal. At small loadings the absolute number of Δ h p ads is then large and almost constant but it significantly decreases at higher loadings. This shows that the arrangement of antibody molecules on the absorber material depends on the loading and is less favourable at higher loadings. Despite the high positive charge of the antibody at pH values of about 5 the value of Δ h p ads is almost zero along the entire isotherm. Furthermore, at pH 4.5 even endothermal effects are observed, although high binding capacities are found. At these conditions the adsorption process seems to be strongly influenced by the ions bound to the antibody. Their release upon absorption explains the endothermal caloric effect. The adsorption equilibrium constant Keq is calculated from the isotherms. From Δ g p ads and the calorimetric results for Δ h p ads , Δ s p ads , the entropy change upon adsorption of the protein is found for the different studied conditions.