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 degrees 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 Deltah(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 Deltah(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 Deltah(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 K(eq) is calculated from the isotherms. From Deltag(p)(ads) and the calorimetric results for Deltah(p)(ads), Deltas(p)(ads), the entropy change upon adsorption of the protein is found for the different studied conditions.
Dieterle
Michael
M
Institute of Thermodynamics and Thermal Process Engineering, Universität Stuttgart, Germany.
Blaschke
Tim
T
Hasse
Hans
H
D000327:Adsorption; D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D002151:Calorimetry / Q000379:methods; D002411:Cation Exchange Resins; D002852:Chromatography, Ion Exchange / Q000379:methods*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D013816:Thermodynamics
D000911:Antibodies, Monoclonal; D002411:Cation Exchange Resins