Biothermodynamic Studies on the Adsorption of Monoclonal Antibodies to Chromatographic Separation Materials

Adsorption of therapeutic proteins on chromatographic media is a challenging but highly relevant research field, because downstream purification must meet strict purity and aggregate limits. Protein binding on ion exchange and hydrophobic interaction materials is complex, so process design is often still based on trial-and-error choices of parameters such as pH and salt type/concentration. A deeper physical and thermodynamic understanding is needed for rational process optimization. This work focuses on human monoclonal antibodies (hmAbs), which are increasingly important therapeutics for indications such as autoimmune diseases and cancer. Successful chromatography requires understanding both solution behavior and adsorption/desorption, since poor buffer conditions can reduce yield, solubility, and promote aggregation. The adsorption of two industrial hmAbs on strong cation exchange and hydrophobic resins was studied using isothermal titration calorimetry (ITC) and equilibrium adsorption isotherms, while solution properties of these hmAbs plus one mouse antibody were examined via static laser light scattering (SLS). Experiments systematically varied pH (4.5–7.0 for adsorption; 4.5–10 for SLS), buffer systems, and salt concentrations at 25 °C, and equilibrium data were fitted with models such as Langmuir as well as salt-dependence models (SMA and AA) that both described cation-exchange behavior well. ITC directly quantified heat effects during adsorption, enabling determination of the specific adsorption enthalpy and its dependence on loading. Near pH 7.0 adsorption was exothermic, with large, nearly constant enthalpy at low loading that decreased markedly at higher loading, indicating less favorable packing at high surface coverage. At around pH 5.0 the enthalpy was close to zero despite strong positive antibody charge, and at pH 4.5 endothermic effects occurred even with high binding capacities, consistent with ion release from the antibody influencing the heat signal. From isotherms, adsorption equilibrium constants were used to derive Gibbs energy and entropy changes under different conditions. SLS provided second virial coefficients and mass-average molecular masses; positive virial coefficients at low pH/low salt reflected electrostatic repulsion, decreasing toward the isoelectric point where attractions reduce solubility, while higher salt promoted salting-out, negative virial coefficients, and aggregation accompanied by changes in molecular mass.