Dipolar Interactions and Protein Hydration in Highly Concentrated Antibody Formulations.

Molecular interaction mechanisms in high-concentrated protein systems are of fundamental importance for the rational development of biopharmaceuticals such as monoclonal antibody (mAb) formulations. In such high-concentrated protein systems, the intermolecular distances between mAb molecules are reduced to the size of the protein diameter (approx. 10 nm). Thus, protein-protein interactions are more pronounced at high concentrations; so a direct extrapolation of physicochemical properties obtained from measurements at a low protein concentration of the corresponding properties at a high protein concentration is highly questionable. Besides the charge-charge interaction, the effects of molecular crowding, dipolar interaction, changes in protein hydration, and self-assembling tendency become more relevant. Here, protein hydration, protein dipole moment, and protein-protein interactions were studied in protein concentrations up to 200 mg/mL (= 1.3 mM) in different formulations for selected mAbs using dielectric relaxation spectroscopy (DRS). These data are correlated with the second virial coefficient, A 2 , the diffusion interaction parameter, k D , the elastic shear modulus, G' , and the dynamic viscosity, η. When large contributions of dipolar protein-protein interactions were observed, the tendency of self-assembling and an increase in solution viscosity were detected. These effects were examined using specific buffer conditions. Furthermore, different types of protein-water interactions were identified via DRS, whereby the effect of high protein concentration on protein hydration was investigated for different high-concentrated liquid formulations (HCLFs).