ASSESSMENT OF pH AND IONIC STRENGTH ON THE BIOPHYSICAL PROPERTIES OF A MONOCLONAL ANTIBODY SOLUTION

To successfully develop and manufacture stable biologic therapeutics and vaccines, it is paramount to understand the effects of pH and ionic strength on the intra and intermolecular biophysical properties of the protein solution. This work investigates the effects of pH and ionic strength on the biophysical properties of the broadly neutralizing HIV antibody, N6LS, which was recently discovered by the National Institutes of Health. The secondary and tertiary structure were determined by circular dichroism and intrinsic fluorescence spectroscopy and were found to be independent of solution pH and ionic strength. The conformational stability was quantified by differential scanning calorimetry; both solution parameters had pronounced effect on antibody thermal unfolding transitions and enthalpy of unfolding. To quantify protein-protein interactions and colloidal properties of the antibody solution, the diffusion interaction parameter and osmotic second virial coefficient were determined by dynamic light scattering and static light scattering and found to be dependent on pH and ionic strength. Additionally, antibody solution viscosity was measured and used to determine the intrinsic viscosity and Huggins coefficient. Intrinsic viscosity and Huggins coefficient data derived from viscosity experiments showed that N6LS antibody molecules have anisotropic conformation and behaved more like flexible polymer chains than hard spheres at pH ≥ 5.0. This work can help inform future protein biophysical and theoretical models for antibody solutions, as well as inform formulation solution conditions in manufacturing and development of future biologic therapeutics and vaccines.