The effects of intramolecular and intermolecular electrostatic repulsions on the stability and aggregation of <scp>NISTmAb</scp> revealed by <scp>HDX‐MS</scp>, <scp>DSC,</scp> and <scp>nanoDSF</scp>
Yoshitomo Hamuro, M.G. Derebe, Sathya Venkataramani, Jennifer F. Nemeth
Abstract
Abstract The stability and aggregation of NIST monoclonal antibody (NISTmAb) were investigated by hydrogen/deuterium exchange mass spectrometry (HDX‐MS), differential scanning calorimetry (DSC), and nano‐differential scanning fluorimetry (nanoDSF). NISTmAb was prepared in eight formulations at four different pH s ( pH 5, 6, 7, and 8) in the presence and absence of 150 mM NaCl and analyzed by the three methods. The HDX‐MS results showed that NISTmAb is more conformationally stable at a p H near its isoelectric point (p I ) in the presence of NaCl than a p H far from its p I in the absence of NaCl. The stabilization effects were global and not localized. The midpoint temperature of protein thermal unfolding transition results also showed the C H 2 domain of the protein is more conformationally stable at a pH near its p I . On the other hand, the onset of aggregation temperature results showed that NISTmAb is less prone to aggregate at a p H far from its p I , particularly in the absence of NaCl. These seemingly contradicting results, higher conformational stability yet higher aggregation propensity near the p I than far away from the p I , can be explained by intramolecular and intermolecular electrostatic repulsion using Lumry‐Eyring model, which separates folding/unfolding equilibrium and aggregation event. The further a p H from the p I , the higher the net charge of the protein. The higher net charge leads to greater intramolecular and intermolecular electrostatic repulsions. The greater intramolecular electrostatic repulsion destabilizes the protein and the greater intermolecular electrostatic repulsion prevents aggregation of the protein molecules at p H far from the p I .