Effects of Salts and Surface Charge on the Biophysical Stability of a Low pI Monoclonal Antibody

The impact of five representative Hofmeister salts (NaCl, KCl, MgCl2, Na2SO4, and NaSCN) on the thermal stability and aggregation kinetics of a slightly acidic monoclonal antibody (mAb) were investigated under different pH conditions. The thermal stability of the mAb was assessed by measuring the lowest unfolding transition temperature, Tm, with differential scanning fluorimetry. MgCl2 and NaSCN significantly decreased Tm at all three charged states of the mAb, but to the greatest extent when the mAb surface charge was net positive. Non-native aggregation kinetics was monitored by measuring Rayleigh light scattering. When the mAb surface charge was net positive or net neutral, the nucleation rate increased non-monotonically with MgCl2 and NaSCN but decreased monotonically with NaCl, KCl, and Na2SO4. By contrast, when the mAb surface was negatively charged, there were only minor changes in the nucleation rate with all salts tested. Furthermore, there was less structural perturbation and slower aggregation rates when the mAb was net negatively charged than when it was net neutrally or positively charged. The observed salt effects on thermal unfolding are consistent with ion-specific mechanisms dominated by short-range amide backbone binding. On the other hand, the salt effects on nucleation rates appear to be influenced by both amide backbone binding and long-range electrostatic binding of ions to charged amino acid side chains.