Model for Counterion Binding and Charge Reversal on Protein Surfaces

Abstract

The structural stability and solubility of proteins in liquid therapeutic formulations is important, especially since new generations of therapeutics are designed for efficacy before consideration of stability. We introduce an electrostatic binding model to measure the net charge of proteins with bound ions in solution. The electrostatic potential on a protein surface is used to separately group together acidic and basic amino acids into patches, which are then iteratively bound with oppositely charged counterions. This model is aimed toward formulation chemists for initial screening of a range of conditions prior to lab-work. Computed results compare well with experimental zeta potential measurements from the literature covering a range of solution conditions. Importantly, the binding model reproduces the charge reversal phenomenon that is observed with polyvalent ion binding to proteins and its dependence on ion charge and concentration. Intriguingly, protein sequence can be used to give similarly good agreement with experiment as protein structure, interpreted as resulting from the close proximity of charged side chains on a protein surface. Further, application of the model to human proteins suggests that polyanion binding and overcharging, including charge reversal for cationic proteins, is a general feature. These results add to evidence that addition of polyanions to protein formulations could be a general mechanism for modulating solution stability.

Isolation and Self-Association Studies of Beta-Lactoglobulin

“…In this way, citrate anion stabilizes more evidently the β-LG especially close to its electric equivalence. According to the electro kinetic theory, the higher valance ions bind more strongly to the protein surface than the monovalent ones and neutralize the protein charge more effectively [ 69 ]. The thicker Stern layer has impact on the diffuse layer.…”

Formulations That Suppress Aggregation During Long-Term Storage of a Bispecific Antibody are Characterized by High Refoldability and Colloidal Stability

“…The formulations containing 10 mM citrate without added sodium chloride also have relatively low ionic strength but exhibit much lower k D compared to histidine counterparts. These effects can be explained by the binding of the citrate ion to the protein that inverts protein surface charge and diminishes the electrostatic repulsion 33,34 . The addition of 70 mM NaCl levels out the differences in k D of Bis-mAb in the two different buffers (see Figure 2) due to screening of the electrostatic interactions 35 .…”

Thermodynamic Origin of Differential Excipient-Lysozyme Interactions

“…From Figure 5A, both polyanions have similar numbers of contacts in similar regions of positively charged patches on the protein surface. Patches are generated on the protein using Poisson-Boltzmann electrostatics as described in previous work (Kalayan et al, 2020). CIT has overall weaker interactions over the protein surface, while TPP interacts with fewer regions of the protein but with higher occupancy.…”