IgG Charge: Practical and Biological Implications

Abstract

Practically, IgG charge can contribute significantly to thermodynamic nonideality, and hence to solubility and viscosity. Biologically, IgG charge isomers exhibit differences in clearance and potency. It has been known since the 1930s that all immunoglobulins carry a weak negative charge in physiological solvents. However, there has been no systematic exploration of this fundamental property. Accurate charge measurements have been made using membrane confined electrophoresis in two solvents (pH 5.0 and pH 7.4) on a panel of twelve mAb IgGs, as well as their F(ab’)2 and Fc fragments. The following observations were made at pH 5.0: (1) the measured charge differs from the calculated charge by ~40 for the intact IgGs, and by ~20 for the Fcs; (2) the intact IgG charge depends on both Fv and Fc sequences, but does not equal the sum of the F(ab)’2 and Fc charge; (3) the Fc charge is consistent within a class. In phosphate buffered saline, pH 7.4: (1) the intact IgG charges ranged from 0 to −13; (2) the F(ab’)2 fragments are nearly neutral for IgG1s and IgG2s, and about −5 for some of the IgG4s; (3) all Fc fragments are weakly anionic, with IgG1 < IgG2 < IgG4; (4) the charge on the intact IgGs does not equal the sum of the F(ab’)2 and Fc charge. In no case is the calculated charge, based solely on H+ binding, remotely close to the measured charge. Some mAbs carried a charge in physiological salt that was outside the range observed for serum-purified human poly IgG. To best match physiological properties, a therapeutic mAb should have a measured charge that falls within the range observed for serum-derived human IgGs. A thermodynamically rigorous, concentration-dependent protein–protein interaction parameter is introduced. Based on readily measured properties, interaction curves may be generated to aid in the selection of proteins and solvent conditions. Example curves are provided.

Designing Feature-Controlled Humanoid Antibody Discovery Libraries Using Generative Adversarial Networks

“…For antibody therapeutics, the isoelectric point (pI, the pH at which the molecule is neutral) is a key measure of developability since a pI near the formulation pH may lead to high viscosity and aggregation or precipitation. [61][62][63][64] A slightly acidic pH generally results in a higher overall charge leading to more recent formulations centered around pH 5.5. 65 To remain stable in solution, therapeutic antibodies would ideally need to have a pI of greater than 8.5 for the overall molecule.…”

Stress Factors in mAb Drug Substance Production Processes: Critical Assessment of Impact on Product Quality and Control Strategy

“…Nevertheless, depending on temperature, the type of salt (Hofmeister series), salt concentration, and protonation state of the protein (net positive charge at pH < pI; net neutral charge at pHp I; net negative charge at pH > pI), complex scenarios of solubility reduction and solubility enhancement ("salting in") have been reported. [89][90][91][92][93][94] The addition of (chaotropic) salts 86,95,96 or charged excipients, for example, amino acids with a hydrophobic moiety such as histidine and arginine 96,97 are reported to lower the tendency to form associates, which in turn also lowers the viscosity of the solution. Similarly, reversible association that leads to liquidliquid phase separation was recently reported to follow similar trends 92,[98][99][100][101] ; this is seen especially at low temperatures, for example, during intermediate storage during DSP.…”

The Molecular Interaction Process

“…It is important to note that due to monovalent ion binding, charge measurement is required to determine the extent of charge-charge repulsion. 66,70,73 The salt dependence of protein solubility and viscosity studies may seem contradictory at times: some proteins become more soluble or exhibit lower intrinsic viscosity with increasing salt, 7,24 whereas other proteins exhibit lower solubility or higher viscosity or lower solubility with increasing salt. [74][75][76] However, proteinprotein interactions include both repulsive (charge-charge) and attractive (charge-dipole, dipole-dipole) interactions, and these contributions have different distance dependencies.…”

“…Is it possible, however, that the unfavorable process of anion desolvation may contribute significantly to antibody solubility? Perhaps anion binding, which is pronounced for IgGs (~0.7 Cl À bound per H þ ), 73 may account for the relatively high solubility of a protein that carries a very low charge (Z DHH~À 6) in physiological salt. 73,91 Perhaps anion binding explains why all naturally occurring immunoglobulins are anionic in physiological conditions.…”

“…Perhaps anion binding, which is pronounced for IgGs (~0.7 Cl À bound per H þ ), 73 may account for the relatively high solubility of a protein that carries a very low charge (Z DHH~À 6) in physiological salt. 73,91 Perhaps anion binding explains why all naturally occurring immunoglobulins are anionic in physiological conditions. 91,92 Furthermore, unfavorable anion displacement may provide a general mechanism for maintaining the solubility of serum antibodies, which physiologically are heterogeneous, and prone to changing composition and concentrations.…”