Global multi-method analysis of interaction parameters for reversibly self-associating macromolecules at high concentrations

Abstract

Weak macromolecular interactions assume a dominant role in the behavior of highly concentrated solutions, and are at the center of a variety of fields ranging from colloidal chemistry to cell biology, neurodegenerative diseases, and manufacturing of protein drugs. They are frequently measured in different biophysical techniques in the form of second virial coefficients, and nonideality coefficients of sedimentation and diffusion, which may be related mechanistically to macromolecular distance distributions in solution and interparticle potentials. A problem arises for proteins where reversible self-association often complicates the concentration-dependent behavior, such that grossly inconsistent coefficients are measured in experiments based on different techniques, confounding quantitative conclusions. Here we present a global multi-method analysis that synergistically bridges gaps in resolution and sensitivity of orthogonal techniques. We demonstrate the method with a panel of monoclonal antibodies exhibiting different degrees of self-association. We show how their concentration-dependent behavior, examined by static and dynamic light scattering and sedimentation velocity, can be jointly described in a self-consistent framework that separates nonideality coefficients from self-association properties, and thereby extends the quantitative interpretation of nonideality coefficients to probe dynamics in highly concentrated protein solutions.

Competing stress-dependent oligomerization pathways regulate self-assembly of the periplasmic protease-chaperone DegP

“…For self-assembling systems such as DegP, the situation becomes more complicated, as multiple species can coexist in solution. Yet in these cases, D z values provide a rich source of information on the oligomerization process as they are highly sensitive to trace amounts of large particles (30)(31)(32). Most importantly, the nature of an underlying oligomeric distribution can be read out in a model-free manner through variations in D z values that are derived from changes in experimental variables such as M T , temperature, or solution ionic strength.…”

The Relationship between Protein–Protein Interactions and Liquid–Liquid Phase Separation for Monoclonal Antibodies

“…In the Supporting Information, we show that the limiting form for the slope of the diffusion coefficient at low protein concentration, which we denote as k D , is given by k D = k − 4 F K dim Here, k D,mm is the interaction parameter between a pair of monomers reflecting only the nonspecific protein–protein interactions, and F is a hydrodynamic factor given by F = 1 – R H,m / R H,d where R H,m and R H,d are the hydrodynamic radii of the monomer and dimer species, respectively. Equation was also derived by Parupudi et al, except in their derivation, k D,mm is replaced by an average k D including monomer–dimer and dimer–dimer interactions. These terms do not appear in our expression because they relate to three-body and four-body interactions.…”

“…There is evidence that some mAb solution properties are controlled by anisotropic protein–protein interactions. Modeling of thermodynamic properties has required invoking existence of an oligomeric equilibrium commonly referred to as reversible self-association, which is only possible if protein–protein interactions are highly directional. − The presence of oligomers has been detected from neutron spin echo measurements , or from analytical ultracentrifugation experiments complemented with dynamic and static light scattering. − In addition, in many instances, coarse-grained Y-shaped bead models of antibodies with bead-specific interactions provide better fits to structure factor profiles and osmotic compressibility data than isotropic spherical models or Y-shaped bead models with uniform bead–bead attractions. ,− The anisotropic models form transient protein clusters at higher protein concentrations, whose properties have been correlated with the concentrated solution viscosity. ,,,− The molecular basis for anisotropic interactions has been elucidated particularly in solutions at low ionic strength and pH near the isoelectric point. Under these conditions, many mAbs exhibit anisotropic electrostatic attractions, ,,,, which have been characterized through rational mutagenesis to disrupt charged patches on mAbs causing significant changes to measured values of either b 22 or the diffusion interaction parameter k D . − This would only be possible if b 22 or k D is determined by a small number of directional protein–protein interactions.…”

“… 42 − 45 The presence of oligomers has been detected from neutron spin echo measurements 46 , 47 or from analytical ultracentrifugation experiments complemented with dynamic and static light scattering. 48 − 50 In addition, in many instances, coarse-grained Y-shaped bead models of antibodies with bead-specific interactions provide better fits to structure factor profiles and osmotic compressibility data than isotropic spherical models or Y-shaped bead models with uniform bead–bead attractions. 41 , 51 − 54 The anisotropic models form transient protein clusters at higher protein concentrations, whose properties have been correlated with the concentrated solution viscosity.…”

Protein–protein interactions in solutions of monoclonal antibodies probed by the dependence of the high-frequency viscosity on temperature and concentration

“…[34][35][36] In concentrated solutions of mAbs, short-ranged attractions lead to cluster formation, which is not well captured by B 22 or k D . 37,38 In this range, the viscosity is often used to probe PPIs, recently reviewed by in ref. 15.…”