Beyond uniform screening: electrostatic heterogeneity dictates solution structure of complex macromolecules

TL;DR

This work tackles how electrostatic heterogeneity, beyond net charge, governs the solution structure of complex macromolecules, focusing on two monoclonal antibodies with uniform and patchy surface charges. It couples atomistic detail to a two-tier coarse-grained model and directly compares implicit Yukawa-based descriptions to explicit-ion simulations against SAXS, SLS, and DLS data, as well as PMF analyses. The key finding is that implicit, screened-Coulomb models fail for strongly heterogeneous charge patterns, while explicit ions reproduce both single-molecule and collective solution properties, providing a quantitative framework for predicting antibody behavior in solution. The results have practical implications for therapeutic formulation and the broader design of predictive models for patchy colloids and biomolecules, highlighting the need to incorporate higher-order electrostatic effects and concentration-dependent screening when charge heterogeneity is significant.

Abstract

The complexity of biomolecular interactions necessitates advanced methodologies to accurately capture their behavior in solution. In this work, we focus on monoclonal antibodies and adopt a multi-scale coarse-graining strategy for their modeling, with particular emphasis on the role of electrostatic interactions. Using scattering experiments, theoretical analysis, and large-scale computer simulations, we explicitly compare two selected case studies-markedly different in their charge distributions. Through mutually corroborating lines of evidence, we demonstrate that conventional approaches relying on electrostatic screening and implicit charge representations fail to capture the structural and thermodynamic properties of antibody solutions when strong charge heterogeneity is present, even at a moderate (amino acid) level of coarse-graining. These findings highlight the importance of a correct treatment of electrostatic interactions and ion screening for heterogeneously- and oppositely-charged colloidal and protein systems. Such considerations are essential to move beyond descriptive models towards a truly predictive framework, with direct implications for the formulation of therapeutics and the treatment of other complex soft-matter systems.

Figures (12)

• Figure 1: Antibodies under investigation. (a) Amino acid representation of the antibody and (b) respective electrostatic isopotential surface (EIS) at $\pm 0.75 k_\mathrm{B}T/e$ calculated with a Poisson-Boltzmann solver for (I) mAb-U and (II) mAb-H from two different perspectives, indicated by $\otimes z$ and $\odot z$. (c) Representative simulation snapshot showing multiple antibodies at the amino acid level. Colored are the beads with charges $\pm 1$, while different shades of grey are for different molecules. For visual clarity, ions and counterions are not drawn to scale, and their number is reduced relative to the actual simulations. The inset shows two representative antibodies taken from the simulation box. In all panels, blue (red) represents positive (negative) charges.
• Figure 2: Experimental characterization. (Left) Osmotic compressibility $S(0)$ and (right) apparent hydrodynamic radius $R_\mathrm{h,app}$ as a function of the antibody concentration $c$ for mAb-U (squares) and mAb-H (circles). Also reported is the theoretical prediction for excluded volume interactions (hs, dashed line).
• Figure 3: Solution behavior for the implicit ions model. Static structure factors $S(q)$ for (a) mAb-U and (b) mAb-H for simulations (sim) with implicit ions models (Yukawa) and for SAXS experiments (exp) for $c=20$ and $100$ mg/ml.
• Figure 4: Solution behavior for the explicit ions model. Static structure factors $S(q)$ for mAb-H for simulations (sim) with explicit ions models (Coulomb) and for SAXS experiments (exp) for $c=20$ and $100$ mg/ml. The experimental data points are the same as in Fig. \ref{['fig:sq_yuk']}b.
• Figure 5: Comparison between PB and Yukawa representation. Mesh representation of the electrostatic isopotential surfaces (EIS) at $\pm 0.75 k_\mathrm{B}T/e$ accounting for (top) Poisson-Boltzmann and (bottom) Yukawa representations for (left) mAb-U and (right) mAb-H from two different perspectives, indicated by $\otimes z$ and $\odot z$.