Coacervation and Binding Affinity of Poly(glutamate)-Monoclonal Antibody Complexes: Insights from Experiments and Theoretical Modeling.

The complex coacervation of poly(glutamate) (PRE) with monoclonal IgG antibodies (mAbs) enables the formulation of stable, concentrated therapeutic antibody preparations with reduced viscosity [1]. Determining whether the molecular interactions responsible for complexation are specific to the antibody sequence or if PRE can broadly bind to various monoclonal antibodies (mAbs) is essential for pharmaceutical applications. To address this, we measured the binding affinities of cetuximab as a function of pH and ionic strength and compared the results with a semi-qualitative theoretical analysis based on data from constant-pH simulations. Cetuximab, a commercial IgG with a publicly available sequence, serves as a representative therapeutic antibody. At low mAb concentrations (< 0.1 g/L), a simple 1:1 stoichiometry was expected and confirmed through electrophoretic mobility measurements at varying PRE concentrations (via capillary zone electrophoresis). The absence of oligomers (indicating the predominance of single mAb:PRE complexes without multiple binding) was verified using Taylor dispersion analysis. Experimental data reveal a clear maximum in binding strength at acidic pH, well below the pI of cetuximab (pH 8.6), suggesting the involvement of counterion release (1–2 per binding event).

Using AlphaFold2-generated structural models of cetuximab, we computed key physicochemical parameters, including net charge and charge regulation capacity, as a function of pH. Coulombic association energy was estimated analytically based on these parameters. The sharp decrease in binding affinity above pH 6 is attributed to a dominant Coulombic mechanism, with small contributions from charge regulation under conditions of maximal association (acidic pH). At higher mAb concentrations (> 1 g/L), we observed phase separation, leading to mAb:PRE coacervate formation with a similar pH optimum.

[1] A. Lapenna et al., Mol. Pharm. 2024, 21(2), 982 ; Tsumura, K. et al., J. Biosci. Bioeng. 133(1): 17.

Fundings: LabEx DYNAMO ANR-11-LABEX-0011-01 Fapesp-ANR (2020/07158-2) and CNPq (305393/2020-0).