Structure of a Therapeutic Full-Length Anti-NPRA IgG4 Antibody: Dissecting Conformational Diversity.

We report the x-ray crystal structure of intact, full-length human immunoglobulin (IgG4) at 1.8 Å resolution. The data for IgG4 (S228P), an antibody targeting the natriuretic peptide receptor A, show a previously unrecognized type of Fab-Fc orientation with a distorted λ-shape in which one Fab-arm is oriented toward the Fc portion. Detailed structural analysis by x-ray crystallography and molecular simulations suggest that this is one of several conformations coexisting in a dynamic equilibrium state. These results were confirmed by small angle x-ray scattering in solution. Furthermore, electron microscopy supported these findings by preserving molecule classes of different conformations. This study fosters our understanding of IgG4 in particular and our appreciation of antibody flexibility in general. Moreover, we give insights into potential biological implications, specifically for the interaction of human anti-natriuretic peptide receptor A IgG4 with the neonatal Fc receptor, Fcγ receptors, and complement-activating C1q by considering conformational flexibility.

Improved Solution-State Properties of Monoclonal Antibodies by Targeted Mutations.

Monoclonal antibody (mAb)-based therapeutics often require high-concentration formulations. Unfortunately, highly concentrated antibody solutions often have biophysical properties that are disadvantageous for therapeutic development, such as high viscosity, solubility limitations, precipitation issues, or liquid-liquid phase separation. In this work, we present a computational rational design principle for improving the thermodynamic stability of mAb solutions through targeted point mutations. Two publicly available IgG1 monoclonal antibodies that exhibit high viscosity at high concentrations were used as model systems. Guided by a computationally efficient approach that combines molecular dynamics simulations with three-dimensional reference interaction site model theory, point mutations of charged residues were introduced in the variable Fv regions in such a manner that the hydration free energy was optimized. Two selected point mutants were then produced by transient expression and characterized experimentally. Both engineered mAbs have reduced viscosity at high concentration, less negative second virial coefficient, and improved solubility compared to the respective wild-types. The results obtained with the suggested straightforward design principle underline the relevance of solvation effects for understanding, and ultimately optimizing, the properties of highly concentrated mAb solutions, with possible implications also for other biomolecular systems.

High-concentration protein formulations: How high is high?

High-concentration protein formulation (HCPF) is a term that is used to describe protein formulations, mostly monoclonal antibody (mAb) drugs, at high protein concentration. The concentration is rarely defined, with typical ranges varying between 50 and 150mg/ml for mAbs. The term HCPF is meant to include and express specific solution properties of formulations that are prone to appear at high protein concentrations such as high viscosity, high opalescence, phase separation, gel formation or the increased propensity for protein particle formation. Thus the term HCPF can be understood as a descriptor of protein formulations, usually at high protein (monoclonal antibody) concentrations, which have specific solution, stability and colloidal properties that differ from formulations at low protein concentration (e.g. at 10mg/ml). The current paper highlights in brief the development challenges that might occur for high-concentration protein/monoclonal antibody formulations. In particular, the maximum concentration regimes achievable in HCPF remained unclear. Based on geometrical considerations involving packing of monoclonal antibodies in a lattice we map out a maximum concentration range that might be theoretically achievable. Different geometrical assumptions and packing models are compared and their relevance is critically discussed, in particular concerning the influence of the physicochemical properties of the monoclonal antibodies on their solubility, which is neglected in the simple geometrical model. According to our estimates, monoclonal antibody concentration above 500mg/ml will be very challenging to achieve. Our results have implications for setting up realistic drug product development strategies and for preparing convincing drug target product profiles for development.

On Using Atomistic Solvent Layers in Hybrid All-Atom/Coarse-Grained Molecular Dynamics Simulations.

Hybrid all-atom/coarse-grained (AA-CG) simulations in which AA solutes are embedded in a CG environment can provide a significant computational speed-up over conventional fully atomistic simulations and thus alleviate the current length and time scale limitations of molecular dynamics (MD) simulations of large biomolecular systems. On one hand, coarse graining the solvent is particularly appealing, since it typically constitutes the largest part of the simulation system and thus dominates computational cost. On the other hand, retaining atomic-level solvent layers around the solute is desirable for a realistic description of hydrogen bonds and other local solvation effects. Here, we devise and systematically validate fixed resolution AA-CG schemes, both with and without atomistic water layers. To quantify the accuracy and diagnose possible pitfalls, Gibbs free energies of solvation of amino acid side chain analogues were calculated, and the influence of the nature of the CG solvent surrounding (polarizable vs nonpolarizable CG water) and the size of the AA solvent region was investigated. We show that distance restraints to keep the AA solvent around the solute lead to too high of a density in the inner shell. Together with a long-ranged effect due to orientational ordering of water molecules at the AA-CG boundary, this affects solvation free energies. Shifting the onset of the distance restraints slightly away from the central solute significantly improves solvation free energies, down to mean unsigned errors with respect to experiment of 2.3 and 2.6 kJ/mol for the polarizable and nonpolarizable CG water surrounding, respectively. The speed-up of the nonpolarizable model renders it computationally more attractive. The present work thus highlights challenges, and outlines possible solutions, involved with modeling the boundary between different levels of resolution in hybrid AA-CG simulations.

Systematic evaluation of bundled SPC water for biomolecular simulations.

In bundled SPC water models, the relative motion of groups of four water molecules is restrained by distance-dependent potentials. Bundled SPC models have been used in hybrid all-atom/coarse-grained (AA/CG) multiscale simulations, since they enable to couple atomistic SPC water with supra-molecular CG water models that effectively represent more than a single water molecule. In the present work, we systematically validated and critically tested bundled SPC water models as solvent for biomolecular simulations. To that aim, we investigated both thermodynamic and structural properties of various biomolecular systems through molecular dynamics (MD) simulations. Potentials of mean force of dimerization of pairs of amino acid side chains as well as hydration free energies of single side chains obtained with bundled SPC and standard (unrestrained) SPC water agree closely with each other and with experimental data. Decomposition of the hydration free energies into enthalpic and entropic contributions reveals that in bundled SPC, this favorable agreement of the free energies is due to a larger degree of error compensation between hydration enthalpy and entropy. The Ramachandran maps of Ala3, Ala5, and Ala7 peptides are similar in bundled and unrestrained SPC, whereas for the (GS)2 peptide, bundled water leads to a slight overpopulation of extended conformations. Analysis of the end-to-end distance autocorrelation times of the Ala5 and (GS)2 peptides shows that sampling in more viscous bundled SPC water is about two times slower. Pronounced differences between the water models were found for the structure of a coiled-coil dimer, which is instable in bundled SPC but not in standard SPC. In addition, the hydration of the active site of the serine protease α-chymotrypsin depends on the water model. Bundled SPC leads to an increased hydration of the active site region, more hydrogen bonds between water and catalytic triad residues, and a significantly slower exchange of water molecules between the active site and the bulk. Our results form a basis for assessing the accuracy that can be expected from bundled SPC water models. At the same time, this study also highlights the importance of evaluating beforehand the effects of water bundling on the biomolecular system of interest for a particular multiscale simulation application.