Unraveling the Microscopic Mechanism of Molecular Ion Interaction with Monoclonal Antibodies: Impact on Protein Aggregation.

Understanding and predicting protein aggregation represents one of the major challenges in accelerating the pharmaceutical development of protein therapeutics. In addition to maintaining the solution pH, buffers influence both monoclonal antibody (mAb) aggregation in solution and the aggregation mechanisms since the latter depend on the protein charge. Molecular-level insight is necessary to understand the relationship between the buffer-mAb interaction and mAb aggregation. Here, we use all-atom molecular dynamics simulations to investigate the interaction of phosphate (Phos) and citrate (Cit) buffer ions with the Fab and Fc domains of mAb COE3. We demonstrate that Phos and Cit ions feature binding mechanisms, with the protein that are very different from those reported previously for histidine (His). These differences are reflected in distinctive ion-protein binding modes and adsorption/desorption kinetics of the buffer molecules from the mAb surface and result in dissimilar effects of these buffer species on mAb aggregation. While His shows significant affinity toward hydrophobic amino acids on the protein surface, Phos and Cit ions preferentially bind to charged amino acids. We also show that Phos and Cit anions provide bridging contacts between basic amino acids in neighboring proteins. The implications of such contacts and their connection to mAb aggregation in therapeutic formulations are discussed.

Mechanistic Insights into the Adsorption of Monoclonal Antibodies at the Water/Vapor Interface.

Monoclonal antibodies (mAbs) are active components of therapeutic formulations that interact with the water-vapor interface during manufacturing, storage, and administration. Surface adsorption has been demonstrated to mediate antibody aggregation, which leads to a loss of therapeutic efficacy. Controlling mAb adsorption at interfaces requires a deep understanding of the microscopic processes that lead to adsorption and identification of the protein regions that drive mAb surface activity. Here, we report all-atom molecular dynamics (MD) simulations of the adsorption behavior of a full IgG1-type antibody at the water/vapor interface. We demonstrate that small local changes in the protein structure play a crucial role in promoting adsorption. Also, interfacial adsorption triggers structural changes in the antibody, potentially contributing to the further enhancement of surface activity. Moreover, we identify key amino acid sequences that determine the adsorption of antibodies at the water-air interface and outline strategies to control the surface activity of these important therapeutic proteins.

Understanding the Stabilizing Effect of Histidine on mAb Aggregation: A Molecular Dynamics Study.

Histidine, a widely used buffer in monoclonal antibody (mAb) formulations, is known to reduce antibody aggregation. While experimental studies suggest a nonelectrostatic, nonstructural (relating to secondary structure preservation) origin of the phenomenon, the underlying microscopic mechanism behind the histidine action is still unknown. Understanding this mechanism will help evaluate and predict the stabilizing effect of this buffer under different experimental conditions and for different mAbs. We have used all-atom molecular dynamics simulations and contact-based free energy calculations to investigate molecular-level interactions between the histidine buffer and mAbs, which lead to the observed stability of therapeutic formulations in the presence of histidine. We reformulate the Spatial Aggregation Propensity index by including the buffer-protein interactions. The buffer adsorption on the protein surface leads to lower exposure of the hydrophobic regions to water. Our analysis indicates that the mechanism behind the stabilizing action of histidine is connected to the shielding of the solvent-exposed hydrophobic regions on the protein surface by the buffer molecules.

Breaking Isolation to Form New Networks: pH-Triggered Changes in Connectivity inside Lipid Nanoparticles.

There is a growing demand to develop smart nanomaterials that are structure-responsive as they have the potential to offer enhanced dose, temporal and spatial control of compounds and chemical processes. The naturally occurring pH gradients found throughout the body make pH an attractive stimulus for guiding the response of a nanocarrier to specific locations or (sub)cellular compartments in the body. Here we have engineered highly sensitive lyotropic liquid crystalline nanoparticles that reversibly respond to changes in pH by altering the connectivity within their structure at physiological temperatures. At pH 7.4, the nanoparticles have an internal structure consisting of discontinuous inverse micellar "aqueous pockets" based on space group Fd3m. When the pH is ≤6, the nanoparticles change from a compartmentalized to an accessible porous internal structure based on a 2D inverse hexagonal phase (plane group p6mm). We validate the internal symmetry of the nanoparticles using small-angle X-ray scattering and cryogenic transmission electron microscopy. The high-resolution electron microscopy images obtained have allowed us for the first time to directly visualize the internal structure of the Fd3m nanoparticles and resolve the two different-sized inverse micelles that make up the structural motif within the Fd3m unit cell, which upon structural analysis reveal excellent agreement with theoretical geometrical models.

The effect of headgroup methylation on polymorphic phase behaviour in hydrated N-methylated phosphoethanolamine:palmitic acid membranes.

Mixtures of fatty acids and phospholipids can form hexagonal (HII) and inverse bicontinuous cubic phases, the latter of which are implicated in various cellular processes and have wide-ranging biotechnological applications in protein crystallisation and drug delivery systems. Therefore, it is vitally important to understand the formation conditions of inverse bicontinuous cubic phases and how their properties can be tuned. We have used differential scanning calorimetry and synchrotron-based small angle and wide angle X-ray scattering (SAXS/WAXS) to investigate the polymorphic phase behaviour of palmitic acid/partially-methylated phospholipid mixtures, and how headgroup methylation impacts on inverse bicontinuous cubic phase formation. We find that upon partial methylation of the phospholipid headgroup (1 or 2 methyl substituents) inverse bicontinuous cubic phases are formed (of the Im3m spacegroup), which is not the case with 0 or 3 methyl substituents. This shows how important headgroup methylation is for controlling phase behaviour and how a change in headgroup methylation can be used to controllably tune various inverse bicontinuous phase features such as their lattice parameter and the temperature range of their stability.

Microfluidic technologies for the synthesis and manipulation of biomimetic membranous nano-assemblies.

Microfluidics has been proposed as an attractive alternative to conventional bulk methods used in the generation of self-assembled biomimetic structures, particularly where there is a desire for more scalable production. The approach also allows for greater control over the self-assembly process, and parameters such as particle architecture, size, and composition can be finely tuned. Microfluidic techniques used in the generation of microscale assemblies (giant vesicles and higher-order multi-compartment assemblies) are fairly well established. These tend to rely on microdroplet templation, and the resulting structures have found use as comparmentalised motifs in artificial cells. Challenges in generating sub-micron droplets have meant that reconfiguring this approach to form nano-scale structures is not straightforward. This is beginning to change however, and recent technological advances have instigated the manufacture and manipulation of an increasingly diverse repertoire of biomimetic nano-assemblies, including liposomes, polymersomes, hybrid particles, multi-lamellar structures, cubosomes, hexosomes, nanodiscs, and virus-like particles. The following review will discuss these higher-order self-assembled nanostructures, including their biochemical and industrial applications, and techniques used in their production and analysis. We suggest ways in which existing technologies could be repurposed for the enhanced design, manufacture, and exploitation of these structures and discuss potential challenges and future research directions. By compiling recent advances in this area, it is hoped we will inspire future efforts toward establishing scalable microfluidic platforms for the generation of biomimetic nanoparticles of enhanced architectural and functional complexity.

Luminescent silicon nanostructures and COVID-19.

This Faraday Discussion volume is unique in the hundred plus year history of the Faraday Discussion series, being produced at a time of unprecedented circumstances worldwide and without the preceding Faraday Discussion conference having taken place.

Flip-flop asymmetry of cholesterol in model membranes induced by thermal gradients.

Lipid asymmetry is a crucial property of biological membranes and significantly influences their physical and mechanical properties. It is responsible for maintaining different chemical environments on the external and internal surfaces of cells and organelles and plays a vital role in many biological processes such as cell signalling and budding. In this work we show, using non-equilibrium molecular dynamics (NEMD) simulations, that thermal fields can induce lipid asymmetry in biological membranes. We focus our investigation on cholesterol, an abundant lipid in the plasma membrane, with a rapid flip-flop rate, significantly influencing membrane properties. We demonstrate that thermal fields induce membrane asymmetry with cholesterol showing thermophobic behaviour and therefore accumulating on the cold side of the membrane. This work highlights a possible experimental route to preparing and controlling asymmetry in synthetic membranes.

Measurement of Forces between Supported Cationic Bilayers by Colloid Probe Atomic Force Microscopy: Electrolyte Concentration and Composition.

The interactions between supported cationic surfactant bilayers were measured by colloidal probe atomic force spectroscopy, and the effect of different halide salts was investigated. Di(alkylisopropylester)dimethylammonium methylsulfate (DIPEDMAMS) bilayers were fabricated by the vesicle fusion technique on muscovite mica. The interactions between the bilayers were measured in increasing concentrations of NaCl, NaBr, NaI, and CaCl2. In NaCl, the bilayer interactions were repulsive at all concentrations investigated, and the Debye length and surface potential were observed to decrease with increasing concentration. The interactions were found to follow the electrical double layer (EDL) component of DLVO theory well. However, van der Waals forces were not detected; instead, a strong hydration repulsion was observed at short separations. CaCl2 had a similar effect on the interactions as NaCl. NaBr and NaI were observed to be more efficient at decreasing surface potential than the chloride salts, with the efficacy increasing with the ionic radius.

Engineering Swollen Cubosomes Using Cholesterol and Anionic Lipids.

Dispersions of nonlamellar lipid membrane assemblies are gaining increasing interest for drug delivery and protein therapeutic application. A key bottleneck has been the lack of rational design rules for these systems linking different lipid species and conditions to defined lattice parameters and structures. We have developed robust methods to form cubosomes (nanoparticles with porous internal structures) with water channel diameters of up to 171 Å, which are over 4 times larger than archetypal cubosome structures. The water channel diameter can be tuned via the incorporation of cholesterol and the charged lipid DOPA, DOPG, or DOPS. We have found that large molecules can be incorporated into the porous cubosome structure and that these molecules can interact with the internal cubosome membrane. This offers huge potential for accessible encapsulation and protection of biomolecules and development of confined interfacial reaction environments.