Evaluating the Effects of Hinge Flexibility on the Solution Structure of Antibodies at Concentrated Conditions

Employing 2 different coarse-grained models, we evaluated the effect of intramolecular domain-domain distances and hinge flexibility on the general solution structure of monoclonal antibodies (mAbs), within the context of protein-protein steric repulsion. These models explicitly account for the hinge region, and represent antibodies at either domain or subdomain levels (i.e., 4-bead and 7-bead representations, respectively). Additionally, different levels of mAb flexibility are also considered. When evaluating mAbs as rigid structures, analysis of small-angle scattering profiles showed that changes in the relative internal distances between Fc and Fab domains significantly alter the local arrangement of neighboring molecules, as well as the molecular packing of the concentrated mAb solutions. Likewise, enabling hinge flexibility in either of the mAb models led to qualitatively similar results, where flexibility increases the spatial molecular arrangement at elevated concentrations. This occurs because fluctuations in mAb quaternary structure are modulated by the close proximity between molecules at elevated concentrations (>50 mg mL^?1), yielding an increased molecular packing and osmotic compressibility. However, our results also showed that the mechanism behind this synergy between flexibility and packing strongly depends on both the level of structural detail and the number of degrees-of-freedom considered in the coarse-grained model.     

A Nonionic Polyethylene Oxide (PEO) Surfactant Model: Experimental and Molecular Dynamics Studies of Kolliphor EL

Polyethoxylated, nonionic surfactants are important constituents of many drug formulations, including lipid-based formulations. In an effort to better understand the behavior of formulation excipients at the molecular level, we have developed molecular dynamics (MD) models for the widely used surfactant Kolliphor EL (KOL), a triricinoleate ester of ethoxylated glycerol. In this work, we have developed models based on a single, representative molecular component modeled with 2 force field variations based on the GROMOS 53A6_DBW and 2016H66 force field parameters for polyethoxylate chains. To compare the computational models to experimental measurements, we investigated the phase behavior of KOL using nephelometry, dynamic light scattering, cross-polarized microscopy, small-angle X-ray scattering, and cryogenic transmission electron microscopy. The potential for digestion of KOL was also evaluated using an in vitro digestion experiment. We found that the size and spherical morphology of the KOL colloids at low concentrations was reproduced by the MD models as well as the growing interactions between the aggregates to from rod-like structures at high concentrations. We believe that this model reproduces the phase behavior of KOL relevant to drug absorption and that it can be used in whole formulation simulations to accelerate the formulation development.     

Conjugation of Emtansine Onto Trastuzumab Promotes Aggregation of the Antibody–Drug Conjugate by Reducing Repulsive Electrostatic Interactions and Increasing Hydrophobic Interactions

The impact of drug conjugation on intra- and intermolecular interactions of trastuzumab (TmAb) was determined by comparing the conformational and colloidal stabilities of TmAb and trastuzumab emtansine (T-DM1). In low ionic strength formulations, drug conjugation to native lysine residues of TmAb significantly reduced the repulsive electrostatic interactions between T-DM1 molecules. When these electrostatic interactions were screened in solutions with high ionic strength, intermolecular interactions between T-DM1 molecules were found to be more attractive than those between TmAb molecules. Drug conjugation lowered the colloidal stability of T-DM1 compared to TmAb, making T-DM1 more susceptible to agitation-induced aggregation. The presence of polysorbate-20 in the formulations inhibited aggregation of TmAb and T-DM1 induced by the hydrophobic air-water interface. Furthermore, the effect of increased hydrophobic interactions between T-DM1 molecules was studied by monitoring aggregation in TmAb and T-DM1 solutions that were incubated at 4°C, 25°C, and 50°C. Conjugating DM1 to TmAb increased the hydrophobicity of the molecule, and faster aggregation of T-DM1 at 50°C could be attributed to a temperature-dependent increase in hydrophobic interactions between T-DM1 molecules.     

Hard sphere-like glass transition in eye lens α-crystallin solutions

We study the equilibrium liquid structure and dynamics of dilute and concentrated bovine eye lens α-crystallin solutions, using small-angle X-ray scattering, static and dynamic light scattering, viscometry, molecular dynamics simulations, and mode-coupling theory. We find that a polydisperse Percus–Yevick hard-sphere liquid-structure model accurately reproduces both static light scattering data and small-angle X-ray scattering liquid structure data from α-crystallin solutions over an extended range of protein concentrations up to 290 mg/mL or 49% vol fraction and up to ca. 330 mg/mL for static light scattering. The measured dynamic light scattering and viscosity properties are also consistent with those of hard-sphere colloids and show power laws characteristic of an approach toward a glass transition at α-crystallin volume fractions near 58%. Dynamic light scattering at a volume fraction beyond the glass transition indicates formation of an arrested state. We further perform event-driven molecular dynamics simulations of polydisperse hard-sphere systems and use mode-coupling theory to compare the measured dynamic power laws with those of hard-sphere models. The static and dynamic data, simulations, and analysis show that aqueous eye lens α-crystallin solutions exhibit a glass transition at high concentrations that is similar to those found in hard-sphere colloidal systems. The α-crystallin glass transition could have implications for the molecular basis of presbyopia and the kinetics of molecular change during cataractogenesis.The cytoplasm of the tightly packed fiber cells of the eye lens contains concentrated aqueous protein mixtures that have high refractive indexes, while normally remaining clear enough for vision. Lens clarity depends on short-range order between lens proteins (1, 2) and can be disrupted by both protein aggregation and liquid–liquid phase separation in cataract, a leading cause of blindness. At the high protein concentrations of lens cytoplasm, 25–60% by weight in mammals, small changes in interprotein interactions can disrupt transparency. For human lens proteins with cataractogenic point mutations, and for high-concentration lens protein mixtures, protein interaction changes as small as a fraction of thermal energy, k_BT, can induce phase separation and thus lead to opacification (3–7).In addition to equilibrium phase transitions, dynamical transitions including glass formation and gelation can also occur at high protein concentrations like those of the eye lens (8, 9). Relatively abrupt viscoelastic changes associated with glass formation or gelation could harden the lens and contribute to presbyopia and could alter cataract formation rates by affecting aggregation and phase separation kinetics.Here we study the equilibrium liquid structure and dynamics of concentrated solutions of eye-lens α-crystallin protein solutions, using small-angle X-ray scattering (SAXS), static light scattering (SLS) and dynamic light scattering (DLS), viscometry, liquid-state theory, event-driven molecular dynamics (MD) simulations, and mode-coupling theory (MCT). α-Crystallin is a polydisperse protein with about 40 subunits of two types, αA and αB, and accounts for up to 50% of lens protein mass. The forward scattering intensity from SAXS and light scattering experiments with concentrated α-crystallin solutions can be well represented by monodisperse hard-sphere liquid-structure models that have been key for understanding short-range order needed for lens transparency (1, 2). However, in physiological conditions α-crystallin ranges in molecular weight from 3 × 10⁵ to 2 × 10⁶, with an average near 8 × 10⁵ (10). There have indeed been reports that structure factors obtained from SAXS and small-angle neutron scattering data deviate from predictions for monodisperse hard spheres (11, 12).Accordingly, we first show that a polydisperse hard-sphere liquid-structure model (PHSM) based on the Percus–Yevick (PY) approximation (13) can accurately model SAXS data obtained from the present bovine α-crystallin preparations for volume fractions up to 49%, using a polydispersity of 20%. In contrast, polydispersity did not strongly influence model predictions for the observed SLS, which unlike SAXS probes length scales much larger than molecular sizes.We then test for glassy dynamics of α-crystallin solutions, using DLS and viscometry, and find glass transition features like those previously found for hard spheres. In particular, with increasing α-crystallin concentration, DLS intermediate scattering functions show expected progressively slower relaxations along with fast decays, and the viscosity diverges, both when approaching volume fractions in the vicinity of 58%. Using MCT (14), we obtain semiquantitative models of DLS data and corresponding data from MD simulations of high-concentration polydisperse sphere systems, in which MD polydispersity values were obtained from fits of the PHSM to the SAXS data. We note that for MCT we have used the theory for monodisperse systems, because the majority of the MCT work to date has focused on such systems. Recent results for polydisperse systems (15) support a similar overall picture.In brief, the data show that the present α-crystallin solutions have liquid structure and glassy dynamics similar to those of polydisperse hard-sphere solutions. Thus, eye lens protein solutions show analogs to ordinary glass in linking short-range order, transparency, and arrested dynamics.     

年龄相关性白内障眼内散射光的检测及临床意义

目的：探讨不同类型年龄相关性白内障眼内散射光的检测及其临床意义。方法回顾性分析单纯年龄相关性白内障患者50例（100眼）的临床资料,另选择健康者30例（60眼）作为对照组。比较不同类型白内障患者眼内散射光值,分析眼内散射光值相关因素。结果白内障组患者眼内散射光平均值显著高于对照组（P＜0.01）。后囊下白内障显著高于核性白内障组、皮质性白内障组和混合性白内障组（P＜0.01）。而混合性白内障组显著高于皮质性白内障组（P＜0.01）。结论核性和皮质性白内障患者眼内散射光与最佳矫正视力负相关。     

Connectivity Defects and Collective Assemblies in Model Metallo‐Supramolecular Dual‐Network Hydrogels

Double network hydrogels are composed of chemical and physical bonds, whose influences on the macroscopic material properties are convoluted. To decouple these, a model dually crosslinked network with independently tunable permanent and reversible crosslinks is introduced. This is realized by interlinking linear and tetra‐arm poly(ethylenegycol) (PEG) precursors with complementary reactive terminal groups. The former also carries a terpyridine ligand at each end, which forms reversible metallo‐supramolecular bonds upon addition of metal ions. These dual networks display different types and amounts of network defects, as studied by light scattering and proton double‐quantum (DQ) NMR. Dynamic light scattering suggests that the network mesh size decreases upon introduction of metal ions, as supported by a decrease of the residual dipolar coupling constant in NMR. Static light scattering indicates larger static inhomogeneities in those networks composed of stronger ions. This is complemented by a fast solid‐like component in the DQ buildup in NMR, attributed to the formation of nanoscopic clusters of charged complexes. The DQ buildup curves also suggest that the presence of strong physical bonds increases the fraction of mobile segments, like loops and dangling ends. This combined study unveils the interplay of chemical and physical bonds toward the formation of a hierarchical structure.     

Making yttria-stabilized tetragonal zirconia translucent

ObjectiveThe aim of this study was to provide a design guideline for developing tetragonal yttria-stabilized zirconia with improved translucency.MethodsThe translucency, the in-line transmission in particular, of 3 mol.% yttria-stabilized tetragonal zirconia (3Y-TZP) has been examined using the Rayleigh scattering model. The theory predicts that the in-line transmission of 3Y-TZP can be related to its thickness with grain size and birefringence the governing parameters. To achieve a threshold value of translucency, the critical grain size of 3Y-TZP was predicted for various thicknesses (0.3–2.0 mm). The threshold value was defined by a measured average in-line transmission value of a suite of dental porcelains with a common thickness of 1 mm. Our theoretical predictions were calibrated with one of the very few experimental data available in the literature.ResultsFor a dense, high-purity zirconia, its in-line transmission increased with decreasing grain size and thickness. To achieve a translucency similar to that of dental porcelains, a nanocyrstalline 3Y-TZP structure was necessitated, due primarily to its large birefringence and high refractive index. Such a grain size dependence became more pronounced as the 3Y-TZP thickness increased. For example, at a thickness of 1.3 mm, the mean grain size of a translucent 3Y-TZP should be 82 nm. At 1.5 mm and 2 mm thicknesses, the mean grain size needed to be 77 nm and 70 nm, respectively.SignificanceA promising future for zirconia restorations, with combined translucency and mechanical properties, can be realized by reducing its grain size.     

Banded structures in collagen vitrigels for corneal injury repair

There is a growing interest in using collagen vitrigels for corneal injury repair. We recently reported the synthesis and thermal denaturation behavior of these gels. In this paper, the banded structure in these vitrified gels is studied by small-angle X-ray scattering (SAXS) one-dimensional (1-D) correlation function analysis and transmission electron microscopy (TEM). Results demonstrate that the collagen vitrigel possess banded structures similar to those of the starting type I collagen, with an average D-spacing of 64 nm (by SAXS) or 57 nm (by TEM). A combination of SAXS 1-D correlation function analyses and TEM show that overlap and gap distances ranged from 30 to 33 nm and from 23 to 25 nm, respectively. Changing the vitrification condition does not impact on the banded structure significantly.     

Metasurfaces for Far-Field Radiation Pattern Correction of Antennas under Dielectric Seamed-Radomes

A high-index dielectric radome seam is camouflaged with respect to a low-index dielectric radome panel by tuning the seam with carefully engineered metasurfaces. A transmission-line approach is used to model the metasurface-tuned seam and analytically retrieve the corresponding surface impedance, from which the unit-cell design is then tailored. Full-wave simulations and microwave antenna measurements performed on a proof-of-concept prototype validate the undesired scattering suppression effect in the case of normally and obliquely incident transverse electric and transverse magnetic wave illuminations. Robustness of the proposed solution to fabrication tolerances is also reported. The study presents metasurface-tuning as an easily implementable, frequency adjustable, and polarization insensitive solution to reduce the scattering of dielectric mechanical seams and improve the overall transparency performance of radome structures.