Optimization of vapor diffusion conditions for anti-CD20 crystallization and scale-up to meso batch

© 2019, MDPI AG. All rights reserved. The crystal form is one of the preferred formulations for biotherapeutics, especially thanks to its ability to ensure high stability of the active ingredient. In addition, crystallization allows the recovery of a very pure drug, thus facilitating the manufacturing process. However, in many cases, crystallization is not trivial, and other formulations, such as the concentrate solution, represent the only choice. This is the case of anti-cluster of differentiation 20 (anti-CD20), which is one of the most sold antibodies for therapeutic uses. Here, we propose a set of optimized crystallization conditions for producing anti-CD20 needle-shaped crystals within 24 h in a very reproducible manner with high yield. High crystallization yield was obtained with high reproducibility using both hanging drop vapor diffusion and meso batch, which is a major step forward toward further scaling up the crystallization of anti-CD20. The influence of anti-CD20 storage conditions and the effect of different ions on the crystallization processes were also assessed. The crystal quality and the high yield allowed the first crystallographic investigation on anti-CD20, which positively confirmed the presence of the antibody in the crystals

Optimization of vapor diffusion conditions for anti-CD20 crystallization and scale-up to meso batch

© 2019, MDPI AG. All rights reserved. The crystal form is one of the preferred formulations for biotherapeutics, especially thanks to its ability to ensure high stability of the active ingredient. In addition, crystallization allows the recovery of a very pure drug, thus facilitating the manufacturing process. However, in many cases, crystallization is not trivial, and other formulations, such as the concentrate solution, represent the only choice. This is the case of anti-cluster of differentiation 20 (anti-CD20), which is one of the most sold antibodies for therapeutic uses. Here, we propose a set of optimized crystallization conditions for producing anti-CD20 needle-shaped crystals within 24 h in a very reproducible manner with high yield. High crystallization yield was obtained with high reproducibility using both hanging drop vapor diffusion and meso batch, which is a major step forward toward further scaling up the crystallization of anti-CD20. The influence of anti-CD20 storage conditions and the effect of different ions on the crystallization processes were also assessed. The crystal quality and the high yield allowed the first crystallographic investigation on anti-CD20, which positively confirmed the presence of the antibody in the crystals

Local Fluctuations and Conformational Transitions in Proteins

The intrinsic plasticity of protein residues, along with the occurrence of transitions between distinct residue conformations, plays a pivotal role in a variety of molecular recognition events in the cell. Analysis aimed at identifying both of these features has been limited so far to protein-complex structures. We present a computationally efficient tool (T-pad), which quantitatively analyzes protein residues’ flexibility and detects backbone conformational transitions. T-pad is based on directional statistics of NMR structural ensembles or molecular dynamics trajectories. T-pad is here applied to human ubiquitin (hU), a paradigmatic cellular interactor. The calculated plasticity is compared to hU’s Debye–Waller factors from the literature as well as those from experimental work carried out for this paper. T-pad is able to identify most of the key residues involved in hU’s molecular recognition, also in the absence of its cellular partners. Indeed, T-pad identified as many as 90% of ubiquitin residues interacting with their cognate proteins. Hence, T-pad might be a useful tool for the investigation of interactions between proteins and their cellular partners at the genome-wide level

A Molecular Dynamics Simulation-Based Interpretation of Nuclear Magnetic Resonance Multidimensional Heteronuclear Spectra of α‑Synuclein·Dopamine Adducts

Multidimensional heteronuclear nuclear magnetic resonance (NMR) spectroscopy provides valuable structural information about adducts between naturally unfolded proteins and their ligands. These are often highly pharmacologically relevant. Unfortunately, the determination of the contributions to observed chemical shifts changes upon ligand binding is complicated. Here we present a tool that uses molecular dynamics (MD) trajectories to help interpret two-dimensional (2D) NMR data. We apply this tool to the naturally unfolded protein human α-synuclein interacting with dopamine, an inhibitor of fibril formation, and with its oxidation products in water solutions. By coupling 2D NMR experiments with MD simulations of the adducts in explicit water, the tool confirms with experimental data that the ligands bind preferentially to ¹²⁵YEMPS¹²⁹ residues in the C-terminal region and to a few residues of the so-called NAC region consistently. It also suggests that the ligands might cause conformational rearrangements of distal residues located at the N-terminus. Hence, the performed analysis provides a rationale for the observed changes in chemical shifts in terms of direct contacts with the ligand and conformational changes in the protein

Selecting the Desired Solid Form by Membrane Crystallizers: Crystals or Cocrystals

This work aims to describe a systematic study on the conditions promoting the selective formation of carbamazepine-saccharin cocrystals or single component crystals from water/ethanol solvent mixtures, by using a membrane crystallization process. Results revealed the ability to operate in the proper zone of the phase diagram of the system when opportunely choosing the initial solution conditions and limiting the maximum level of supersaturation by using the membrane-based technology. Control in the selective crystallization of a specific solid form can be achieved by adjusting the solvent evaporation through the micropores of the membrane. Furthermore, the direct correlation between transmembrane flow and polymorphic composition in the case of carbamazepine precipitation confirmed the possibility to produce particular metastable phases upon increasing the supersaturation rate

How a β‑d‑Glucoside Side Chain Enhances Binding Affinity to Thrombin of Inhibitors Bearing 2‑Chlorothiophene as P1 Moiety: Crystallography, Fragment Deconstruction Study, and Evaluation of Antithrombotic Properties

The β-d-glucose-containing compound <b>3</b>, bearing 2-chlorothiophene and 1-isopropylpiperidine moieties as binders of the S1 and S4 pockets, respectively, proved to be potent competitive inhibitor of factor Xa (fXa, <i>K</i>_i = 0.090 nM) and thrombin (fIIa, <i>K</i>_i = 100 nM). The potency of <b>3</b> increases, over the parent compound <b>1</b>, against fIIa (110-fold), much more than against fXa (7-fold). Experimental deconstruction of <b>3</b> into smaller fragments revealed a binding cooperativity of the P3/P4 and propylene-linked β-d-glucose fragments, stronger in fIIa (15.5 kJ·mol^–1) than in fXa (2.8 kJ·mol^–1). The crystal structure of human fIIa in complex with <b>3</b> revealed a binding mode including a strong H-bond network between the glucose O1′, O3′, and O5′ and two critical residues, namely R221a and K224, belonging to the Na⁺-binding site which may allosterically perturb the specificity sites. The potential of <b>3</b> as antithrombotic agent was supported by its ability to inhibit thrombin generation and to stimulate fibrinolysis at submicromolar concentration

Synthesis and Biological Evaluation of Direct Thrombin Inhibitors Bearing 4‑(Piperidin-1-yl)pyridine at the P1 Position with Potent Anticoagulant Activity

The design and synthesis of a new class of nonpeptide direct thrombin inhibitors, built on the structure of 1-(pyridin-4-yl)­piperidine-4-carboxamide, are described. Starting from a strongly basic 1-amidinopiperidine derivative (<b>6</b>) showing poor thrombin (fIIa) and factor Xa (fXa) inhibition activities, anti-fIIa activity and artificial membrane permeability were considerably improved by optimizing the basic P1 and the X-substituted phenyl P4 binding moieties. Structure–activity relationship studies, usefully complemented with molecular modeling results, led us to identify compound <b>13b</b>, which showed excellent fIIa inhibition (<i>K</i>_i = 6 nM), weak anti-Xa activity (<i>K</i>_i = 5.64 μM), and remarkable selectivity over other serine proteases (e.g., trypsin). Compound <b>13b</b> showed in vitro anticoagulant activity in the low micromolar range and significant membrane permeability. In mice (ex vivo), <b>13b</b> demonstrated anticoagulant effects at 2 h after oral dosing (100 mg·kg^–1), with a significant 43% prolongation of the activated partial thromboplastin time (aPTT), over controls (<i>P</i> < 0.05)

Hydrogel Composite Membranes Incorporating Iron Oxide Nanoparticles as Topographical Designers for Controlled Heteronucleation of Proteins

In this study, we exploited the possibility of tuning physical–chemical properties of hydrogel composite membranes (HCMs) surfaces, by using iron oxide nanoparticles (NPs) as topographical designers, with the aim of examining the effect of surface topography and wettability on the heterogeneous nucleation of protein crystals. On the basis of roughness and contact angle measurements, it was found that surface structural characteristics, in addition to chemical interactions between the surface and protein molecules, have influence on the heterogeneous nucleation of lysozyme and thermolysin crystals to different extents. We demonstrated that increasing the amount of NPs incorporated in the hydrogel matrix promotes protein nucleation to a higher extent, potentially due to the increase of local solute concentration, arising from the enhanced wetting tendency in the Wenzel regime, and physical confinement at rougher hydrophilic surfaces. An extensive crystallographic analysis suggested the tendency of the growing crystals to incorporate hydrogel materials, which allows inducement of protein conformational states slightly different from those covered by standard crystallization methods. Protein flexibility can be thus sampled by changing the amount of NPs in the HCMs, with negligible influence on the quantity and quality of X-ray diffraction data

Light-Induced Formation of Pb³⁺ Paramagnetic Species in Lead Halide Perovskites

Hybrid halide perovskites are soft materials processed at room temperature, revolutionary players in the photovoltaic field. Nowadays, investigation of the nature and role of defects is seen as one of the key challenges toward full comprehension of their behavior and achievement of high device stability under working conditions. We reveal the reversible generation, under illumination, of paramagnetic Pb³⁺ defects in CH₃NH₃PbI₃, synthesized in ambient conditions, induced by the presence of Pb–O defects in the perovskite structure that may trap photogenerated holes, possibly mediated by the concomitant oxidation and migration of ions. According to the mechanism that we hypothesize, one charge is trapped for each paramagnetic center generated; thus, it does not contribute to the photocurrent, potentially limiting the solar cell performance. Our study, based on combined experimental/theoretical approach, reveals the dynamic evolution of the perovskite characteristics under illumination that needs to be considered when investigating the material physical–chemical properties

Direct Band Gap Chalcohalide Semiconductors: Quaternary AgBiSCl₂ Nanocrystals

Heavy pnictogen chalcohalide semiconductors are coming under the spotlight for energy conversion applications. Here we present the colloidal synthesis of phase pure AgBiSCl2 nanocrystals. This quaternary chalcohalide compound features a quasi-two-dimensional crystal structure and a direct band gap, in contrast with the monodimensional structure and the indirect band gap peculiar to the orthorhombic, ternary Bi chalcohalides. Consistently, colloidal AgBiSCl2 nanocrystals exhibit photoinduced luminescence compatible with both band edge excitons and midgap states. This is the first observation of band edge emission in chalcohalide nanomaterials at large, although exciton recombination in our AgBiSCl2 nanocrystals mostly occurs via nonradiative pathways. This work further advances our knowledge on this class of mixed anion semiconductor nanomaterials and provides a contribution to establishing chalcohalides as a reliable alternative to metal chalcogenides and halides