A workflow for the development of template-assisted membrane crystallization downstream processing for monoclonal antibody purification.

Monoclonal antibodies (mAbs) are commonly used biologic drugs for the treatment of diseases such as rheumatoid arthritis, multiple sclerosis, COVID-19 and various cancers. They are produced in Chinese hamster ovary cell lines and are purified via a number of complex and expensive chromatography-based steps, operated in batch mode, that rely heavily on protein A resin. The major drawback of conventional procedures is the high cost of the adsorption media and the extensive use of chemicals for the regeneration of the chromatographic columns, with an environmental cost. We have shown that conventional protein A chromatography can be replaced with a single crystallization step and gram-scale production can be achieved in continuous flow using the template-assisted membrane crystallization process. The templates are embedded in a membrane (e.g., porous polyvinylidene fluoride with a layer of polymerized polyvinyl alcohol) and serve as nucleants for crystallization. mAbs are flexible proteins that are difficult to crystallize, so it can be challenging to determine the optimal conditions for crystallization. The objective of this protocol is to establish a systematic and flexible approach for the design of a robust, economic and sustainable mAb purification platform to replace at least the protein A affinity stage in traditional chromatography-based purification platforms. The procedure provides details on how to establish the optimal parameters for separation (crystallization conditions, choice of templates, choice of membrane) and advice on analytical and characterization methods.

Oxidation of Human Copper Chaperone Atox1 and Disulfide Bond Cleavage by Cisplatin and Glutathione.

Cancer cells cope with high oxidative stress levels, characterized by a shift toward the oxidized form (GSSG) of glutathione (GSH) in the redox couple GSSG/2GSH. Under these conditions, the cytosolic copper chaperone Atox1, which delivers Cu(I) to the secretory pathway, gets oxidized, i.e., a disulfide bond is formed between the cysteine residues of the Cu(I)-binding CxxC motif. Switching to the covalently-linked form, sulfur atoms are not able to bind the Cu(I) ion and Atox1 cannot play an antioxidant role. Atox1 has also been implicated in the resistance to platinum chemotherapy. In the presence of excess GSH, the anticancer drug cisplatin binds to Cu(I)-Atox1 but not to the reduced apoprotein. With the aim to investigate the interaction of cisplatin with the disulfide form of the protein, we performed a structural characterization in solution and in the solid state of oxidized human Atox1 and explored its ability to bind cisplatin under conditions mimicking an oxidizing environment. Cisplatin targets a methionine residue of oxidized Atox1; however, in the presence of GSH as reducing agent, the drug binds irreversibly to the protein with ammine ligands trans to Cys12 and Cys15. The results are discussed with reference to the available literature data and a mechanism is proposed connecting platinum drug processing to redox and copper homeostasis.

Quantum Nature of Light in Nonstoichiometric Bulk Perovskites.

Sources of single photons are a fundamental brick in the development of quantum information technologies. Great efforts have been made so far in the realization of reliable, highly efficient, and on demand quantum sources that could show an easy integration with quantum devices. This has recently culminated in the use of solid state quantum dots as promising candidates for future sources of quantum technologies. However, some challenges, like their complex fabrication, random distribution, and difficult integrability with silicon technology, could hinder their broad application, making necessary the study of alternative systems. In this work, we clearly demonstrate single photon emission from quantum dots formed in nonstoichiometric bulk perovskites. Their simple growing procedures, exceptional stability under constant illumination, easy control of their optical properties, as well as ease of integrability make these materials very interesting candidates for the development of quantum light sources in the near-infrared.

Oxaliplatin Binding to Human Copper Chaperone Atox1 and Protein Dimerization.

Copper trafficking proteins have been implicated in the cellular response to platinum anticancer drugs. We investigated the reaction of the chaperone Atox1 with an activated form of oxaliplatin, the third platinum drug to reach worldwide approval. Unlike cisplatin, which contains monodentate ammines, oxaliplatin contains chelated 1,2-diaminocyclohexane (DACH), which is more resistant to displacement by nucleophiles. In solution, one or two {Pt(DACH)(2+)} moieties bind to the conserved CXXC metal-binding motif of Atox1; in the latter case the two sulfur atoms likely bridging the two platinum units. At longer reaction times, a dimeric species is formed whose composition, Atox12·Pt(2+)2, indicates complete loss of the diamine ligands. Such a dimerization process is accompanied by partial unfolding of the protein. Crystallization experiments aiming at the characterization of the monomeric species have afforded, instead, a dimeric species resembling that already obtained by Boal and Rosenzweig in a similar reaction performed with cisplatin. However, while in the latter case there was only one Pt-binding site (0.4 occupancy) made of four sulfur atoms of the CXXC motifs of the two Atox1 chains in a tetrahedral arrangement, we found, in addition, a secondary Pt-binding site involving Cys41 of the B chain (0.25 occupancy). Moreover, both platinum atoms have lost their diamines. Thus, there appears to be little relationship between what is observed in solution and what is formed in the solid state. Since full occupancy of the tetrahedral cavity is a common feature of all Atox1 dimeric structures obtained with other metal ions (Cu(+), Cd(2+), and Hg(2+)), we propose that in the case of platinum, where the occupancy is only 0.4, the remaining cavities are occupied by Cu(+) ions. Experimental evidence is reported in support of the latter hypothesis. Our proposal represents a meeting point between the initial proposal of Boal and Rosenzweig (0.4 Pt occupancy) and the reinterpretation of the original crystallographic data put forward by Shabalin et al. (1 Cu occupancy), and could apply to other cases.

How a ß-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 ß-d-glucose-containing compound 3, 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, Ki = 0.090 nM) and thrombin (fIIa, Ki = 100 nM). The potency of 3 increases, over the parent compound 1, against fIIa (110-fold), much more than against fXa (7-fold). Experimental deconstruction of 3 into smaller fragments revealed a binding cooperativity of the P3/P4 and propylene-linked ß-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 3 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 3 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 (6) 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 13b, which showed excellent fIIa inhibition (Ki = 6 nM), weak anti-Xa activity (Ki = 5.64 µM), and remarkable selectivity over other serine proteases (e.g., trypsin). Compound 13b showed in vitro anticoagulant activity in the low micromolar range and significant membrane permeability. In mice (ex vivo), 13b 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 (P < 0.05).

Cobalt binding in the photosynthetic bacterium R. sphaeroides by X-ray absorption spectroscopy.

Cobalt is an important oligoelement required for bacteria; if present in high concentration, exhibits toxic effects that, depending on the microorganism under investigation, may even result in growth inhibition. The photosynthetic bacterium Rhodobacter (R.) sphaeroides tolerates high cobalt concentration and bioaccumulates Co(2+) ion, mostly on the cellular surface. Very little is known on the chemical fate of the bioaccumulated cobalt, thus an X-ray absorption spectroscopy investigation was conducted on R. sphaeroides cells to gain structural insights into the Co(2+) binding to cellular components. X-ray absorption near-edge spectroscopy and extended X-ray absorption fine structure measurements were performed on R. sphaeroides samples containing whole cells and cell-free fractions obtained from cultures exposed to 5 mM Co(2+). An octahedral coordination geometry was found for the cobalt ion, with six oxygen-ligand atoms in the first shell. In the soluble portion of the cell, cobalt was found bound to carboxylate groups, while a mixed pattern containing equivalent amount of two sulfur and two carbon atoms was found in the cell envelope fraction, suggesting the presence of carboxylate and sulfonate metal-binding functional groups, the latter arising from sulfolipids of the cell envelope.