Plutonium Hybrid Materials: A Platform to Explore Assembly and Metal-Ligand Bonding.

We report the synthesis of five new hybrid materials containing the [PuCl6]2- anion and charge-balancing, noncovalent interaction donating 4-X-pyridinium (X = H, Cl, Br, I) cations. Single crystals of the title compounds were grown and harvested from acidic, chloride-rich, aqueous media, and their structures were determined via X-ray diffraction. Compounds 1-4, (4XPyH)2[PuCl6], and 5, (4IPyH)4[PuCl6]·2Cl, exhibit two distinct sheet-like structure types. Structurally relevant noncovalent interactions were tabulated from crystallographic data and verified computationally using electrostatic surface potential maps and the quantum theory of atoms in molecules (QTAIM). The strength of the hydrogen and halogen bonds was quantified using Kohn-Sham density functional theory, and a hierarchy of acceptor-donor pairings was established. The PuIV-Cl bonds were studied using QTAIM and natural localized molecular orbital (NLMO) analyses to delineate the underlying bond mechanism and hybrid atomic orbital contributions therein. The results of the PuIV-Cl bond analyses were compared across compositions via analogous treatments of previously reported [PuO2Cl4]2- and [PuCl3(H2O)5] molecular units. The Pu-Cl bonds are predominately ionic yet exhibit small varying degrees of covalent character that increases from [PuCl3(H2O)5] and [PuO2Cl4]2- to [PuCl6]2-, while the participation of the Pu-based s/d and f orbitals concurrently decreases and increases, respectively.

A novel symmetric pyrazine (pyz)-bridged uranyl dimer [UO2Cl3(H2O)(Pyz)0.5]22-: synthesis, structure and computational analysis.

Herein we report on the synthesis of (HPyz+)2[UO2Cl3(H2O)(Pyz)0.5]2·2H2O which features a novel pyrazine-bridged uranyl dimer, [UO2Cl3(H2O)(Pyz)0.5]22-. A rigorous computational and experimental analysis of this compound was performed to fully explore the influence of coordination on the electronic structure and potential charge-transfer characteristics of this dimer, revealing a delocalized π-system across the bridging pyrazine and the axial components of both uranyl centers. Electrostatic surface potentials, used to rationalize the observed assembly, indicate a decreased basicity of the uranyl oxo versus [UO2Cl4]2-, and signify a lessened capacity for the terminal -yl oxos of the [UO2Cl3(H2O)(Pyz)0.5]22- dimer to participate in supramolecular assembly. A combined density functional theory (DFT) and quantum theory of atoms in molecules (QTAIM) analysis further evidenced an increase in UO bond strengths within the dimer, which is supported by a blue shift in the characteristic Raman-active uranyl symmetric stretch (ν1) with respect to the more typically observed [UO2Cl4]2-.

Novel anion exchange membrane chromatography method for the separation of empty and full adeno-associated virus.

A challenge in the production of recombinant Adeno-Associated Virus (AAV) for gene therapies is the presence of capsids that lack the required gene of interest. The impact of these empty vectors in therapies is not fully understood, however the ability to control the ratio of empty to full particles, which contain the genetic payload, is a necessary step in the purification of these viruses. In this study, a novel anion exchange chromatography elution method for enrichment of full AAV particles is demonstrated. A step gradient with small conductivity increases of around 1 mS cm-1 provides more efficient separation of empty and full AAV serotype 5 across membrane media as compared to conventional linear gradient method. The use of this approach in optimizing a simpler method for manufacturing processes and scalability to a larger chromatographic volume is explored. With this approach, the authors achieved greater than 4-fold enrichment of full capsids, to give a total of ≈50%-60% full capsids, using a 25 mM Bis-Tris Propane pH 9.0 buffer system with NaCl as the eluting salt. Results suggest that this elution method can be implemented into a scalable process and can provide insight into development of elution methods for other AAV serotypes.

Pb-Oxo Interactions in Uranyl Hybrid Materials: A Combined Experimental and Computational Analysis of Bonding and Spectroscopic Properties.

Reported are the syntheses and characterization of six new heterometallic UO22+/Pb2+ compounds. These materials feature rare instances of M-oxo interactions, which influence the bonding properties of the uranyl cation. The spectroscopic effects of these interactions were measured using luminescence and Raman spectroscopy. Computational density functional theory-based natural bonding orbital and quantum theory of atoms in molecules methods indicate interactions arise predominantly through charge transfer between cationic units via the electron-donating uranyl O spx lone pair orbitals and electron-accepting Pb2+ p orbitals. The interaction strength varies as a function of Pb-oxo interaction distance and angle with energy values ranging from 0.47 kcal/mol in the longer contacts to 21.94 kcal/mol in the shorter contacts. Uranyl units with stronger interactions at the oxo display an asymmetric bond weakening and a loss of covalent character in the U═O bonds interacting closely with the Pb2+ ion. Luminescence quenching is observed in cases in which strong Pb-oxo interactions are present and is accompanied by red-shifting of the uranyl symmetric Raman stretch. Changes to inner sphere uranyl bonding manifest as a weakening of the U═O bond as a result of interaction with the Pb2+ ion. Comprehensive evaluation of the effects of metal ions on uranyl spectra supports modeling efforts probing uranyl bonding and may inform applications such as forensic signatures.

Bimetallic uranyl/cobalt(II) isothiocyanates: structure, property and spectroscopic analysis of homo- and heterometallic phases.

We report the synthesis and characterization of a family of UO22+/Co2+ isothiocyanate materials containing [UO2(NCS)5]3- and/or [Co(NCS)4]2- building units charged balanced by tetramethylammonium cations and assembled via SS or SOyl non-covalent interactions (NCIs), namely (C4H12N)3[UO2(NCS)5], (C4H12N)2[Co(NCS)4], and (C4H12N)5[Co(NCS)4][UO2(NCS)5]. The homometallic uranyl phase preferentially assembles via SS interactions, whereas in the heterometallic phase SOyl interactions are predominant. The variation in assembly mode is explored using electrostatic surfaces potentials, revealing that the pendant -NCS ligands of the [Co(NCS)4]2- anion is capable of outcompeting those of the [UO2(NCS)5]3- anion. Notably, the heterometallic phase displays atypical blue shifting of the uranyl symmetric stretch in the Raman spectra, which is in contrast to many other compounds featuring non-covalent interactions at uranyl oxygen atoms. A combined experimental and computational (density functional theory and natural bond orbital analyses) approach revealed that coupling of the uranyl symmetric stretch with isothiocyanate modes of equatorial -NCS ligands was responsible for the atypical blue shift in the heterometallic phase.

Validation and optimization of viral clearance in a downstream continuous chromatography setting.

Continuous bioprocessing holds the potential to improve product consistency, accelerate productivity, and lower cost of production. However, switching a bioprocess from traditional batch to continuous mode requires surmounting business and regulatory challenges. A key regulatory requirement for all biopharmaceuticals is virus safety, which is assured through a combination of testing and virus clearance through purification unit operations. For continuous processing, unit operations such as capture chromatography have aspects that could be impacted by a change to continuous multicolumn operation, for example, do they clear viruses as well as a traditional batch single column. In this study we evaluate how modifying chromatographic parameters including the linear velocity and resin capacity utilization could impact virus clearance in the context of moving from a single column to multicolumn operation. A Design of Experiment (DoE) approach was taken with two model monoclonal antibodies (mAbs) and two bacteriophages used as mammalian virus surrogates. The DoE enabled the identification of best and worst-case scenario for virus clearance overall. Using these best and worst-case conditions, virus clearance was tested in single column and multicolumn modes and found to be similar as measured by Log Reduction Values (LRV). The parameters identified as impactful for viral clearance in single column mode were predictive of multicolumn modes. Thus, these results support the hypothesis that the viral clearance capabilities of a multicolumn continuous Protein A system may be evaluated using an appropriately scaled-down single mode column and equipment.

Modeling the Downstream Processing of Monoclonal Antibodies Reveals Cost Advantages for Continuous Methods for a Broad Range of Manufacturing Scales.

The biopharmaceutical industry is evolving in response to changing market conditions, including increasing competition and growing pressures to reduce costs. Single-use (SU) technologies and continuous bioprocessing have attracted attention as potential facilitators of cost-optimized manufacturing for monoclonal antibodies. While disposable bioprocessing has been adopted at many scales of manufacturing, continuous bioprocessing has yet to reach the same level of implementation. In this study, the cost of goods of Pall Life Science's integrated, continuous bioprocessing (ICB) platform is modeled, along with that of purification processes in stainless-steel and SU batch formats. All three models include costs associated with downstream processing only. Evaluation of the models across a broad range of clinical and commercial scenarios reveal that the cost savings gained by switching from stainless-steel to SU batch processing are often amplified by continuous operation. The continuous platform exhibits the lowest cost of goods across 78% of all scenarios modeled here, with the SU batch process having the lowest costs in the rest of the cases. The relative savings demonstrated by the continuous process are greatest at the highest feed titers and volumes. These findings indicate that existing and imminent continuous technologies and equipment can become key enablers for more cost effective manufacturing of biopharmaceuticals.

Optimized Continuous Multicolumn Chromatography Enables Increased Productivities and Cost Savings by Employing More Columns.

The advantages of continuous chromatography with respect to increased capacity are well established. However, the impact of different loading scenarios and total number of columns on the process economics has not been addressed. Here four different continuous multicolumn chromatography (MCC) loading scenarios are evaluated for process performance and economics in the context of a Protein A mAb capture step. To do so, a computational chromatography model is validated experimentally. The model is then used to predict process performance for each of the loading methods. A wide range of feed concentrations and residence times are considered, and the responses of operating binding capacity, specific productivity, and the number of process columns are calculated. Processes that are able to add more columns proved to be up to 65% more productive, especially at feed concentrations above 5 g L-1 . An investigation of the operating costs shows that discrete column sizing and process performance metrics do not always correlate and that the most productive process is not necessarily the most cost effective. However, adding more columns for the non-load steps at higher feed concentrations allows for overall cost savings of up to 32%.

A Direct Approach for Process Development Using Single Column Experiments Results in Predictable Streamlined Multi-Column Chromatography Bioprocesses.

An emphasis on continuous monoclonal antibody (mAb) bioprocessing in the pharmaceutical industry necessitates effective approaches for downstream process development (PD). With a PD strategy, the process parameters are optimized to directly develop streamlined three-step continuous chromatography processes. A design of experiment (DoE) approach with single column (batch mode) is used to simulate a multi-column (continuous mode) purification method and characterize each chromatography step: Protein A capture, anion exchange, and mixed mode cation exchange. A novel and targeted approach to quickly characterize a DoE design space was employed and empirical modeling was used to define the capacity for multi-column chromatography to accurately transfer the batch process to continuous mode of purification. This PD approach effectively mimics the continuous mode of operation and provides the flexibility to develop multiple continuous processes with target mAb recovery and purity. By implementing this PD strategy and the process parameters defined in batch mode, two robust and predictable continuous bioprocesses were developed within 7 weeks of investigation, which resulted in a total product yield of recovery at or above 74%, host cell protein (HCP) content below 5 ppm, and aggregate content below 1%.

Current state of the art in continuous bioprocessing.

There is an upsurge of interest in continuous bioprocessing, but currently continuous downstream bioprocessing has not been implemented to generate clinical material. This review focusses on the current state of the art of continuous downstream processing, highlighting the key advantages over traditional batch manufacturing. This allows the identification of scenarios where continuous downstream processing may be critical for commercial manufacturing success.

Transfer of a three step mAb chromatography process from batch to continuous: Optimizing productivity to minimize consumable requirements.

The goal of this study was to adapt a batch mAb purification chromatography platform for continuous operation. The experiments and rationale used to convert from batch to continuous operation are described. Experimental data was used to design chromatography methods for continuous operation that would exceed the threshold for critical quality attributes and minimize the consumables required as compared to batch mode of operation. Four unit operations comprising of Protein A capture, viral inactivation, flow-through anion exchange (AEX), and mixed-mode cation exchange chromatography (MMCEX) were integrated across two Cadence BioSMB PD multi-column chromatography systems in order to process a 25L volume of harvested cell culture fluid (HCCF) in less than 12h. Transfer from batch to continuous resulted in an increase in productivity of the Protein A step from 13 to 50g/L/h and of the MMCEX step from 10 to 60g/L/h with no impact on the purification process performance in term of contaminant removal (4.5 log reduction of host cell proteins, 50% reduction in soluble product aggregates) and overall chromatography process yield of recovery (75%). The increase in productivity, combined with continuous operation, reduced the resin volume required for Protein A and MMCEX chromatography by more than 95% compared to batch. The volume of AEX membrane required for flow through operation was reduced by 74%. Moreover, the continuous process required 44% less buffer than an equivalent batch process. This significant reduction in consumables enables cost-effective, disposable, single-use manufacturing.

A straightforward methodology for designing continuous monoclonal antibody capture multi-column chromatography processes.

A simple process development strategy for continuous capture multi-column chromatography (MCC) is described. The approach involves a few single column breakthrough experiments, based on several simplifying observations that enable users to rapidly convert batch processes into well-designed multi-column processes. The method was validated using a BioSMB(®) (Pall Life Sciences) lab scale multi-column system and a mAb capture process employing Protein A resin. The approach enables users to optimize MCC processes based on their internal preferences and constraints without requiring any mathematical modeling expertise.

Combined Protein A and size exclusion high performance liquid chromatography for the single-step measurement of mAb, aggregates and host cell proteins.

Quantification of monoclonal antibody (mAb) monomer, mAb aggregates, and host cell proteins (HCPs) is critical for the optimization of the mAb production process. The present work describes a single high throughput analytical tool capable of tracking the concentration of mAb, mAb aggregate and HCPs in a growing cell culture batch. By combining two analytical HPLC methods, Protein A affinity and size-exclusion chromatography (SEC), it is possible to detect a relative increase or decrease in the concentration of all three entities simultaneously. A comparison of the combined Protein A-SEC assay to SEC alone was performed, demonstrating that it can be useful tool for the quantification of mAb monomer along with trending data for mAb aggregate and HCP. Furthermore, the study shows that the Protein A-SEC method is at least as accurate as other commonly used analytical methods such as ELISA and Bradford.

Development of a tetrameric streptavidin mutein with reversible biotin binding capability: engineering a mobile loop as an exit door for biotin.

A novel form of tetrameric streptavidin has been engineered to have reversible biotin binding capability. In wild-type streptavidin, loop(3-4) functions as a lid for the entry and exit of biotin. When biotin is bound, interactions between biotin and key residues in loop(3-4) keep this lid in the closed state. In the engineered mutein, a second biotin exit door is created by changing the amino acid sequence of loop(7-8). This door is mobile even in the presence of the bound biotin and can facilitate the release of biotin from the mutein. Since loop(7-8) is involved in subunit interactions, alteration of this loop in the engineered mutein results in an 11° rotation between the two dimers in reference to wild-type streptavidin. The tetrameric state of the engineered mutein is stabilized by a H127C mutation, which leads to the formation of inter-subunit disulfide bonds. The biotin binding kinetic parameters (k(off) of 4.28×10(-4) s(-1) and K(d) of 1.9×10(-8) M) make this engineered mutein a superb affinity agent for the purification of biotinylated biomolecules. Affinity matrices can be regenerated using gentle procedures, and regenerated matrices can be reused at least ten times without any observable reduction in binding capacity. With the combination of both the engineered mutein and wild-type streptavidin, biotinylated biomolecules can easily be affinity purified to high purity and immobilized to desirable platforms without any leakage concerns. Other potential biotechnological applications, such as development of an automated high-throughput protein purification system, are feasible.

TagA is a secreted protease of Vibrio cholerae that specifically cleaves mucin glycoproteins.

Vibrio cholerae is a human diarrhoeal pathogen that is a major cause of gastrointestinal disease and death worldwide. Pathogenic V. cholerae strains are characterized by the presence of a Vibrio pathogenicity island (VPI) that encodes virulence factors, including the toxin co-regulated pilus (TCP). TagA is encoded within the VPI and is positively co-regulated with cholera toxin and TCP. TagA is a sequelogue of the StcE mucinase of Escherichia coli O157 : H7. We investigated whether this sequence homology reflected a conserved enzymic substrate profile. TagA exhibited metalloprotease activity toward crude purified mucins, salivary mucin and LS174T goblet cell surface mucin. Like StcE, TagA did not cleave general protease substrates, but unlike StcE, TagA did not cleave the mucin-like serpin C1 esterase inhibitor. Both proteins cleaved the immune cell surface mucin CD43, but TagA demonstrated reduced enzymic efficiency relative to StcE. TagA was expressed and secreted by V. cholerae under ToxR-dependent conditions. A tagA-deficient V. cholerae strain showed no defect in a model of in vitro attachment to the HEp-2 cell line; however, overexpression of a proteolytically inactive mutant, TagA(E433D), caused a significant increase in attachment. The increased attachment was reduced by pretreatment of epithelial monolayers with active TagA. Our results indicate that TagA is a mucinase and suggest that TagA may directly modify host cell surface molecules during V. cholerae infection.

Substituent effects on (15)N and (13)C NMR chemical shifts of 5-phenyl-1,3,4-oxathiazol-2-ones: a theoretical and spectroscopic study.

The synthesis and assignment of (15)N and (13)C NMR signals of the 1,3,4-oxathiazol-2-one ring in a series of para-substituted 5-phenyl derivatives are reported. DFT calculations of (15)N and (13)C chemical shifts correspond closely to observed values. Substituent effects are interpreted in terms of the Hammett correlation and calculated bond orders.

Substituent effects on 15N and 13C NMR chemical shifts of 3-phenylisoxazoles: a theoretical and spectroscopic study.

The synthesis and assignment of 15N and 13C NMR signals of the isoxazole ring in a series of para-substituted 3-phenyl derivatives are reported. DFT calculations of 15N and 13C chemical shifts are presented and compared to observed values. Substituent effects are interpreted in terms of the Hammett correlation and calculated bond orders.

Formation of a DNA mismatch repair complex mediated by ATP.

The mismatch repair proteins, MutS and MutL, interact in a DNA mismatch and ATP-dependent manner to activate downstream events in repair. Here, we assess the role of ATP binding and hydrolysis in mismatch recognition by MutS and the formation of a ternary complex involving MutS and MutL bound to a mismatched DNA. We show that ATP reduces the affinity of MutS for mismatched DNA and that the modulation of DNA binding affinity by nucleotide is even more pronounced for MutS E694A, a protein that binds ATP but is defective for ATP hydrolysis. Despite the ATP hydrolysis defect, E694A, like WT MutS, undergoes rapid, ATP-dependent dissociation from a DNA mismatch. Furthermore, MutS E694A retains the ability to interact with MutL on mismatched DNA. The recruitment of MutL to a mismatched DNA by MutS is also observed for two mutant MutL proteins, E29A, defective for ATP hydrolysis, and R266A, defective for DNA binding. These results suggest that ATP binding in the absence of hydrolysis is sufficient to trigger formation of a MutS sliding clamp. However, recruitment of MutL results in the formation of a dynamic ternary complex that we propose is the intermediate that signals subsequent repair steps requiring ATP hydrolysis.

DNA bending and unbending by MutS govern mismatch recognition and specificity.

DNA mismatch repair is central to the maintenance of genomic stability. It is initiated by the recognition of base-base mismatches and insertion/deletion loops by the family of MutS proteins. Subsequently, ATP induces a unique conformational change in the MutS-mismatch complex but not in the MutS-homoduplex complex that sets off the cascade of events that leads to repair. To gain insight into the mechanism by which MutS discriminates between mismatch and homoduplex DNA, we have examined the conformations of specific and nonspecific MutS-DNA complexes by using atomic force microscopy. Interestingly, MutS-DNA complexes exhibit a single population of conformations, in which the DNA is bent at homoduplex sites, but two populations of conformations, bent and unbent, at mismatch sites. These results suggest that the specific recognition complex is one in which the DNA is unbent. Combining our results with existing biochemical and crystallographic data leads us to propose that MutS: (i) binds to DNA nonspecifically and bends it in search of a mismatch; (ii) on specific recognition of a mismatch, undergoes a conformational change to an initial recognition complex in which the DNA is kinked, with interactions similar to those in the published crystal structures; and (iii) finally undergoes a further conformational change to the ultimate recognition complex in which the DNA is unbent. Our results provide a structural explanation for the long-standing question of how MutS achieves mismatch repair specificity.

DNA mismatch repair: molecular mechanisms and biological function.

DNA mismatch repair (MMR) guards the integrity of the genome in virtually all cells. It contributes about 1000-fold to the overall fidelity of replication and targets mispaired bases that arise through replication errors, during homologous recombination, and as a result of DNA damage. Cells deficient in MMR have a mutator phenotype in which the rate of spontaneous mutation is greatly elevated, and they frequently exhibit microsatellite instability at mono- and dinucleotide repeats. The importance of MMR in mutation avoidance is highlighted by the finding that defects in MMR predispose individuals to hereditary nonpolyposis colorectal cancer. In addition to its role in postreplication repair, the MMR machinery serves to police homologous recombination events and acts as a barrier to genetic exchange between species.