One-Step Purification Strategy for Cowpea Chlorotic Mottle Virus-Like Particles Produced by the IC-BEVS.

Virus-like particles (VLP) of the cowpea chlorotic mottle virus (CCMV), a plant virus, have been shown to be safe and noncytotoxic vehicles for delivering various cargos, including nucleic acids and peptides, and as scaffolds for presenting epitopes. Thus, CCMV-VLP have acquired increasing attention to be used in fields such as gene therapy, drug delivery, and vaccine development. Regardless of their production method, most reports purify CCMV-VLP through a series of ultracentrifugation steps using sucrose density gradient ultracentrifugation, which is a complex and time-consuming process. Here, the use of anion exchange chromatography is described as a one-step protocol for purification of CCMV-VLP produced by the insect cell-baculovirus expression vector system (IC-BEVS).

Using the reactive/transport dispersive models to simulate a monolithic anion exchanger: Experimental parameter determination, simultaneous model evaluation, and validation.

Selecting an adequate model to represent the mass transfer mechanisms occurring in a chromatographic process is generally complicated, which is one of the reasons why monolithic chromatography is scarcely simulated. In this study, the chromatographic separation of model proteins bovine serum albumin (BSA), ß-lactoglobulin-A, and ß-lactoglobulin-B on an anion exchange monolith was simulated based on experimental parameter determination, simultaneous model testing, and validation under three statistical criteria: retention time, dispersion accuracies, and Pearson correlation coefficient. Experimental characterization of morphologic, physicochemical, and kinetic parameters was performed through volume balances, pressure drop analysis, breakthrough curve analysis, and batch adsorptions. Free Gibbs energy indicated a spontaneous adsorption process for proteins and counterions. Dimensionless numbers were estimated based on height equivalent to a theoretical plate analysis, finding that pore diffusion controlled ß-lactoglobulin separation, whereas adsorption/desorption kinetics was the dominant mechanism for BSA. The elution profiles were modeled using the transport dispersive model and the reactive dispersive model coupled with steric mass action (SMA) isotherms because these models allowed to consider most of the mass transport mechanisms that have been described. RDM-SMA presented the most accurate simulations at pH 6.0 and at low (250 mM) and high (400 mM) NaCl concentrations. This simulation will be used as reference to forecast the purification of these proteins from bovine whey waste and to extrapolate this methodology to other monolith-based separations using these three statistical criteria that have not been used previously for this purpose.

Purificação de imunoglobulinas de plasma humano empregando Cromatografia de troca iônica

Derivatives of human plasma are essential for use in public health in the prevention and treatment of different diseases. Worldwide, the blood products market is leading and competitive. IgG is the blood product of greatest interest and accounts for most of the world market, however IgM has also become a target of interest, due to the efficiency of the pentamer form contributing with more benefits compared to IgG. In this project, the main objective was to separate the fractions containing IgG and IgM immunoglobulins from human plasma using liquid chromatography. In the first stage, the ANX Sepharose FF anion exchange resin was used, in which 98 % of the proteins present in human plasma were collected in the FT, and this fraction was applied to the second anion column in Q Sepharose FF, using a gradient of pH and saline. It was possible to obtain IgG in the FT, albumin in pH 5.5 and IgM in pH 4.5. The 3 fractions obtained require additional purification steps to obtain the final product.

Os derivados do plasma humano são indispensáveis para utilização no âmbito da saúde pública na prevenção e tratamento de distintas doenças. Mundialmente o mercado de hemoderivados são liderados e competitivos. A IgG é o hemoderivado de maior interesse e corresponde a maior parte do mercado mundial, entretanto a IgM também se tornou alvo de interesse, devido a eficiência da forma pentâmera contribuir com mais benefícios em relação a IgG. Neste projeto, o objetivo principal foi separar as frações contendo as imunoglobulinas IgG e IgM de plasma humano empregando cromatografia líquida. Na primeira etapa utilizou-se a resina de troca aniônica ANX Sepharose FF em que 98 % das proteínas presentes no plasma humano foram recolhidas no FT, e esta fração foi aplicada na segunda coluna aniônica em Q Sepharose FF empregando um gradiente de pH e salina. Foi possível obter IgG no FT, albumina em pH 5,5 e IgM em pH 4,5. As 3 frações obtidas requerem etapas adicionais de purificação para se obter o produto final.

Non-Cryoprecipitation Separation of Coagulation FVIII and Prothrombin Complex Proteins by Pseudoaffinity Calcium Elution Chromatography Using Anion Exchange Resin.

Hemophilia A is treated with human plasma coagulation factor VIII (FVIII) replacement therapy and Hemophilia B with coagulation factor IX, which is purified from prothrombin complex concentrate (PCC). In this paper we evaluated the separation of FVIII and PCC by directly loading raw thawed plasma to an anion exchange resin (AEX). Under this relatively high ionic strength, most of the plasma proteins such as albumin, immunoglobulins and others were not adsorbed. Five resins commonly used in protein purification (plasma fractionation) were tested. With all resins, PCC was eluted by pseudoaffinity in a calcium gradient step. Afterwards, FVIII could be recovered with a good yield and high purification factor in the salt gradient step with 400-500 mM NaCl. Using ANX Sepharose FF and Q Sepharose FF, the CaCl2 elution step was introduced after the intermediate wash with 200 mM NaCl, whereas using DEAE Sepharose FF, Fractogel EMD TMAE and Fractogel EMD DEAD, PCC eluted after the wash of the unbound proteins. Our results indicate that three important fractions: (1) albumin, immunoglobulin etc.; (2) PCC; and (3) FVIII can be separated in one chromatographic AEX column and the delicate and troublesome cryoprecipitation can be eliminated, making the purification of blood products faster and cheaper.

Synthesis of Anion Exchange Membranes Containing PVDF/PES and Either PEI or Fumion®.

In this work, the preparation of dense blended membranes, from blends of poly(vinylidene fluoride) (PVDF), poly(ether sulfone) (PES) and polyethyleneimine (PEI) or Fumion®, with possible applications in alkaline fuel cell (AEMFC) is reported. The blended PEI/Fumion® membranes were prepared under a controlled air atmosphere by a solvent evaporation method, and were characterized regarding water uptake, swelling ratio, thermogravimetric analysis (TGA), infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), ion exchange capacity (IEC), OH- conductivity and novel hydroxide ion exchange rate (HIER), which is related to the mass transport capacity of the OH- ions through the membrane. The effect of the chemical composition on its morphological and anion exchange properties was evaluated. It was expected that the usage of a commercial ionomer Fumion®, in the blended membranes would result in better features in the electrical/ionic conductivity behaviour. However, two of the membranes containing PEI exhibited a higher HIER and OH- conductivity than Fumion® membranes, and were excellent option for potential applications in AEMFC, considering their performance and the cost of Fumion®-based membranes.

A simple and economic three-step process for producing highly purified Fab' fragments directly from the egg yolk water-soluble fraction.

Although mammals are still the main source of polyclonal antibodies production, the use of egg yolk immunoglobulins (IgY) is increasing, and thus improvement of its safety to reduce undesired reactions is needed. Removal of IgY constant domains by pepsin enzymatic treatment is expected to reduce potential adverse effects derived from the parenteral administration of these antibodies, while also increasing its distribution volume. Current Fab' production requires the previous purification of IgY to be used as starting material. In this context, relevant economic benefits may be gained if initial IgY purification could be avoided, by using rawer starting materials. In this work, a three-step process for Fab' production from crude egg yolk water-soluble fractions is described using scalable and simple low-cost technologies such as ultrafiltration and anion exchange chromatography. The overall process yield of 33% highly pure Fab' from water-soluble fractions favorably compares to the manufacture of related medicinal products from mammalian antibodies, such as antivenoms.

A comprehensive method for modeling and simulating ion exchange chromatography of complex mixtures

In recent years, many biological-based products have been developed, representing a significant fraction of income in the pharmaceutical market. Ion exchange chromatography is an important downstream step for the purification of target recombinant proteins present in clarified cell extracts, together with many other unknown impurities. This work develops a robust approach to model and simulate the purification of untagged heterologous proteins, so that the improved conditions to carry out an ion exchange chromatography are identified in a rational basis prior to the real purification run itself. Purification of the pneumococcal surface protein A (PspA4Pro) was used as a case study. This protein is produced by recombinant Escherichia coli and is a candidate for the manufacture of improved pneumococcal vaccines. The developed method combined experimental and computational procedures. Different anion exchange operating conditions were mapped in order to gather a broad range of representative experimental data. The equilibrium dispersive and the steric mass action equations were used to model and simulate the process. A training strategy to fit the model and separately describe the elution profiles of PspA4Pro and other proteins of the cell extract was applied. Based on the simulation results, a reduced ionic strength was applied for PspA4Pro elution, leading to increases of 14.9% and 11.5% for PspA4Pro recovery and purity, respectively, compared to the original elution profile. These results showed the potential of this method, which could be further applied to improve the performance of ion exchange chromatography in the purification of other target proteins under real process conditions.

Non-cryoprecipitation separation of coagulation FVIII and prothrombin complex proteins by pseudoaffinity calcium elution chromatography using anion exchange resin

Hemophilia A is treated with human plasma coagulation factor VIII (FVIII) replacement therapy and Hemophilia B with coagulation factor IX, which is purified from prothrombin complex concentrate (PCC). In this paper we evaluated the separation of FVIII and PCC by directly loading raw thawed plasma to an anion exchange resin (AEX). Under this relatively high ionic strength, most of the plasma proteins such as albumin, immunoglobulins and others were not adsorbed. Five resins commonly used in protein purification (plasma ractionation) were tested. With all resins, PCC was eluted by pseudoaffinity in a calcium gradient step. Afterwards, FVIII could be recovered with a good yield and high purification factor in the salt gradient step with 400–500 mM NaCl. Using ANX Sepharose FF and Q Sepharose FF, the CaCl2 elution step was introduced after the intermediate wash with 200 mM NaCl, whereas using DEAE Sepharose FF, Fractogel EMD TMAE and Fractogel EMD DEAD, PCC eluted after the wash of the unbound proteins. Our results indicate that three important fractions: (1) albumin, immunoglobulin etc.; (2) PCC; and (3) FVIII can be separated in one hromatographic AEX column and the delicate and troublesome cryoprecipitation can be eliminated, making the purification of blood products faster and cheaper.

Electrospinning of Polyepychlorhydrin and Polyacrylonitrile Anionic Exchange Membranes for Reverse Electrodialysis.

The saline gradient present in river mouths can be exploited using ion-exchange membranes in reverse electrodialysis (RED) for energy generation. However, significant improvements in the fabrication processes of these IEMs are necessary to increase the overall performance of the RED technology. This work proposes an innovative technique for synthesizing anion exchange membranes (AEMs) via electrospinning. The AEM synthesis was carried out by applying a high voltage while ejecting a mixture of polyepichlorohydrin (PECH), 1,4-diazabicyclo [2.2.2] octane (DABCO® 33-LV) and polyacrylonitrile (PAN) at room temperature. Different ejection parameters were used, and the effects of various thermal treatments were tested on the resulting membranes. The AEMs presented crosslinking between the polymers and significant fiber homogeneity with diameters between 1400 and 1510 nm, with and without thermal treatment. Good chemical resistance was measured, and all synthesized membranes were of hydrophobic character. The thickness, roughness, swelling degree, specific fixed-charge density and ion-exchange capacity were improved over equivalent membranes produced by casting, and also when compared with commercial membranes. Finally, the results of the study of the electrospinning parameters indicate that a better performance in electrochemical properties was produced from fibers generated at ambient humidity conditions, with low flow velocity and voltage, and high collector rotation velocity.

Study of Polyaniline/Poly(Sodium 4-Styrenesulfonate) Composite Deposits Using an Electrochemical Quartz Crystal Microbalance for the Modification of a Commercial Anion Exchange Membrane.

One of the intended applications for the modification of ion exchange membranes with polyaniline (PAni) is to use it as a matrix to include chemical species that confer a special property such as resistance to fouling or ion selectivity. In particular, the inclusion of polyelectrolyte molecules into the PAni matrix appears to be the way to modulate these properties of selective membranes. Therefore, it must be clearly understood how the polyelectrolyte is incorporated into the matrix of polyaniline. Among the results obtained in this paper using poly(sodium 4-styrenesulfonate) (PSS) and an electrochemical quartz crystal microbalance, the amount of polyelectrolyte incorporated into PAni is found to be proportional to the PSS concentration in solution if its value is between 0 and 20 mM, while it reaches a maximum value when the PSS in solution is greater than 20 mM. When the anion exchange membranes are modified with these composite deposits, the transport number of chloride was found to decrease progressively (when the PSS concentration in solution is between 0 and 20 mM) to reach a practically constant value when a concentration of PSS greater than 20 mM was used.

Feasibility of quaternary ammonium and 1,4-diazabicyclo[2.2.2]octane-functionalized anion-exchange membranes for biohydrogen production in microbial electrolysis cells.

In this work, two commercialized anion-exchange membranes (AEMs), AMI-7001 and AF49R27, were applied in microbial electrolysis cells (MECs) and compared with a novel AEM (PSEBS CM DBC, functionalized with 1,4-diazabicyclo[2.2.2]octane) to produce biohydrogen. The evaluation regarding the effect of using different AEMs was carried out using simple (acetate) and complex (mixture of acetate, butyrate and propionate to mimic dark fermentation effluent) substrates. The MECs equipped with various AEMs were assessed based on their electrochemical efficiencies, H2 generation capacities and the composition of anodic biofilm communities. pH imbalances, ionic losses and cathodic overpotentials were taken into consideration together with changes to substantial AEM properties (particularly ion-exchange capacity, ionic conductivity, area- and specific resistances) before and after AEMs were applied in the process to describe their potential impact on the behavior of MECs. It was concluded that the MECs which employed the PSEBS CM DBC membrane provided the highest H2 yield and lowest internal losses compared to the two other separators. Therefore, it has the potential to improve MECs.

Unravelling the Photocatalytic Behavior of All-Inorganic Mixed Halide Perovskites: The Role of Surface Chemical States.

Within the most mesmerizing materials in the world of optoelectronics, mixed halide perovskites (MHPs) have been distinguished because of the tunability of their optoelectronic properties, balancing both the light-harvesting efficiency and the charge extraction into highly efficient solar devices. This feature has drawn the attention of analogous hot topics as photocatalysis for carrying out more efficiently the degradation of organic compounds. However, the photo-oxidation ability of perovskite depends not only on its excellent light-harvesting properties but also on the surface chemical environment provided during its synthesis. Accordingly, we studied the role of surface chemical states of MHP-based nanocrystals (NCs) synthesized by hot-injection (H-I) and anion-exchange (A-E) approaches on their photocatalytic (PC) activity for the oxidation of ß-naphthol as a model system. We concluded that iodide vacancies are the main surface chemical states that facilitate the formation of superoxide ions, O2●-, which are responsible for the PC activity in A-E-MHP. Conversely, the PC performance of H-I-MHP is related to the appropriate balance between band gap and a highly oxidizing valence band. This work offers new insights on the surface properties of MHP related to their catalytic activity in photochemical applications.

Functionality of Agave Bagasse as Supplement for the Development of Prebiotics-Enriched Foods.

Agave bagasse is a fibrous-like material obtained during aguamiel extraction, which is also in contact with indigenous microbiota of agave plant during aguamiel fermentation. This plant is a well-known carrier of the prebiotic fructan-type carbohydrates, which have multiple ascribable health benefits. In the present work, the potential of ashen and green agave bagasse as functional ingredients in supplemented cookies was studied. For its application, the chemical, functional, properties of agave bagasses and formulated cookies were evaluated, as well as the physical properties of cookies. Chemical characterization was carried out by the proximate analysis of both bagasses and cookies, besides, the analysis of oligosaccharides was made by thin-layer chromatography and high-performance anion-exchange chromatography. In the same way, functional properties such as oil holding capacity, organic molecule absorption capacity, swelling capacity, and water holding capacity were analyzed in both agave bagasses and supplemented cookies. Finally, modifications in color and texture due to bagasse addition was studied through an analysis of total color difference and a penetrometric test, respectively. In this sense, ashen and green agave bagasses demonstrated chemical and functional properties for use in the food industry, since they increased oil holding capacity of cookies and transferred prebiotic fructooligosaccharides to both agave bagasse formulations, which remain active as a prebiotic ingredient in cookies after in vitro digestion and cookie manufacture, including thermal treatment. Hence, agave bagasse could be considered a valuable alternative for the addition of the nutritionally-relevant dietary fiber in healthier foods.

Modeling and simulation of anion exchange chromatography for purification of proteins in complex mixtures

Ion exchange chromatography is extensively used in the purification of biological compounds. Reliable mathematical models describing this chromatographic technique are available and can be used to improve the performance of this separation step. However, the use of synthetic mixtures for model development hampers the application of this approach with real cell extracts processed in downstream operations. This work presents an original approach for handling non-synthetic genuine mixtures of proteins, which was applied in the purification of an untagged recombinant pneumococcal surface protein A (PspA4Pro). First, evaluation was made of the efficiency of steric mass action (SMA) and modified Langmuir isotherms, which were separately used together with the equilibrium dispersive model (EDM). The data used for parameter estimation and model validation were obtained from anion exchange chromatography runs (employing Q-Sepharose FF), applied to real cell extracts produced by different cultivation strategies. Simulations showed that the models were able to describe the complex mixtures of unknown proteins. Next, the EDM and SMA approaches were used to separately describe the profile of PspA4Pro and the pool of protein impurities eluted together. The simulations showed that PspA4Pro tended to elute at the beginning of the peak, enabling the establishment of an alternative elution schedule that provided a 34% increase in the purity achieved using the anion exchange chromatography.

Phosphorus availability and dynamics in soil affected by long-term ruzigrass cover crop.

The use of grasses as cover crops in the off-season of cash crops under no-till has been largely adopted. However, soil phosphorus (P) uptake was previously shown to be reduced when ruzigrass is introduced in the rotation, affecting the viability and sustainability of this cropping system. The objective of this study was to assess the effect of ruzigrass on soil P availability and desorption kinetics under different P fertilizer application rates. A long-term field experiment where soybean (Glycine max) has been grown in rotation with ruzigrass (Urochloa ruziziensis) or fallow for 10 years, with the application of 0, 13, and 26 kg ha-1 of P, was evaluated for two consecutive years. Soil P desorption kinetics was assessed using diffusive equilibrium (DET) and gradient in thin films (DGT) techniques, as well as the DGT-induced fluxes in soils model (DIFS). Microbial biomass P (MBP) was assessed to verify if soil solution P (PDET) was reduced due to immobilization by microorganisms. Ruzigrass reduced MBP and PDET especially when P fertilizer was applied. The concentration of labile P (PDGT) was also lower after ruzigrass than in fallow. The soil ability to resupply P to soil solution was lower after ruzigrass regardless of P rates due to a slower desorption in response to the perturbation imposed by DGT. Growing ruzigrass as cover crop in the soybean off-season decreases soil P availability regardless of P fertilizer application rates by fundamentally reducing P mobility and P resupply from soil solid phase into soil solution.

Membrane electrolysis for the removal of Mg2+ and Ca2+ from lithium rich brines.

Lithium is today an essential raw material for renewable energy technologies and electric mobility. Continental brines as present in the Lithium Triangle are the most abundant and the easiest to exploit lithium sources. Lithium is present in diluted concentrations together with different ions, and it is imperative to fully remove both magnesium and calcium before lithium carbonate can be precipitated. Here we use membrane electrolysis as a novel method to generate hydroxyl groups in situ in a two-chamber electrochemical cell with a side crystallizer, omitting the need for chemical addition and not leading to substantial loss of lithium rich brine. Batch electrolysis experiments fully removed more than 99.99% of both Mg2+ and Ca2+ for three different native South-American brines treated at current densities ranging from 27 to 350 A m-2 (final concentrations were below ICP detection limit: < 0.05 mg L-1). For a brine containing 3090 mg L-1 of Mg2+ and 685 mg L-1 of Ca2+, 62 kWh m-3 are needed for the full removal of both cations when a current density of 223 A m-2 is employed. Most importantly, the Li+ concentration in the brine is not affected. The removed cations are precipitated as Mg(OH)2 and Ca(OH)2. Our process has the potential to simultaneously recover lithium, magnesium, and calcium compounds, minimizing waste production.

Removal of both N-nitrosodimethylamine and trihalomethanes precursors in a single treatment using ion exchange resins.

Drinking water utilities are relying more than ever on water sources impacted by wastewater effluents. Disinfection/oxidation of these waters during water treatment may lead to the formation of several disinfection by-products, including the probable human carcinogen N-nitrosodimethylamine (NDMA) and the regulated trihalomethanes (THMs). In this study, the potential of ion exchange resins to control both NDMA and THMs precursors in a single treatment is presented. Two ion exchange resins were examined, a cation exchange resin (Plus) to target NDMA precursors and an anion exchange resin (MIEX) for THMs precursors control. We applied the resins, individually and combined, in the treatment of surface and wastewater effluent samples. The treatment with both resins removed simultaneously NDMA (43-85%) and THMs (39-65%) precursors. However, no removal of NDMA precursors was observed in the surface water with low initial NDMA FP (14 ng/L). The removals of NDMA FP and THMs FP with Plus and MIEX resins applied alone were (49-90%) and (41-69%), respectively. These results suggest no interaction between the resins, and thus the feasibility of effectively controlling NDMA and THMs precursors concomitantly. Additionally, the effects of the wastewater impact and the natural attenuation of precursors were studied. The results showed that neither the wastewater content nor the attenuation of the precursor affected the removals of NDMA and THMs precursors. Finally, experiments using a wastewater effluent sample showed that an increase in the calcium concentration resulted in a reduction in the removal of NDMA precursors of about 50%.

Dataset on protein composition of a human plasma sub-proteome able to modulate the Dengue 2 virus infection in Huh 7.5 cells.

The four serotypes of dengue virus (DENV1-4) are the causal agents of the emerging disease Dengue Fever and its severe forms. DENV is inoculated into human blood through a mosquito bite. Thus, plasma is an important media for DENV dissemination in infected persons and several important interactions should take place for the virus with human plasma proteins that strongly influence or may determine the course of the infection. This dataset contains 239 proteins identified in the elution fractions of human plasma subjected to DE-52 anion exchange chromatography. Data on DENV2 infection of Huh 7.5 cells in presence of the human plasma fraction is also presented.

Synthesis of tunable-band-gap "Open-Box" halide perovskites by use of anion exchange and internal dissolution procedures.

We demonstrate the synthesis of cuboid MAPbBr3 (MA=CH3NH3) microcrystals and subsequent conversion into open-box-like MAPb(Br(1-x)I(x))3 (0⩽x⩽1) microcrystals by anion exchange in MAI solution. During the substitution of Br(-) with I(-), the initial cuboid framework of MAPbBr3 crystals is retained. The preferential internal dissolution of MAPbBr3 due to the surface coverage and protection of MAPb(Br(1-x)I(x))3 induces voids inside the cuboid crystals, finally leading to open-box-like iodide-rich MAPb(Br(1-x)I(x))3. By controlling the degree of anion exchange, the intense light absorption of the product is able to be tuned in specific wavelengths throughout the visible range. This solution-phase anion exchange approach provides a synthetic strategy in designing sophisticated organolead halide perovskites structures as well as tuning the band gaps for further applications across a range of possible domains.

Purification of coagulation factor VIII by immobilized metal affinity chromatography.

Factor VIII (FVIII) is a glycoprotein that plays an essential role in blood coagulation cascade. Purification of plasma-derived coagulation FVIII by direct application of plasma to a chromatographic column is a method of choice. Anion exchange column is a very powerful method because FVIII is strongly adsorbed, resulting in good activity recovery and high purification factor. However, vitamin-K-dependent coagulation factors coelute with FVIII. In the present study, we report the separation of vitamin-K-dependent coagulation proteins from FVIII using immobilized metal affinity chromatography (IMAC) with Cu(2+) as the metal ligand. Plasma was directly loaded to a Q Sepharose Big Beads column, and FVIII was recovered with 65% activity and a purification factor of approximately 50 times. Then, the Q Sepharose eluate was applied to the IMAC-Cu(2+) column, and FVIII was eluted with 200 mM imidazole, with up to 85% recovery of activity. The mass recovery in this fraction was less than 10% of the applied mass of protein. Vitamin-K-dependent proteins elute with imidazole concentrations of lower than 60 mM. Because of the difference in affinity, FVIII could be completely separated from the vitamin-K-dependent proteins in the IMAC column.