Final Purification Step Research Articles

Optimization of VPSA-EHP/C process for high-pressure hydrogen recovery from Coke Oven Gas using CO selective adsorbent

The production of hydrogen through conventional pathways and recovery from by-products typically utilize pressure swing adsorption (PSA) technology as final purification step. Dual-layered PSA columns packed with conventional activated carbon and molecular sieve 5A material exhibit relatively low selectivity for O2, N2 and CO in particular. Therefore, eliminating CO (and other poisons) using conventional PSA to acceptable concentrations for EHP/C is only achievable with lower recovery rates. To improve recovery rates, there is a need for a highly efficient purification process that is highly selective for these hydrogen contaminants without compromising the product quality. Here we report an optimization study where vacuum PSA (VPSA) and electrochemical hydrogen purification and compression (EHP/C) technology is utilized for purification and compression of hydrogen from Coke Oven Gas (COG). The VPSA columns were packed with activated carbon and CuCl(7.0)-activated carbon to selectively retain poisonous CO2 and CO, respectively. The optimal operating conditions were determined with surrogate models produced via non-linear regression of known sample input-output data points, by varying the adsorbent layering ratio (0.30–0.84), adsorption pressure (0.38–0.78 MPa), purge to feed ratio (P/F-ratio) (1–10%), adsorption step time (100–1500 s) and the EHP/C stack current per cell (37–52 A) in the original models. The two-bed VPSA system obtained 90.5% recovery and retained CO and CO2 below their thresholds at 0.84 layering ratio, 0.78 MPa adsorption pressure, 840s adsorption time and 5.3% P/F-ratio, at the expense of H2 purity (77.1%) by breakthrough of CH4, N2 and O2. Hydrogen purity was upgraded to >99.999% by EHP/C, which recovered 90.0% of hydrogen and simultaneously compressed to 20 MPa, which required 3.2 kWh/kg H2. The overall VPSA-EHP/C recovery rate in this configuration was 81.5%. By utilizing the EHP/C retentate gas as VPSA purge gas, overall VPSA-EHP/C recovery rates may reach 87.3% and consume less energy due to a decrease in adsorption pressure. We show that adsorption columns designed to function as poisonous component eliminator are an effective strategy to pre-condition hydrogen synthesis gases prior to further processing with EHP/C. Although the EHP/C was exposed to significant concentrations of methane, nitrogen and oxygen by their advancement through VPSA, the performance was only slightly affected. The VPSA-EHP/C method is applicable to a wide range of hydrogen gas mixtures that require further purification and compression. Traditional PSA for purification from primary and by-product (COG, annealing, chlor-alkali and flat/float glass manufacturing) hydrogen sources can be changed to a VPSA-EHP/C systems for hydrogen purification and simultaneous compression.

Expression and purification of the antimicrobial peptide Bin1b in Escherichia coli tagged with the fusion proteins CusF3H+ and SmbP

We have previously shown that the small metal-binding proteins CusF3H+ and SmbP can be used as fusion proteins for the expression and purification of recombinant proteins in Escherichia coli. Because of their small size, both around 10 kDa, they are suitable for the production of peptides to avoid meager yields after the final purification step of tag removal. Bin1b is a beta-defensin found in the epididymis of rats that has shown to have antimicrobial activity. Previous methodologies used to express this antimicrobial peptide in E. coli involve the expression of the peptide as inclusion bodies followed by in vitro refolding or the supplementation of the proteins necessary for proper folding of the peptide in the cytoplasm via a second plasmid. Here, we developed a methodology that forgoes these approaches and instead uses the fusion proteins CusF3H+ or SmbP and the E. coli strain SHuffle to obtain a soluble recombinant protein that contains the mature Bin1b peptide. The recombinant protein is purified using IMAC chromatography and is subsequently cleaved with enterokinase to separate the fusion protein from Bin1b. The purified peptide displays antimicrobial activity against E. coli, as previously shown. Furthermore, we also tested its antimicrobial activity against the Gram-positive bacteria Staphylococcus aureus and found that Bin1b is also capable of inhibiting the growth of this bacterium. In conclusion, we developed a practical methodology for the expression and purification of the bioactive Bin1b peptide in E. coli using the fusion proteins CusF3H+ and SmbP. This approach could be further applied for the production of more biologically active peptides.

Extraction and Purification of Lipases Enzyme from Germinating Seeds of Four Crops

Lipase enzyme has attracted a lot of attention in recent years because of its diverse biotechnological applications. The present study was conducted to screen germinated seeds of four crops, namely sunflower (Helianthus annuus), flaxor linseed (Linum usitatissimum ), peanut (Arachis hypogaea ) and castor bean (Ricinus communis), for the activity of their lipases. to the study also included the extraction and purification of lipase from the seeds of the most promising crop using different solvents.
 The results indicated that the maximum enzymatic activity (0.669 U/ml) was obtained when 0.1 M Tris-HCl buffer extract was used after 3 days of seed germination of all the tested species, as compared to the other test solvents (acetone and water). Sunflower germinated seeds showed the highest lipase activity, which was higher by 159.67, 185.32, and 285.90 % over the activities of castor bean, flax, and peanut seeds, respectively. Among the used ranges of saturation of ammonium sulfate, the ratio of 70% was the best in precipitating the crude enzyme, showing a highest specific activity of 2.576 U/ mg protein.
 The first stage of gel filtration chromatography column by Sephadex G-200 indicated the presence of two non-identical peaks, one for protein and another for lipase activity, between the fractions of 18 to 23. The active fractions were pooled and loaded again in the Sephadex G-200 column and the eluted fractions showed two identical peaks, one for protein and another for lipase activity, between the fractions of 19 to 23. The final purification step by gel filtration showed a specific activity of 6.482 U/mg proteins with a yield of 38.75 % and 11.33 folds of time of purification. The study revealed that sunflower seeds are a better source of lipase as compared to the other used plant seeds, which can be used in different industries.

Optimizing the α1B-adrenergic receptor for solution NMR studies

Sample preparation for NMR studies of G protein-coupled receptors faces special requirements: Proteins need to be stable for prolonged measurements at elevated temperatures, they should ideally be uniformly labeled with the stable isotopes 13C, 15N, and all carbon-bound protons should be replaced by deuterons. In addition, certain NMR experiments require protonated methyl groups in the presence of a perdeuterated background. All these requirements are most easily satisfied when using Escherichia coli as the expression host. Here we describe a workflow, starting from a temperature-stabilized mutant of the α1B-adrenergic receptor, obtained using the CHESS methodology, into an even more stable species, in which flexible parts from termini were removed and the intracellular loop 3 (ICL3) was stabilized against proteolytic cleavage. The yield after purification corresponds to 1–2 mg/L of D2O culture. The final purification step is ligand-affinity chromatography to ensure that only well-folded ligand-binding protein is isolated. Proper selection of detergent has a remarkable influence on the quality of NMR spectra. All optimization steps of sequence and detergent are monitored on a small scale by monitoring the melting temperature and long-term thermal stability to allow for screening of many conditions. The stabilized mutant of the α1B-adrenergic receptor was additionally incorporated in nanodiscs, but displayed slightly inferior spectra compared to a sample in detergent micelles. Finally, both [15N,1H]- as well as [13C,1H]-HSQC spectra are shown highlighting the high quality of the final NMR sample. Importantly, the quality of [13C,1H]-HSQC spectra indicates that the so prepared receptor could be used for studying side-chain dynamics.

Extraction, Purification and Characterization of Transglutaminase from Silver Beet Leaves and its Effect on Sensory, Chemical and Rheological Properties of Kareish Cheese

Strategy in transgluaminase (TGase) extracted from silver beet leaves (Beta vulgaris L.) performed by three stages. Ammonium sulfate precipitation (40-80%) is an important technique as an initial step in TGase purification, followed by an impure TGase sample is loaded into 2-(Diethylamino)ethyl ether sephadex A50. The final purification step was carried out by eluting dialyzed fractions into Sephadex G-100. The purified TGase was successfully purified by three purification steps with 224.2 U/mg of TGase specific activity, 3.4 fold increase in purification and 20.33% yields. The optimum temperature and pH of silver beet leaves TGase were 50°C and 5.0, respectively. The TGase completely lost its activity after 15 min at 80°C, however recovery of TGase after exposure to sodium chloride concentrations ranging from 2-6% does not affect its activity. The influence of different concentrations (5, 7.5 and 10U/g protein) of TGase on chemical, textural and sensorial of Kareish over storage time were detected. The yield and moisture content were reached the maximum values in cheese treated with TGase (10Ug-1protein), in addition to the same treatment received maximum average scores in sensory evaluation. The concentration of TGase is negatively proportional with the gumminess, adhesiveness and hardnesss values of resulted cheese.

Computation-assisted analyzing and forecasting on impurities removal behavior during zone refining of antimony

High purity antimony (Sb) gains nowadays a rising interest for high-tech applications. Zone refining is commonly used as a reliable final purification step to achieve higher purity levels of Sb. However, the removal of arsenic (As) - the most troublesome impurity in Sb often occurring in quite high amounts - stays as the challenge during zone refining, due to its very unfavorable distribution coefficient. In addition, there are two important factors, essential to predict the zone refining efficiency and the required time (required number of passes and heater movement velocity), which are still seldom explored. These factors are first the effective distribution coefficient (keff) and second the ratio of the diffusion layer thickness to the diffusion constant of each impurity (δ/D). Therefore, in the present work, these two research-demanding aspects have been investigated. Firstly, the possibility of removing vaporized As through introducing an inert carrying gas flux was studied by testing two different initial concentrations of As (670 and 430 ppm) and undergoing different process parameters. Higher initial concentrations of As showed to be slightly (10 %) reduced when the melting time increases. Lower As concentration, however, remains almost stable. On the other hand, using the so-called Spim-model, zone refining of Sb was conducted under 1 L/min Nitrogen gas flux at different heater moving velocities (2, 1 and 0.5mm/min) to calculate the keff of impurities, derived by fitting the experimental impurity distribution with the simulated profiles. Finally and with the known values of keff, the values of δ/D for each impurity could be then retrieved by applying the BPS model. That not only allows predicting keff for any moving velocity based on Spim-model, but also provides an alternative way to calculate these two values when one of them was obtained from other technologies.

Non-chemical water treatment in unified light and sound field and testing on waste water decontamination

The paper presents the results of testing of a high-performance method for decontaminating sewage, involving combination of ultrasonic and ultraviolet exposure at the final step of water purification aimed at destroying pathogenic microorganisms and thereby preventing the spread of infectious diseases. A variation of the method was developed and patented by the authors and passed the test at one of the most advanced water facilities in Russia which is the Vodokanal of St. Petersburg. The combined unit was manufactured at OOO Novotech-EKO and installed at the South-West Wastewater Treatment Plant (SWTP) of Vodokanal of St. Petersburg. The results of the test showed the efficiency of combined ultraviolet and ultrasonic wastewater treatment, both in terms of intensifying disinfection and in terms of the stability of the plant operation by preventing biofouling and salt deposition on the surface of the lamp casings. The latter fact was the basis for the pilot project of improvement of the existing system of UV wastewater disinfection at the SWTP.

Optimization of solid-phase extraction (SPE) in the preparation of [18F]D3FSP: A new PET imaging agent for mapping Aβ plaques

IntroductionAlzheimer's disease is a common neurodegenerative disease that is characterized by the presence of Aβ plaques in the brain. The FDA has approved the use of Amyvid (florbetapir f18, AV-45) as a PET imaging agent for detecting Aβ plaques in the living human brain. In an attempt to reduce N-demethylation in vivo by taking advantage of more stable C-D bonds, an analog of AV-45, [18F]D3FSP ([18F]7), was synthesized to improve image contrast for detecting and monitoring the Aβ plaques. A convenient and improved preparation of [18F]D3FSP ([18F]7) is needed for widespread clinical application. We report herein the optimization of the radiosynthesis and solid-phase extraction (SPE) procedure. MethodsRadiosyntheses of [18F]D3FSP ([18F]7) under different fluorination conditions were evaluated, and the intermediate, containing an N-Boc protecting group, was deprotected using different acids. One of the major objectives was to simplify the final purification step via SPE to avoid the commonly employed HPLC purification and maximize the radiochemical yields of [18F]D3FSP ([18F]7) while simultaneously removing several chemical impurities (pseudocarriers). Washing various solid-phase cartridges with different combinations of ethanol/water and acetonitrile/water was explored to optimize the purification step. To evaluate the potential interference in Aβ plaques imaging from the presence of pseudocarriers, each chemical was identified and quantified by LC/MS and HPLC. An in vitro binding assay was employed to evaluate the binding affinities of [18F]D3FSP ([18F]7) and the pseudocarriers to Aβ plaques using postmortem AD brain tissue. ResultsUsing the optimized radiosynthesis method and SPE purification, the final dose of [18F]D3FSP ([18F]7) was obtained in 50 min with a very low content of pseudocarriers (21.7 ± 5.5 μg). The radiochemical yield was 44.4 ± 5.7% (decay corrected), and the radiochemical purity was >95%. SPE-purified doses of [18F]D3FSP ([18F]7) displayed excellent binding affinity and specificity for Aβ plaques as measured in an in vitro binding assay using AD brain homogenates, and the OH-pseudocarrier, 8 (Ki = 19.5 ± 0.5 nM), and the Cl-pseudocarrier, 10 (Ki = 18.6 ± 3.9 nM), showed lower binding affinities for Aβ plaques than those of AV-45 (Ki = 8.6 ± 0.5 nM) and D3FSP, 7 (Ki = 9.8 ± 0.5 nM). ConclusionsAn optimized radiosynthesis and fast SPE purification method suitable for the preparation of clinical doses of [18F]D3FSP ([18F]7) was accomplished. The results of quality control tests and binding studies suggested that the SPE-purified doses of [18F]D3FSP ([18F]7) are appropriate for imaging Aβ plaques in the human brain.

Characterization of Antifungal Lipopeptide Biosurfactants Produced by Marine Bacterium Bacillus sp. CS30.

This study was initiated to screen for marine bacterial agents to biocontrol Magnaporthe grisea, a serious fungal pathogen of cereal crops. A bacterial strain, isolated from the cold seep in deep sea, exhibited strong growth inhibition against M. grisea, and the strain was identified and designated as Bacillus sp. CS30. The corresponding antifungal agents were purified by acidic precipitation, sequential methanol extraction, Sephadex LH-20 chromatography, and reversed phase high-performance liquid chromatography (RP-HPLC), and two antifungal peaks were obtained at the final purification step. After analysis by mass spectrometry (MS) and tandem MS, two purified antifungal agents were deduced to belong to the surfactin family, and designated as surfactin CS30-1 and surfactin CS30-2. Further investigation showed that although the antifungal activity of surfactin CS30-1 is higher than that of surfactin CS30-2, both of them induced the increased generation of reactive oxygen species (ROS) and caused serious damage to the cell wall and cytoplasm, thus leading to the cell death of M. grisea. Our results also show the differences of the antifungal activity and antifungal mechanism of the different surfactin homologs surfactin CS30-1 and surfactin CS30-2, and highlight them as potential promising agents to biocontrol plant diseases caused by M. grisea.

A comparative study of continuous operation between a dynamic baffle crystallizer and a stirred tank crystallizer

Crystallization, often as the final isolation and purification step in drug substance manufacturing, has substantial impact on downstream efficiency and final drug product quality. It is a critical but challenging step in developing end-to-end continuous manufacturing, which has been identified as an emerging technology in the pharmaceutical manufacturing sector by the U.S. Food and Drug Administration (FDA). A traditional stirred tank crystallizer (STC) operated as a mixed-suspension-mixed-product-removal (MSMPR) system is a popular choice for continuous crystallization for its utilization of existing knowledge and equipment. However, there are disadvantages associated with agitational systems like the STC, such as poor local mixing and high shear rate. A commercial dynamic baffle crystallizer (DBC) was studied here as an alternative unit operation. The DBC in this study consists of a jacketed glass vessel with dynamic ‘donut’ shaped baffles to provide oscillatory mixing to improve heat and mass transfer while exerting less shear. Our goal is to compare continuous operation performances in the DBC with the STC as MSMPR systems. First, residence time distribution (RTD) studies of both systems were performed considering single phase (liquid only) and two-phase (solid-liquid) operation. The RTD gives good insights into the mixing dynamics without computationally heavy fluid dynamic simulations. Continuous cooling crystallization of paracetamol was performed in the DBC and the STC. The DBC showed good potential to be used as an MSMPR system producing more uniform RTDs as well as higher quality crystallization products compared to a traditional STC.

Isolation and Characterization of Cellulase-producing Bacteria from Sugar Industry Waste

Cellulases are inducible enzymes that are synthesized by a large number of microorganisms during their growth on cellulosic materials. This study focuses on the isolation and screening of cellulase-producing bacteria from sugar industry waste (molasses) and characterization by morphological and biochemical analysis. Further, purification of cellulase was carried by ammonium sulfate precipitation and followed by column chromatography and molecular weight determined by SDS-PAGE. The isolated bacterial strains were grown on carboxymethyl cellulose (CMC) agar plate at various optimum conditions like pH, temperature, incubation period, carbon and nitrogen sources and substrate concentration. Three isolated strains showed clear hydrolyzing zone on agar plates containing CMC agar after Congo-red staining were identified as cellulase-producing bacteria. Based on cultural, morphological, and biochemical characteristics, the isolated strains were identified as <i>Paenibacillus sp., Aeromonas sp.</i>, and <i>Bacillus sp</i>. Among the isolated strains, <i>Paenibacillus sp</i>. showed the capability for highest cellulase production (0.89 µmol ml^-1 min^-1) at optimal pH 7.0 and 40°C temperature on 24 hour of the incubation period at 1% CMC substrate concentration and was selected for further cellulase purification. In the final step of cellulase purification, the specific activity, purification fold and recovery were 1720 U/mg, 9.74 and 35.6%, respectively. The molecular weight of the purified enzyme was determined 66.9 kDa and the enzyme showed a high specificity to CMC substrate. The bacterial strains present in molasses have the potential for cellulase production. Substrate specificity of the purified cellulase indicates it to be an endo-β-1, 4-glucanase. The cellulase produced from the selected strain may benefit for industrial application.

Production of high-purity fucose from the seaweed of Undaria pinnatifida through acid-hydrolysis and simulated-moving bed purification

An efficient process suitable for a large-scale production of fucose from the seaweed of Undaria pinnatifida was developed in this study. The developed process consisted of a hydrolysis, a series of pretreatment processing for decolorization and deionization, and a final-stage purification using a well-designed simulated-moving bed (SMB) process. First, the optimal acid concentration and reaction time for the seaweed hydrolysis were investigated. The hydrolysate resulting from such investigation was then used to determine the optimal operating conditions for the pretreatment step consisting of an activated-carbon treatment, an electrodialysis, and an ion-exchange processing, which were performed to accomplish decolorization and deionization. On the basis of the optimal conditions that were obtained from the preceding experiments, a series of hydrolysis and pretreatment operations were carried out to secure a sufficiently large amount of decolorized and deionized hydrolysate. This was then moved on to the final purification step based on the use of the SMB process, which was designed such that the detailed recovery of fucose with a very high purity in a continuous mode could be ensured. The results showed that the purity of fucose, which reached 15.4% after the completion of the pretreatment processing, was markedly increased to nearly 100% through the SMB processing. It was also confirmed that the overall loss of fucose amounted to 22.9%, and most of the fucose loss occurred during the electrodialysis and ion-exchange procedures whereas there was almost no loss during the SMB processing.

Characterization and Interpretation of the Aluminum Zone Refining through Infrared Thermographic Analysis.

High purity metals are nowadays increasingly in demand to serve in electronic, photovoltaic, and target materials industries. The zone refining process is the most common way to achieve high purity in the final step of metal purification. Zone length and crystal growth rate are the main parameters that control the zone refining process. To determine these values, information about temperature profiles in the molten zone is necessary due to its direct correlation with these values. As the determination of this profile is not practically achievable in the present, the novel approach of applying an infrared (IR) camera during the zone refining of 2N8 aluminum is the focus of the investigation in this work. The whole temperature profile of the region near the molten zone was recorded by IR camera during the entire running process. The zone length and the crystal growth rate at each thermographic image shooting moment were successfully extracted by thermographic analysis. Results showed that both factors varied significantly, which is in contrast to the assumption in literature about their stability while running under constant input power and heater movement velocity, though noticeable purification took place in all of these experiments. However, the impurity concentration during refinement fluctuated remarkably. This was well-demonstrated by the tendency of variation in crystal growth rate attained in this work. These results provide a better understanding of the mechanisms of zone refining with an inductive heater and contributes to the optimization of the process.

Purification of recombinant trichodysplasia spinulosa–associated polyomavirus VP1-derived virus-like particles using chromatographic techniques

Trichodysplasia spinulosa-associated polyomavirus (TSPyV) has been linked to a rare and recently characterized skin disease occurring in immunocompromised patients. In analogy with other polyomaviruses, the major capsid protein VP1 of TSPyV can self-assemble into virus-like particles (VLPs). VLPs are increasingly applied for the vaccination and diagnostics. Mostly, non-scalable and labor intensive ultracentrifugation-based techniques are used for the purification of VLPs. In this work, we developed a purification procedure for TSPyV VP1 VLPs based on two chromatographic steps, ion-exchange monolith and core bead chromatography. Prior to chromatography, ammonium sulfate precipitation was used for the initial purification of TSPyV VP1 VLPs from yeast lysate. The VLPs were further purified using CIMmultus QA ion-exchange monolith in bind-elute mode. Most of TSPyV VP1 VLPs bound to the monolith and were subsequently eluted by a linear NaCl gradient. After ion-exchange monolith chromatography, the purity of TSPyV VP1 protein was about 75%. The final purification step of TSPyV VP1 VLPs was core bead chromatography using Capto Core 700 resin in flow-through mode. After core bead chromatography, 42% of TSPyV VP1 protein was recovered with a purity of 93%. The assembly of purified TSPyV VP1 protein into VLPs approximately 45–50 nm in diameter was confirmed by electron microscopy analysis. The purification procedure for TSPyV VP1 VLPs described here could be a scalable alternative to the conventional ultracentrifugation-based purification methods.

Glycosyltransferase cascades made fit for chemical production: Integrated biocatalytic process for the natural polyphenol C-glucoside nothofagin.

Glycosyltransferase cascades are promising tools of biocatalysis for natural product glycosylation, but their suitability for actual production remains to be shown. Here, we demonstrate at a scale of 100 g isolated product the integrated biocatalytic production of nothofagin, the natural 3'-C-β-D-glucoside of the polyphenol phloretin. A parallel reaction cascade involving coupled C-glucosyltransferase and sucrose synthase was optimized for the one-pot glucosylation of phloretin from sucrose via an UDP/UDP-glucose shuttle. Inclusion complexation with the highly water soluble 2-hydroxypropyl-β-cyclodextrin pushed the phloretin solubility to its upper practical limit (∼120 mM) and so removed the main bottleneck on an efficient synthesis of nothofagin. The biotransformation thus intensified had excellent performance metrics of 97% yield and ∼50 gproduct /L at a space-time yield of 3 g/L/hr. The UDP-glucose was regenerated up to ∼220 times. A scalable downstream process for efficient recovery of nothofagin (≥95% purity; ≥65% yield) was developed. A tailored anion-exchange chromatography at pH 8.5 was used for capture and initial purification of the product. Recycling of the 2-hydroxypropyl-β-cyclodextrin would also be possible at this step. Product precipitation at a lowered pH of 6.0 and re-dissolution in acetone effectively replaced desalting by size exclusion chromatography in the final step of nothofagin purification. This study therefore, reveals the potential for process intensification in the glycosylation of polyphenol acceptors by glycosyltransferase cascades. It demonstrates that, with up- and downstream processing carefully optimized and suitably interconnected, a powerful biocatalytic technology becomes available for the production of an important class of glycosides difficult to prepare otherwise.

Optimized purification of a fusion protein by reversed-phase high performance liquid chromatography informed by the linear solvent strength model

Fusion protein systems are commonly used for expression of small proteins and peptides. An important criterion for a fusion protein system to be useful is the ability to separate the protein of interest from the tag. Additionally, because no protease cleaves fusion proteins with 100% efficiency, the ability to separate the desired peptide from any remaining uncleaved protein is also necessary. This is likely to be the more difficult task as at least a portion of the sequence of the fusion protein is identical to that of the protein of interest. When a high level of purity is required, gradient elution reversed-phase HPLC is frequently used as a final purification step. Shallow gradients are often advantageous for maximizing both the purity and yield of the final product; however, the relationship between relative retention times at shallow gradients and those at steeper gradients typically used for analytical HPLC are not always straightforward. In this work, we report reversed-phase HPLC results for the fusion protein system consisting of the N-terminal domain of ribosomal protein L9 (NTL9) and the 36-residue villin headpiece subdomain (HP36) linked by a recognition sequence for the protease factor Xa. This system represents an excellent example of the difficulties in purification that may arise from this unexpected elution behavior at shallow gradients. Additionally, we report on the sensitivity of this elution behavior to the concentration of the additive trifluoroacetic acid in the mobile phase and present optimized conditions for separating HP36 from the full fusion protein by reversed-phase HPLC using a shallow gradient. Finally, we suggest that these findings are relevant to the purification of other fusion protein systems, for which similar problems may arise, and support this suggestion using insights from the linear solvent strength model of gradient elution liquid chromatography.

Risk Considerations on Developing a Continuous Crystallization System for Carbamazepine

Continuous manufacturing (CM) is an emerging technology in the pharmaceutical manufacturing sector, and the understanding of the impact on product quality is currently evolving. As the final purification and isolation step, crystallization has a significant impact on the final physicochemical properties of drug substance and is considered a critical process step in achieving the continuous manufacturing of drug substances. Although many publications previously focused on various innovative techniques to continuously make crystals with desired properties, engineering difficulties such as system design, automation, and integration with process analytical technology (PAT) tools have not been thoroughly discussed. Here, we focus on how to develop a continuous crystallization system, from the perspective of process engineering, and the related risk considerations on product quality. Specifically, we designed and built an automated two-stage mixed suspension mixed product removal (MSMPR) crystallization platform for a model compound (carbamazepine, CBZ) that exhibits multiple polymorphs. The crystallization process includes the integration of PAT tools (online Raman microscopy and focused beam reflectance microscopy, FBRM) for real-time monitoring. A series of case studies were done to evaluate the performance of the continuous system and PAT tools. Specifically, the drawing schemes, slurry transport, and variations on process variables are considered as the three key risk areas for continuous crystallization process development. Our proof-of-concept continuous crystallization system uses feedback/feedforward controls to achieve constant levels in crystallizers, a centralized automation program coded in LabVIEW, and PAT monitoring for polymorphs and particle size distribution (Raman and FBRM). To the best of our knowledge, this is also the first study on continuous crystallization of carbamazepine for form III and its polymorphic transition (between form II and form III).

Characterization, purification, and temperature/pressure stability of polyphenol oxidase extracted from plums ( Prunus domestica )

Abstract In this study, polyphenol oxidase (PPO) was extracted from Prunus domestica and partially purified by ammonium sulfate precipitation, hydrophobic interaction chromatography, and ion exchange chromatography. The final purification step revealed a 32.81-fold purification, and the molecular mass was estimated to be 65 kDa by SDS-PAGE. The purified PPO showed enzymatic activity mainly toward five substrates, namely catechol, catechin, 4-methyl catechol, chlorogenic acid, and L-3,4-dihydroxyphenylalanine, whereas it showed no activity toward caffeic acid, ferulic acid, p-coumaric acid, p-cresol, and l -tyrosine. The optimum pH and temperature values were 6.0 and 25 °C, respectively. The enzyme showed high stability in the pH range of 5.0–7.0 and in the temperature range of 25–65 °C. The most effective inhibitors of this enzyme were found to be ascorbic acid and l -cysteine. The thermal inactivation followed a first-order kinetic model, with activation energy of Ea 150.46 ± 1.29 kJ/mol. PPO extracted from plum showed stability at high pressure, with enzyme activation at 500 MPa.

Plasmid pVAX1-NH36 purification by membrane and bead perfusion chromatography.

The demand for plasmid DNA (pDNA) has increased in response to the rapid advances in vaccines applications to prevent and treat infectious diseases caused by virus, bacteria or parasites, such as Leishmania species. The immunization protocols require large amounts of supercoiled plasmid DNA (sc-pDNA) challenging the development of efficient and profitable processes for capturing and purified pDNA molecules from large volumes of lysates. A typical bioprocess involves four steps: fermentation, primary recovery, intermediate recovery and final purification. Ion-exchange chromatography is one of the key operations in the purification schemes of pDNA owing the chemical structure of these macromolecules. The goal of this research was to compare the performance of the final purification step of pDNA using ion-exchange chromatography on columns packed with Mustang Q membranes or perfusive beads POROS 50 HQ. The experimental results showed that both matrixes could separate the plasmid pVAX1-NH36 (3936bp) from impurities in clarified Escherichia coli lysates with an adequate resolution. In addition, a 24- and 21-fold global purification factor was obtained. An 88 and 63% plasmid recuperation was achieved with ion-exchange membranes and perfusion beads, respectively. A better understanding of perfusion-based matrices for the purification of pDNA was developed in this research.

Monocationic salts of carrageenans: Preparation and physico-chemical properties

Highly pure monocationic forms of κ-, ι-, λ-carrageenans and furcellaran were prepared with the aid of successive dialysis and ion exchange steps. Two approaches were used to obtain the samples: direct exchange of the galactan-bound cations with the aid of ion-exchange resin and generation of H+-forms of the polysaccharides followed by neutralization process. The galactan samples were characterized by 1H-NMR, 13C-NMR, pulsed-field-gradient stimulated echo 1H-NMR, FTIR, FT-Raman spectroscopy, ICP-OES, size exclusion chromatography, viscometry, static and dynamic rheometry methods. The ability of cations to enhance gelation follows the order of Li+ < Na+ ≪ Mg2+, Ca2+ < NH4+ < K+ for κ-type carrageenan and NH4+, Li+ < Na+ < K+ < Ca2+ for ι-carrageenan samples prepared by ion exchange. Pure K+-forms of κ-carrageenan and furcellaran form gels of moderate gel strength at 1.5%, but also Ca2+-ι-carrageenan affords workable gels. Mixed cationic content using both mono- and divalent cations promotes gel formation of κ-carrageenan more effectively compared to furcellaran. κ-Carrageenan forms exceptionally strong gels (G′ > 1 × 105 Pa) in the presence of equal amounts of Rb+ and Sr2+ ions. The rheological properties are notably pH-dependent. Among the monocationic samples, Ba2+-forms of κ-carrageenan and furcellaran yield the strongest gels in alkaline media, whereas Rb+- and Cs+-forms show the highest gelling capacities under acidic conditions. The widely employed method involving excessive dialysis against water as the final purification step causes cation depletion, acidification of the carrageenan sample and significant degradation during the freeze-drying step.