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Abstract: Fusion M13 phage with disulfide constrained heptapeptide, C-WSFFSNI-C, inserted into the minor coat protein (gpIII), has been selected in the current study as ligand in direct purification of hepatitis B core antigen (HBcAg) from unclarified Escherichia coli (E. coli) feedstock. The selected fusion phage showed strong association with the surface of the core particle. In the present study, this fusion M13 phage was immobilized onto Streamline base matrix via epoxy activation and used as adsorbent to capture HBcAg from crude E. coli homogenate. The maximum binding capacity for the adsorbent was 3.76mg/mL with equilibrium coefficient of 1.83mg/mL. Due to the slow uptake rate of HBcAg by M13 phage-immobilized adsorbents, a modified EBAC operation with recirculation of feedstock into the expanded bed has been investigated in this study. The introduction of feedstock recirculation has led to an 18% increase in yield; however, the purity of the eluted product was reduced by 15% compared with typical EBAC operation. The level of antigenicity exhibited by the core particles purified by both EBAC operations employed in the present study was comparable to that purified using sucrose ultracentrifugation. [Copyright &y& Elsevier]

Abstract: An extracellular aspartyl proteinase from Mucor mucedo DSM 809 submerged cultures was purified by a two-steps chromatographic procedure. The enzyme had a molecular weight (MW) of 32.7kDa, and an isoelectric point (pI) value of 4.29; no evidence of N-linked glycosylation was found. As judged by mass spectrometry, the primary structure of the M. mucedo enzyme presented homology with Rhizopus spp. proteinases. The secondary structure showed 4% α-helix, 48% β-sheet and 48% random coil structure in 20mM phosphate buffer (pH 5.8), as evidenced by circular dichroism spectroscopy. When acting on milk to provoke curd formation, the proteinase showed maximum potency at pH 5.0 and at 40°C. The enzyme was heat-sensitive and became completely inactivated after incubation at 55°C for 10min. These results indicate that the milk-clotting enzyme from M. mucedo can be considered as a potential substitute for bovine chymosin in cheese manufacturing. [Copyright &y& Elsevier]

Recombinant polyesterase (Est119) from Thermobifida alba AHK119 was purified by two chromatography steps. The final protein was observed as a single band in SDS-PAGE, and the specific activity of Est119 for p-nitrophenyl butyrate was 2.30 u/mg. Purified Est119 was active with aliphatic and aliphatic- co-aromatic polyesters. Kinetic data indicated that p-nitrophenyl butyrate (pNPB) or hexanoate was the best substrate for Est119 among p-nitrophenyl acyl esters. Calcium was required for full activity and thermostability of Est119, which was stable at 50 °C for 16 h. Three-dimensional modeling and biochemical characterization showed that Est119 is a typical cutinase-type enzyme that has the compact ternary structure of an α/β-hydrolase. Random and site-directed mutagenesis of wild-type Est119 resulted in improved activity with increased hydrophobic interaction between the antiparallel first and second β-sheets (A68V had the greatest effect). Introduction of a proline residue (S219P) in a predicted substrate-docking loop increased the thermostability. The specific activity of the A68V/S219P mutant on pNPB was increased by more than 50-fold over the wild type. The mutant was further activated by 2.6-fold (299 u/mg) with 300 mM Ca and was stable up to 60 °C with 150 mM Ca. Another identical gene was located in tandem in the upstream of est119. [ABSTRACT FROM AUTHOR]

Abstract: Recombinant human serum albumin (rHSA) is considered as an alternative of human serum albumin and used to treat patients with severe burn, shock or blood loss. However, separation and purification of rHSA are difficult and have become the bottle neck in industrial production. In this study, ethanol/K2HPO4 aqueous two-phase system (ATPS) and hydrophobic interaction chromatography (HIC) were integrated to provide a new approach for the extraction and purification of rHSA from high density fermentation broth. Using a 0.01–73L ATPS scale, the extraction of rHSA from the fermentation broth attained an average recovery of 100.4%. At the same time, 99.8% of cells and 87.2% of polysaccharides as well as some other protein impurities were also removed. The activity of proteinase A in the broth was also remarkably decreased. The purified rHSA appeared as a single band on reduced SDS–PAGE gel, and it had a purity of 99.1% as determined by HPLC. It was essentially identical to the plasma-derived HSA in terms of molecular weight and circular dichroism spectrum. The total recovery of rHSA was 75.2%, which was 1.1–2.0 times higher than that obtained from conventional processes. Residual polysaccharide was reduced to 1.61μg/mg rHSA and the degree of coloring was lower than that of plasma-derived HSA. The procedure employed in this study has the advantages of simple operation, shorter time, low energy consumption and high yield, and it could produce rHSA with high purity. It is therefore suitable in the production of rHSA and other biological products produced by high-density fermentation. [Copyright &y& Elsevier]

Prenylated isoflavonoids such as 6,8-diprenylorobol, 6,8-diprenylgenistein and 4′-O-methylalpinumisoflavone were purified from n-hexane extract of C. tricuspidata fruits using centrifugal partition chromatography (CPC) with a n-hexane-ethyl acetate-methanol-water (7:3:6:4, v/v, ascending mode) solvent system in a one-step within 200 min. One-step preparative CPC yielded 22.4 mg of 6,8-diprenylorobol and 26.7 mg of 6,8-diprenylgenistein with 95% and 85% purity, respectively, from 350 mg of crude hexane extract. Further purification by crystallization in methanol yielded 4′-O-methylalpinumisoflavone (9.3 mg) at over 95% purity based on the HPLC analysis. The chemical structures of purified compounds were elucidated by 1H, 13C NMR, and ESI-MS. [ABSTRACT FROM AUTHOR]

Abstract: The crude polysaccharides (GFP) were isolated from the fruiting bodies of Grifola frondosa and purified by DEAE cellulose-52 chromatography and Sephadex G-100 size-exclusion chromatography in that order. Three main fractions, GFP-1, GFP-2 and GFP-3, were obtained through the isolation and purification steps. Then the antioxidant activities of these three fractions were investigated in vitro. The results showed that GFP-1, GFP-2 and GFP-3 possessed significant inhibitory effects on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, hydroxyl radical and superoxide radical; their reducing power, ferrous ions chelating effect and the inhibition ability of the rat liver lipid oxidation where also strong. These results suggest that G. frondosa polysaccharides could be a suitable natural antioxidant and may be the functional foods for humans. [Copyright &y& Elsevier]

The article presents a study of the monoprotection of triamines using alkyl phenyl carbonates. It discusses the investigations carried out by Sayre and coworkers and Bruice and Willis regarding the acylation of 1,2-diaminoethane with different acylating reagents and the acylation of one of the amino groups in α,ω-diamine respectively. It prepares a series of monoprotected triamines from the triamines and alkyl phenyl carbonates. It develops a simple method for monoprotection of triamines in methanol with alkyl phenyl carbonates avoiding chromatographic purification.

Abstract: A novel neutral heteropolysaccharide (DDP-1-D) was purified from hot water extracts of dried stem of Dendrobium densiflorum by DEAE-52 and Sephacryl S-200 High-Resolution Chromatography. The heteropolysaccharide had an average molecular weight about 9440Da. It was composed mainly of glucose and mannose in the ratio of 3.01:1. Structural features of DDP-1-D were elucidated by a combination of chemical and instrumental techniques, including FT-IR, GC–MS, periodate oxidation–Smith degradation, 1H and 13C NMR spectroscopies (including COSY, TOCSY, HSQC, and HMBC spectra). The results indicated that DDP-1-D is a mannoglucan and has a backbone consisting of (1→4)-linked α-d-Glcp, (1→6)-linked α-d-Glcp, (1→2)-linked α-d-Manp and (1→4)-linked β-Manp. This is the first study to provide clear evidence for the structure of the polysaccharide in D. densiflorum. [Copyright &y& Elsevier]

Abstract: Sample displacement chromatography (SDC) in reversed-phase and ion-exchange modes was introduced approximately twenty years ago. This method takes advantage of relative binding affinities of components in a sample mixture. During loading, there is a competition among different sample components for the sorption on the surface of the stationary phase. SDC was first used for the preparative purification of proteins. Later, it was demonstrated that this kind of chromatography can also be performed in ion-exchange, affinity and hydrophobic-interaction mode. It has also been shown that SDC can be performed on monoliths and membrane-based supports in both analytical and preparative scale. Recently, SDC in ion-exchange and hydrophobic interaction mode was also employed successfully for the removal of trace proteins from monoclonal antibody preparations and for the enrichment of low abundance proteins from human plasma. In this review, the principals of SDC are introduced, and the potential for separation of proteins and peptides in micro-analytical, analytical and preparative scale is discussed. [Copyright &y& Elsevier]

Abstract: Functionalized magnetic particles are emerging as a reliable and convenient technique in the purification of biomacromolecules (proteins and nucleic acids) and cell separation. In this study, we used novel solid nickel ferromagnetic particles coated with Protein A for the affinity purification of antibody. The study demonstrated that IgG can be purified from undiluted mouse serum in as few as 5min using Protein A-coated nickel particles. Further, protein crosslinking was shown to stabilize the Protein A on the nickel particle surfaces to minimize Protein A leaching during the affinity purification and elution of IgG. The separation procedure is gentle, scalable, automatable, efficient and economical. By modifying the functional groups of amino acids in the protein coating, crosslinked nickel particles can be used not only for protein affinity purification but for other biological sample preparation and chromatographic applications as well. Methods proposed and tested in this study can be easily modified for small and medium scale antibody purification in lab and pre-clinical research. [Copyright &y& Elsevier]

Abstract: A novel heme peroxidase from Jatropha curcas, an important source of bio-diesel, was purified to homogeneity using ammonium sulfate fractionation, desalting chromatography and ion exchange chromatography. Molecular mass of this purified enzyme was around 48kDa as detected by SDS-PAGE. Gel filtration analysis revealed that the enzyme was a monomer under native conditions. The purified enzyme had broad substrate specificity with the ideal substrates of guaiacol and o-phenylenediamine. The optimum temperature, pH and K m value of this peroxidase for guaiacol was 60°C, 5.0 and 0.17mM, respectively. In addition, NaCl (2.5M) significantly enhanced the activity of this peroxidase. The purified enzyme was stable under high temperature (70% activity retained after 1h incubation at 70°C), extreme pH environment (93% or more activity retained after 2h incubation under pH 3–12), high NaCl concentration (88% or more activity retained after 2h incubation with 1–4M NaCl) and organic solvents (95% or more activity retained after 54h incubation with various organic solvents). Moreover, this peroxidase was resistant against 20mM hydrogen peroxide, 8M urea, 3M guanidine hydrochloride and 20mM EDTA. However, the peroxidase activity was significantly inhibited by sodium azide, dithiothreitol, CTAB, β-mercaptoethanol, DMSO, toluene and ferrous ion. The enzyme had long shelf life with 180 days at 4°C and 14 days at room temperature. This new robust peroxidase may bring a better understanding for the high anti-adversity property of J. curcas. Meanwhile, the broad substrate specificity, wide stability against high temperature, extreme pH, organic solvent and hydrogen peroxide suggested that the enzyme could be a potential candidate peroxidase source for industrial and biomedical applications. [Copyright &y& Elsevier]

Abstract: Integration of supercritical fluid/mass spectrometry (SFC/MS) and reversed phase liquid (HPLC/MS) chromatographic screening techniques into a single chromatographic system and utilized in “walk up” mode, enabled us to produce an orthogonal data set for selecting purification conditions for medicinal chemistry compounds. To streamline the overall workflow, we also demonstrate the use of automated batch data processing of individual data files to identify suitable separation conditions without user intervention. We have addressed the chromatographic challenges that hinder the identification of the intended target and thus the selection of ideal purification conditions. For instance, multiple component-of-interest (COI) peaks, co-elution of impurities with the COI, and chromatographic suitability factors such as retention times and peak shapes are all important considerations when selecting appropriate methods for purification and, therefore, are bottlenecks to an automated approach. Since SFC and HPLC data were collected in parallel from separate instruments in our workflow, the time required for the separation scientist to analyze acquired data from both systems was a time-limiting factor. To reduce data processing time and accelerate or “FastTrack” samples to purification, two unique and automated solutions were introduced. We describe the implementation of an integrated, multi-column, walk-up HPLC/SFC/MS system, and the implementation of an intelligent, automated method selection application which uses advanced data evaluation criteria to selectively score and identify the most practical separation conditions for SFC/MS and HPLC/MS methodologies. [Copyright &y& Elsevier]

Abstract: This paper demonstrates a combined approach for separating an active pharmaceutical ingredient (API) from a heavily contaminated waste stream. The approach uses organic solvent nanofiltration (OSN) to improve the application of counter-current chromatography (CCC) in an industrial process. OSN provides an efficient route for exchanging solutes from the process solvent into the desired mobile phase for CCC, generating a CCC feed containing less than 0.01% (area % by GC) of the original process solvents. The high solvent burden of CCC was additionally reduced through recovery of mobile phase using OSN, with the recovered solvent containing less than 1% (area % by HPLC) impurities. The recovered solvent was then successfully recycled into a subsequent CCC run with no indication of impurity build-up. Coupling OSN with CCC improved the mass-intensity of the CCC process, reducing the solvent use by 56%. OSN can be a useful tool in facilitating the application of CCC to pharmaceutical process streams. [Copyright &y& Elsevier]

Abstract: A PID controller able to support the operator in the operation of the Multi-column Countercurrent Solvent Gradient Purification (MCSGP) process which is a continuous, countercurrent chromatographic process has been developed. As measurement, only the online UV signals at each column outlet are used. This guarantees a simple and cheap control implementation and a fast control action. Accordingly, the controller does not guarantee any purity or yield value, but simply that the withdrawn window of the product is centered in a specific region of the UV chromatogram where the purity specifications are expected to be satisfied. This can be determined by the operator based on the batch chromatogram selected for designing the MCSGP operating conditions. Thus the controller provides a reliable and efficient tool for the operator to run properly a MCSGP unit in combination with suitable offline analytics for the quantification of purity and yield. The applications are discussed involving the purification of a model protein and a peptide. It is shown that the developed controller is effective in driving the unit to steady state during start up and in keeping a stable steady state while rejecting external disturbances. [Copyright &y& Elsevier]

Abstract: A novel glycosaminoglycan-like sulfated polysaccharide (AAP) from the pleopods of Haliotis discus hannai Ino was purified by DEAE ion exchange chromatography followed with S-300 HR geltrion chromatography. Chemical composition analysis indicated that AAP was composed of galactosamine, glucuronic acid, fucose, galactose with a ratio of 2.14:2.37:2.94:1, the content of sulfate was 15.5%. The average molecular weight (M w) was 56.2kDa in a TSK G4000 column. Further IR, 1D, 2D NMR spectroscopic analysis of the correlation signals of different sugar units gave a proposal repeating structure, as follows: Display Omitted In vitro anticoagulant assay indicated AAP prolonged both the activated partial thromboplastin time (APTT) and thrombin time (TT), with a 22.5U/mg and 72.0U/mg compared with standard heparin, respectively. The anticoagulant property of AAP was mainly attributed to powerful potentiation thrombin by anti-thrombin III. [Copyright &y& Elsevier]

Abstract: Today more and more attentions had been attracted by many nutritionists and pharmacologists on polysaccharides from natural plants or animals due to their significant biological activities. In this research three polysaccharides (BRR-W1, BRR-WA1 and BRR-WA2) were isolated and purified from the water extract of Boschniakia rossica by DEAE Sepharose Fast Flow and Sepharose 6 Fast Flow column chromatography. Chemical and physical characteristics of three polysaccharides were investigated by a combination of chemical and instrumental analysis methods. The assays of their effect on macrophages activation were also investigated in vitro, including phagocytosis of macrophages, detections for NO production and TNF-α secretion. The results indicated that the effect of polysaccharides on macrophages activation was influenced by their respective physicochemical properties. [Copyright &y& Elsevier]

Abstract: A family of linear hexamer peptide ligands HWRGWV, HYFKFD and HFRRHL, initially identified for their affinity to the Fc portion of human immunoglobulin G (hIgG), also have potential for use in the purification of human immunoglobulins A (hIgA) and M (hIgM). HWRGWV demonstrated the strongest binding affinity to hIgM, followed by hIgA and hIgG respectively. The effects of N-terminal acetylation of the peptide, as well as elution buffer pH, on the chromatographic elution of human IgG, IgA and IgM from HWRGWV resins at various peptide densities (0.04–0.55meq/g) were investigated. Over 80% recovery and 90% purity were achieved for human IgG and IgA isolation from complete minimum essential medium (cMEM) using HWRGWV resin at optimum peptide densities. For human IgM, 75.7% recovery and 86.0% purity were achieved by using HWRGWV at a low peptide density of 0.04meq/g. Although HYFKFD and HFRRHL exhibited their ability for isolation of human IgG, IgA and IgM from cMEM as well, HWRGWV is the best option among them for large-scale purification of human IgG, IgA and IgM based on conditions tested. [Copyright &y& Elsevier]

Abstract: The growing demand on plasmid DNA (pDNA) manufacture for therapeutic applications requires a final product with higher quality and quantity, spending the least time. Most of the current processes for pDNA production use at least one chromatographic step, which often constitutes a key-step in the purification sequence. Monolithic stationary phases are new alternatives to the conventional matrices, which offer fast separation of pDNA due to their excellent mass transfer properties and their high binding capacity for large molecules, as pDNA. However, the efficient recovery of pure pDNA focuses on a suitable balance of the feedstock, adsorbent and mobile phase properties. To satisfy the increasing demand for pharmaceutical grade plasmids, we developed a novel downstream process which overcomes the bottlenecks of common lab-scale techniques while complying with all regulatory requirements. This work reports an integrative approach using the carbonyldiimidazole monolith to efficiently purify the supercoiled (sc) pDNA active conformation from other plasmid topologies and Escherichia coli impurities present in a clarified lysate. The monolith specificity and selectivity was also assessed by performing experiments with plasmids of several sizes of 2.7, 6.05 and 7.4 kilo base pairs (kbp), verifying the applicability to purify different plasmids. Hence, the process yield of the pDNA purification step using the CDI monolith was 89%, with an extremely reduced level of impurities (endotoxins and gDNA), which was reflected in good transfection experiments of the sc plasmid DNA sample. Overall, the analytical results and transfection studies performed with the pDNA sample purified with this monolithic enabling technology, confirmed the suitability of this pDNA to be used in pharmaceutical applications. [Copyright &y& Elsevier]

A practical protocol for the large-scale preparation of 2-diazo-1,3-dicarbonyl compounds is described by diazo-transfer reactions with tosyl azide followed by efficient chromatographic purifications on silica gel and/or alumina. [ABSTRACT FROM AUTHOR]

Abstract: A microwave-assisted extraction (MAE) method is presented for the extraction of xanthones, α-mangostin and γ-mangostin from Garcinia mangostana. The MAE conditions including extraction temperature, liquid/solid ratio, extraction time and concentration of ethanol were optimized with an orthogonal test, and 5g sample was extracted with the optimized conditions. The crude extraction of MAE was successfully isolated and purified by high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of petroleum ether–ethyl acetate–methanol–water (0.8:0.8:1:0.6, v/v) in one-step separation. The separation yielded 75mg of α-mangostin at 98.5% purity, and 16mg of γ-mangostin at 98.1% purity from 360mg crude extract of G. mangostana in less than 7h. The purity of the two xanthones was determined by HPLC. Their structures were further identified by ESI-MS, 1H NMR and 13C NMR. [Copyright &y& Elsevier]

Abstract: The design of gradient simulated moving bed (SMB) chromatographic processes requires an appropriate selection of the chromatographic system followed by the determination of adsorption isotherm parameters in the relevant range of mobile phase conditions. The determination of these parameters can be quite difficult for recombinant target proteins present in complex protein mixtures. The first part of this work includes the estimation of adsorption isotherm parameters for streptokinase and a lumped impurity fraction present in an Escherichia coli cell lysate for a hydrophobic interaction chromatography (HIC) matrix. Perturbation experiments were carried out using a Butyl Sepharose matrix with purified recombinant protein on buffer equilibrated columns as well as with crude cell lysate saturated columns. The Henry constants estimated for streptokinase were found to exhibit in a wide range a linear dependence on the salt concentration in the mobile phase. These parameters were applied in subsequent investigations to design a simulated moving bed (SMB) process capable to purify in a continuous manner recombinant streptokinase from the E. coli cell lysate. [Copyright &y& Elsevier]

Abstract: Acid/base mobile phase modifiers affect enantioseparations in ways that are not fully understood yet, for the lack of systematic studies. This makes chiral analysis of some pharmaceuticals difficult to reproduce. Once a column has been exposed to a modifier, the selectivity of certain pairs of enantiomers may change, for the better or the worse. We study the behavior of five enantiomeric pairs, three which are highly sensitive to the addition of certain modifiers and two that have little sensitivity to these modifiers. Their use permits the determination of the extent of the memory effect response on individual columns. The selectivity of 4-chlorophenylalanine methyl and ethyl ester, and of ketoprofen improve as a solution of ethanesulfonic acid is percolated through the column. As a result, these pairs are most useful for the determination of the extent of acid memory effect on a column. The selectivity of propranolol HCl and, to a lesser degree, Tröger''s base increases as a solution of diisopropylethylamine is percolated through the column. The separation of each one of these five pairs is inversely affected by the percolation of the opposite acid/base solution. We used trans-stilbene oxide (TSO) as a ‘standard’ to determine the column stability because no memory effect is observed for it (its retention, enantioselectivity, and resolution remain constant). Understanding whether a column is under the influence of the memory effect is critical to both the analysis of pharmaceutical ingredients and to the development of preparative purification techniques for racemic mixtures. Thus, columns that were unreliable for method development and method transfer, due to the memory effect and a lack of proper solvent exposure records, can now be used. [Copyright &y& Elsevier]

Abstract: In the first part of this work adsorption isotherm parameters were acquired to describe the migration of recombinant streptokinase in Butyl Sepharose columns at different salt concentrations. Based on these results, a simulated moving bed (SMB) chromatographic process was designed and realised, which exploits a two-step salt gradient and allows the continuous separation of streptokinase from contaminants present in a clarified Escherichia coli cell lysate solution. This second part describes the design of the three-zone open-loop gradient SMB process applying both equilibrium theory and an equilibrium stage model and presents results of a series of experiments aiming to obtain pure streptokinase. Moreover, the potential of the SMB process and the design approach are evaluated. [Copyright &y& Elsevier]

Semi-preparative high-speed counter-current chromatography (HSCCC) was successfully used for isolation and purification of oridonin from Isodon rubescens by using a two-phase-solvent system composed of n-hexane-ethyl acetate-methanol-water (2.8:5:2.8:5, v/v/v/v). The targeted compound isolated, collected and purified by HSCCC was analyzed by high performance liquid chromatography (HPLC). A total of 40.6 mg of oridonin with the purity of 73.5% was obtained in less than 100 min from 100 mg of crude Isodon rubescens extract. The chemical structure of the compound was identified by IR, ¹H-NMR and 13C-NMR. [ABSTRACT FROM AUTHOR]

New regio- and chemoselective [4+2+1] domino cyclization consisting of the formation of two spiro rings and five σ bonds has been developed for the synthesis of spiro-substituted benzo[b]furo[3,4-e][1,4]diazepine derivatives. The reaction is a multicomponent domino green process and can be readily performed by reacting inexpensive starting materials of benzene-1,2-diamines, tetronic acid and 2,2-dihydroxy-2H-indene-1,3-dione in aqueous solution under microwave irradiation. The present synthesis shows attractive characteristics, such as the use of water as reaction media, convenient one-pot operation, short reaction periods of 10–18 min and reduced waste production without the use of any strong acids or metal promoters. This synthesis serves as a nice addition to GAP (Group-Assistant-Purification) chemistry in which purification viachromatography and recrystallization can be avoided, and the pure products were obtained simply by washing the crude products with 95% EtOH. [ABSTRACT FROM AUTHOR]

A facile synthesis of 1,3,4-thiadiazine-5-yl-pyran-2-one derivatives is achieved via a three-component reaction involving 3-(2-bromoacetyl)-4-hydroxy-6-methyl-2H-pyran-2-one, thiocarbohydrazide with various carbonyl compounds in one-pot under stirring. The main advantage of this procedure is the short reaction time, high yields, simple workup, and purification of products by non-chromatographic methods, i.e. by simple recrystallization from ethanol. The structures of newly prepared compounds have been established by elemental analysis and spectral data. [image omitted] [ABSTRACT FROM AUTHOR]

Abstract: Given an existing demand to establish a process of tetanus vaccine production in a way that allows its complete validation and standardization, this paper focuses on tetanus toxoid purification step. More precisely, we were looking at a possibility to replace the widely used ammonium-sulphate precipitation by a chromatographic method. Based on the tetanus toxin''s biochemical characteristics, we have decided to examine the possibility of tetanus toxoid purification by hydrophobic chromatography, and by chromatographic techniques based on interaction with immobilized metal ions, i.e. chelating chromatography and immobilized metal affinity chromatography. We used samples obtained from differently fragmented crude tetanus toxins by formaldehyde treatment (assigned as TTd-A and TTd-B) as starting material for tetanus toxoid purification. Obtained results imply that purification of tetanus toxoid by hydrophobic chromatography represents a good alternative to ammonium-sulphate precipitation. Tetanus toxoid preparations obtained by hydrophobic chromatography were similar to those obtained by ammonium-sulphate precipitation in respect to yield, purity and immunogenicity. In addition, their immunogenicity was similar to standard tetanus toxoid preparation (NIBSC, Potters Bar, UK). Furthermore, the characteristics of crude tetanus toxin preparations had the lowest impact on the final purification product when hydrophobic chromatography was the applied method of tetanus toxoid purification. On the other hand, purifications of tetanus toxoid by chelating chromatography or immobilized metal affinity chromatography generally resulted in a very low yield due to not satisfactory tetanus toxoid binding to the column, and immunogenicity of the obtained tetanus toxoid-containing preparations was poor. [Copyright &y& Elsevier]

Abstract: Bio-nanocapsules (BNCs) are hollow particles (approx. 50nm diameter) consisting of hepatitis B virus surface antigen (HBsAg) large (L, pre-S1+pre-S2+S) proteins embedded in a unilamellar liposome, sharing the same transmembrane S region with an immunogen of hepatitis B vaccine (i.e., HBsAg small (S) protein particle). BNCs can incorporate drugs and genes into the hollow space and systemic administration of the BNCs can deliver the products to human liver via the human hepatocyte-specific receptor within the pre-S (pre-S1+pre-S2) region displayed on BNC’s surface. Thus, BNCs are expected to offer efficient and safe non-viral nanocarriers to deliver human liver-specific genes and drugs. To date, BNCs have been purified from the crude extract of BNC-overexpressing yeast cells by fractionation with polyethylene glycol followed by one CsCl equilibrium and two sucrose density gradient ultracentrifugation steps. However, the process was inefficient in terms of yield and time, and was not suitable for mass production because of the ultracentrifugation step. Furthermore, trace contamination with yeast-derived proteinases degraded the pre-S region, which is indispensable for liver-targeting, during long-term storage. In this study, we developed a new purification method involving heat treatment and sulfated cellulofine column chromatography to facilitate rapid purification, completely remove proteinases, and enable mass production. In addition, the BNCs were functional for at least 14months after lyophilization with 5% (w/v) sucrose as an excipient. This new process will significantly contribute to the development of forthcoming BNC-based nanomedicines as well as hepatitis B vaccines. [Copyright &y& Elsevier]

Abstract: A water-soluble polysaccharide obtained from Acanthopanax senticosus leaves (ASL), was fractionated by DEAE–Sepharose fast-flow column chromatography, and purified by Sephadex G-75 gel-permeation column chromatography. The characteristics of ASP-2-1 were determined by chemical analysis, high-performance capillary electrophoresis (HPCE), high-performance gel-permeation chromatography (HPGPC). The results show that ASP-2-1 contained 89.47% carbohydrate, 7.45% uronic acid, 2.16% protein and seven kinds of monosaccharides including rhamnose, xylose, glucose, mannose, arabinose, galactose and glucuronic acid in a molar ratio of 7.45:18.63:25.15:0.93:8.35:2.79:5.69, with an average molecular weight of about 14,573Da. Furthermore, the immunobiological and antioxidant activities, in vitro, of ASP-2-1 were evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and ferric-reducing antioxidant power assay (FRAP), 2,2-diphenyl-1-picrylhydrazyl (DPPH ), superoxide radical and hydroxyl radical ( OH) free radical-scavenging assay, respectively. The results showed that ASP-2-1 exhibited significantly higher immunomodulatory activities against the lymphocyte proliferation in vitro, pronounced reductive power (FRAP value: 785.1μM at 0.2mg/ml), strong hydroxyl radical (89.56% at 1mg/ml) scavenging activity, moderate superoxide radicals (65.32% at 1mg/ml) and DPPH radicals (68.9% at 1mg/ml) scavenging activities. ASP-2-1 should be explored as a novel and potential natural antioxidant and immunostimulating agent for use in functional foods or medicine. [Copyright &y& Elsevier]

Abstract: Ureases are used in beverage, fertilizer and pharmaceutical industries. We report purification, characterization and a simple method for immobilization of urease form Momordica charantia seeds. The purification of the enzyme is a two steps process involving anion exchange chromatography using DEAE–Sepharose matrix followed by size exclusion chromatography. The fold purification of the enzyme was found to be 6.5. The estimated molecular mass and K m of the enzyme were 174.5kDa and 34±0.5mM, respectively. The purified urease was immobilized on chitosan beads activated with glutaraldehyde. The activation of chitosan beads and immobilization were confirmed by Fourier Transform Infrared (FTIR) spectrometry and Scanning Electron Microscope (SEM), respectively. Moreover, Energy Dispersive X-ray (EDX) analysis shows increased percentage of nitrogen which further confirms immobilization. The efficiency of immobilization was found to be approximately 97%. The immobilized enzyme retains 60% activity till fourteen successive batches of enzymatic reactions which makes it a highly stable product. Comprehensively, the enzyme shows desirable properties for various applications. [Copyright &y& Elsevier]

Abstract: Proteins conjugated to neutral biopolymers are of keen interest to the food and pharmaceutical industries. Conjugated proteins are larger and more charge shielded than un-reacted proteins, making purification difficult using conventional beaded chromatographic supports because of slow mass transfer rates, weak binding, and viscous solutions. Past methods developed for pharmaceuticals are unsuitable for foods. In this work, a food-grade whey protein–dextran conjugate was purified from a feed solution also containing un-reacted protein and dextran using either a column packed with 800mL of a beaded support that was specifically designed for purification of conjugated proteins or an 8mL tube monolith. The monolith gave a similar dynamic binding capacity as the beaded support (4–6g/L), at a 42-fold greater mass productivity, and 48-fold higher flow rate, albeit at somewhat lower conjugate purity. Performance of the monolith did not depend on flow rate. In conclusion, monoliths were found to be well suited for the purification of whey protein–dextran conjugates. [Copyright &y& Elsevier]

Three flavonoid glycosides including quercetin-3-O-[2''-O-(6'''-O-E-sinapoyl)-β-D-glucopyranosyl]-β-D-glucopyranoside(I), quercetin-3-O-[2''-O-(6'''-O-E-feruloyl)-β-D-glucopyranosyl]-β-D-glucopyranoside(II) and quercetin-3-O-[2''-O-(6'''-O-E-feruloyl)-β-D-glucopyranosyl]-β-D-galactopyranoside(III) were isolated and purified from Hedyotis diffusa Willd by high-speed counter-current chromatography (HSCCC). This run was carried out with a two-phase solvent system composed of n-hexane-ethyl acetate-n-butanol-methanol-1.0% acetic acid (1:1:3.5:1:4.5, v/v) by eluting the lower phase as the mobile phase with a flow-rate at 2.0 ml/min. Consequently, 29.6 mg of I, 35.1 mg of II, 41.3 mg of III with purities of over 95% were obtained from 200 mg of the crude extracts in a single run in less than 130 min. The structure of the isolated compounds was confirmed by MS, 1H NMR, and 13C NMR analysis. [ABSTRACT FROM AUTHOR]

Preparative separation of quinolone alkaloids in Evodia rutaecarpa (Juss.) Benth was conducted by high performance counter-current chromatography (HPCCC) with a pair of two solvent systems consisting of n-hexane-methanol-water-acetic acid (2:1:1:0.2, v/v) and (5:4:2:0.1, v/v). Consequently, 31.78 mg 1-methyl-2-nonyl-4 (1H)-quinolone (I), 59.25 mg 1-methyl-2-(6-undecenyl)-4 (1H)-quinolone (II), 333.27 mg evocarpine (III), 101.13 mg 1-methyl-2-(6,9-pentadecadienyl)-4(1H)-quinolone (IV), 132.17 mg dihydroevocarpine (V), and 86.99 mg 1-methyl-2-(10-pentadecenyl)-4(1H)-quinolone (VI) were obtained from 1.3 g of the crude extract. The structures of these compounds were identified by mass spectrometer (MS), nuclear magnetic resonance (1H NMR and 13C NMR). [ABSTRACT FROM AUTHOR]

Abstract: An extracellular thermostable lipase from Amycolatopsis mediterranei DSM 43304 has been purified to homogeneity using ammonium sulphate precipitation followed by anion exchange chromatography and hydrophobic interaction chromatography. This protocol resulted in a 398-fold purification with 36% final recovery. The purified A. mediterranei DSM 43304 lipase (AML) has an apparent molecular mass of 33kDa. The N-terminal sequence, AANPYERGPDPTTASIEATR, showed highest similarity to a lipase from Streptomyces exfoliatus. The values of and for p-nitrophenyl palmitate (p-NPP) at the optimal temperature (60°C) and pH (8.0) were 0.099±0.010mM and 2.53±0.06mmol/minmg, respectively. The purified AML displayed significant activity towards a range of short and long chain triglyceride substrates and p-nitrophenyl esters. Hydrolysis of glycerol ester bonds occurred non-specifically. The purified AML displayed significant stability in the presence of organic solvents (40%, v/v) and catalyzed the synthesis of the flavour ester isoamyl acetate in free and immobilized states. [ABSTRACT FROM AUTHOR]

Abstract: Activated media allow the user to easily synthesize a variety of affinity media. We have developed a novel activated medium based on porous silica modified with phosphorylcholine (PC) and N-hydroxysuccinimide (NHS) groups for the purpose of high-throughput purification and reducing nonspecific protein adsorption. The PC groups function as suppressors of nonspecific protein adsorption, whereas the NHS groups are able to covalently bind to the primary amino groups of ligands. Because protein A affinity medium is the most frequently used affinity medium, we prepared protein A media in which a recombinant protein A was bound to the NHS groups of the activated media and evaluated its utility. After optimizing various factors in the synthetic process, the resultant protein A medium showed improved durability at a high flow rate over 300 purification cycles and reduced nonspecific protein adsorption compared with commercially available protein A media. [ABSTRACT FROM AUTHOR]

Abstract: A novel NADH-dependent dehydrogenases/reductases (SDRs) superfamily reductase (PsCRII) was isolated from Pichia stipitis. It produced ethyl (S)-4-chloro-3-hydroxybutanoate [(S)-CHBE] in greater than 99% enantiomeric excess. This enzyme was purified to homogeneity by ammonium sulfate precipitation followed by Q-Sepharose chromatography. Compared to similar known reductases producing (S)-CHBE, PsCR II was more suitable for production since the purified PsCRII preferred the inexpensive cofactor NADH to NADPH as the electron donor. Furthermore, the K m of PsCRII for ethyl 4-chloro-3-oxobutanoate (COBE) was 3.3mM, and the corresponding V max was 224μmol/mg protein/min. The catalytic efficiency is the highest value ever reported for NADH-dependent reductases from yeasts that produce CHBE with high enantioselectivity. In addition, this enzyme exhibited broad substrate specificity for several β-keto esters using NADH as the coenzyme. The properties of PsCRII with those of other carbonyl reductases from yeasts were also compared in this study. [ABSTRACT FROM AUTHOR]

Abstract: Histidine-tagged proteins have been purified with immobilized nickel affinity chromatography. This method, however, bears some serious disadvantages, including carcinogenicity and potential leakage of the nickel and the handling and disposal costs. We developed a novel purification process for water-soluble histidine-tagged green fluorescent protein from the cocoons of transgenic silkworms, unlike conventional E. coli expression system which sometimes produced insoluble and inactive inclusion bodies. 60.3% extraction efficiency was achieved with 50mM Tris containing 150mM NaCl, pH 7.5. The extract was purified in one step on zinc-immobilized hydroxyapatite eluted with phosphate buffer. Purity was 91.2% with a recovery of 30.0μg from 160mg of silk fiber. This purification method may provide a simple non-toxic alternative to nickel-immobilized metal affinity chromatography for purification of other histidine-tagged proteins. [Copyright &y& Elsevier]

Abstract: Dityrosine (DY) can be used as a biomarker to detect oxidative protein damage and selective proteolysis. It is generally prepared by horseradish peroxidase (HRP)-catalyzed oxidation of l-tyrosine (Y) followed by multistep chromatographic separations. In this study, we present an alternative method for the preparation of DY by HRP-catalyzed synthesis of N,N′-diBoc-dityrosine (DBDY) from N-Boc-l-tyrosine (BY). The presence of the tert-butoxycarbonyl (Boc) group ensured that the fraction of further oxidized by-products (e.g., trimers and pulcherosine) was quite low. The yield of DBDY (37.5%) was comparable to that reported for DY (> 26%). DBDY could be purified by a simple one-step silica column chromatography procedure that resulted in a purity of 89.7%. DBDY is considered to be better than DY for subsequent chemical reactions (for binding to polymers, amino acids, drugs, antibodies, etc.) because such reactions can be selectively performed by using the carboxylic acid and amine groups in the following sequence: first, the carboxylic acid groups are used; next, the Boc groups are removed; and finally, the amino groups are used. To prepare DY, the Boc groups in DBDY were simply and completely removed by treatment with trifluoroacetic acid. [ABSTRACT FROM AUTHOR]

Abstract: A preparative high-speed counter-current chromatography (HSCCC) method for isolation and purification of four prenylflavanones from the crud extract after microbial biotransformation of kurarinone was developed successfully using a stepwise elution with two-phase solvent system composed n-hexane–ethyl acetate–methanol–water at the volume ratios of 1:1:0.7:1 (v/v) and 1:1:1.2:1 (v/v). A total of 7mg of 4a,5a-dihydroxy norkurarinone (1), 11mg of 7-methoxyl-4a,5a-dihydroxy norkurarinone (2), 14mg of 6a-hydroxykurarinone (3) and 10mg of norkurarinone (4) were obtained in one-step separation from 520mg of the crude biotransformation sample with purities of 94.0%, 97.8%, 99.5% and 98.7%, respectively. Among them, compounds 1–3 are novel compounds, and their chemical structures were identified on the basis of the extensive spectral methods such as UV, IR, HR-MS and NMR. [ABSTRACT FROM AUTHOR]

All therapeutic antibodies that are currently approved or under development are IgGs, with IgG1 and IgG2 being the most prevalent subclasses. The present study investigates the effect of antibody subclass on its chromatographic properties. To this end, two sets of antibodies with identical variable domains and differing subclasses (IgG1 and IgG2) were compared by using purification methods that are most commonly used in industrial antibody manufacturing processes. Each antibody was examined using Protein A chromatography, cation-exchange chromatography, hydrophobic interaction chromatography and salt-induced precipitation studies. The results of these experiments show that the variable region of an antibody has a greater influence on its chromatographic behaviour than antibody subclass. [ABSTRACT FROM AUTHOR]

Abstract: Monacolin K from red yeast rice was extracted by accelerated solvent extraction (ASE). The effects of various extraction parameters including extraction temperature, static extraction time and cycle index on yield were investigated using a DIONEX ASE 300 system to select the optimal conditions by an orthogonal test design L9 (3)3. The optimum extraction conditions were determined as follows: extraction temperature 120°C, static extraction time 7min, and cycle index 3. Under the optimal conditions, the yield of ASE extract and monacolin K was 5.35% and 9.26mg/g of dry red yeast rice, respectively. A separation and purification method of monacolin K was then established using high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of n-hexane–ethyl acetate–methanol–water (8:2:5:5, v/v/v/v). From 300mg of crude extract, 51.2mg of monacolin K was obtained with the purity of 98.7%. The chemical structure of isolated compound was identified by UV, ESI-MS and 1H NMR. [ABSTRACT FROM AUTHOR]

HSP65–MUC1 is a fusion protein between BCG (Bacille Calmette–Guerin)-derived HSP65 (heat-shock protein 65) and human MUC1 (mucin 1) VNTR (variable number of tandem repeats)-domain peptides that has shown antitumour efficacy. China's Food and Drug Administration has recently approved a Phase I clinical trial using HSP65–MUC1 for the treatment of MUC1-positive breast cancer. In order to produce sufficient quantities of clinical-grade HSP65–MUC1, we established a pilot-scale purification scheme comprising two steps of column chromatography: HIC (hydrophobic-interaction chromatography) and IEX (ion-exchange chromatography). The pH values of the buffers used in homogenization and HIC were adjusted to pH 9.0 to maintain protein stability and prevent protein degradation. Using this manufacturing process, we obtained clinical-grade HSP65–MUC1 with a yield of 400 mg per 70 g of wet cell pellet and >96% purity. [ABSTRACT FROM AUTHOR]

Batch isotherm experiments were carried out to select the best chromatographic supports for the purification of beet distillery condensates with the aim of recovering fermentable water. Adsorption parameters of four inhibitory solutes of the condensates chosen as targets were obtained, leading to the selection of a weak anion-exchanger and a polystyrenic resin. These results were further used for the chromatographic scaleup. Dynamic adsorption experiments run on both resins with a synthetic mixture of the four target solutes showed the presence of competition for the adsorption. The extended Langmuir model was then found to be applicable for a first estimation of the solute breakthrough. The treatment efficiencies of the two supports were confirmed with an industrial condensate, indicating components in the effluent that were not considered did not significantly affect the adsorption of the main compounds. Eventually, a combination of the two complementary resins was proposed for complete effluent detoxification. [ABSTRACT FROM AUTHOR]

Potassium nickel hexacyanoferrate(II) matrix was prepared and characterized. Purification of neutron-activation Mo–molybdate(VI) solutes from some residual cross-contaminant radionuclides including Cs, Rb, Sb, Co and Cr was carried out onto small chromatographic columns of the matrix (each of 1.0 g). Gamma-ray spectrometric analysis data showed that the purification process was not affected by either Mo(VI) concentrations (0.13 and 0.02 M Mo) in the feeding solution of 3.5 M NaNO at pH 9.5 or flow rates (0.2 and 5.0 mL/min) at room temperature. Molybdenum(VI) average loss onto the column matrix did not exceed 7.7 × 10 mmol Mo. Except for Cr, the matrix has very high selectivity for Cs, Rb and Sb with ~50% Co elimination. The obtained data were briefly discussed. [ABSTRACT FROM AUTHOR]

Synthetic oligodeoxynucleotides are purified with use of a catching by polymerization, washing, and releasing approach. The method does not require any chromatography, and purification is achieved by simple operations such as shaking, washing, and extraction. It is therefore useful for large-scale purification of synthetic oligonucleotide drugs. In addition to purification of oligonucleotides, this catching by polymerization concept is expected to be equally useful for purification of other synthetic oligomers such as peptides and oligosaccharides. [ABSTRACT FROM AUTHOR]

Abstract: In this paper, a simple method for the rapid extraction, separation and purification of bergenin from Ardisia crenata sims and Rodgersia sambucifolia hemsl by microwave-assisted extraction (MAE) coupled with high-speed counter-current chromatography (HSCCC) was developed. The MAE conditions were optimized and 2.0g sample was extracted using 60% (v/v) aqueous methanol as extraction solvent with liquid/solid ratio of 10/1(mL/g) at 60°C for 15min. The crude extract of MAE was separated and purified directly by HSCCC using ethyl acetate–n-butanol–water (3:2:5, v/v/v) solvent system. In less than 3.5h, 18.6 or 25.0mg of bergenin was obtained from 160mg crude extract of A. creanta or R. sambucifolia in one-step separation, respectively. The purity of bergenin was over 99% determined by HPLC and its chemical structure was further identified by ESI-MS, 1H NMR and UV. The results indicate that microwave-assisted extraction coupled with high-speed counter-current chromatography is very suitable for the extraction, separation and purification of bergenin from A. creanta and R. sambucifolia. [ABSTRACT FROM AUTHOR]

Abstract: The adsorbent ω-aminodecyl-agarose was evaluated as to its feasibility for the adsorption of human serum and plasma proteins, aiming at the purification of immunoglobulin G (IgG). The contribution of electrostatic and hydrophobic interactions (mixed-mode) and the effects of buffer system on the adsorption of serum proteins were also studied. The adsorption isotherm parameters of human serum albumin (HSA) and IgG were evaluated, pointing to the existence of cooperative effects in the process. A positive (n =2.30±0.38) and negative cooperativity (n =0.63±0.12) were observed for IgG and HSA binding, respectively. High purity IgG was obtained (based on total protein concentration and nephelometric analysis of HSA, transferrin, and immunoglobulins A, G, and M) with a 75% recovery in Hepes 25mmolL−1 pH 6.8 feeding human serum. These results indicate that the use of ω-aminodecyl-agarose is a potential technique for purification of IgG from human serum. [Copyright &y& Elsevier]

Abstract: The extraction of lemon balm (Melissa officinalis) leaves with aqueous methanol was optimised using response surface methodology. Fifteen runs were conducted following a Box-Behnken design (BBD) followed by ridge analysis using the concentration of methanol, the extraction temperature and time as the independent variables and taking the extraction yield of RA from lemon balm as the response variable. The optimal extraction conditions were a methanol concentration of 59.0% (v/v), a temperature of 54.8°C and a time of 64.8min, which gave a maximal RA yield of 46.1mg RA/g dry materials. The RA extract was loaded onto a column packed with Sephadex LH-20 and then was eluted with 100% methanol, which resulted in RA with a purity of 38.8% and a yield of 43.8%. The purity of RA increased by 3.1-fold when compared to its initial purity in the extract obtained from extraction. [Copyright &y& Elsevier]

Two alkaline keratinases-I and II secreted by Bacillus halodurans PPKS-2 were purified and characterized. Both the keratinases were purified using ammonium sulfate, DEAE-Sephadex followed by Sephadex G-200 column chromatography. The purification was 21.5-fold and 11.17% yield for keratinase-I and 23.7-fold with yield 18.46 for keratinase-II and its molecular weights 30 and 66 kDa. Both purified enzymes were relatively stable over a broad pH range 7.0–13.0 and optimally active at pH 11.0 and 60–70 °C. Keratinase-II was found to be more stable at 70 °C for 3 h and retained 100% of its activity, whereas keratinase-I lost 10% activity. Keratinase-I had high keratin disulfide reductase activity with low keratinase activity whereas keratinase-II had high keratinase activity with low keratin disulfide reductase activity. Keratinase activities of both the enzymes were completely inhibited by PMSF at 1 mM, whereas keratin disulfide reductase activity of keratinase-I was not affected. Enzymes were active and stable in the presence of the surfactants, bleaching agents (20% H2O2), commercial detergents (1%), and SDS (20%). Both the enzymes were partially sequenced and found that keratinase-I and II had a homology with disulfide reductases and serine type of proteases, respectively. [ABSTRACT FROM AUTHOR]

Abstract: We report an improved procedure for purification of the ω subunit of Escherichia coli RNA polymerase. In contrast to the original procedure, the revised procedure: (i) allows purification of ω entirely from the soluble fraction, obviating the need for denaturation/renaturation, (ii) results in >99% pure ω in only two chromatographic steps, and (iii) improves the yield of purified ω by at least 5-fold. Reconstitution of E. coli RNAP from ω purified by this procedure, as well as purified sigma and core RNAP lacking ω, produces active holoenzyme in vitro, and co-overexpression of ω from a plasmid containing rpoZ and an additional plasmid encoding the other RNAP core subunits results in production of active core enzyme in vivo. [Copyright &y& Elsevier]