Exchange Chromatographic Techniques Research Articles

Comparison of purified and crude extracted coagulants from plant species for turbidity removal

The coagulation properties of crude extracts and purified proteins of seeds of Moringa stenopetala and tubers of Maerua subcordata, which are locally used for purification of turbid water in Ethiopia, were studied. The turbidity removal effectiveness was compared using synthetic turbid and natural river water where the initial turbidity of synthetic water was approximately 260 NTU, whereas, for river water, the initial turbidity was 30 NTU. The active coagulants from the extracts were purified by the single-step and the two-step ion exchange (IEX) chromatography technique using 0.1, 0.3 and 0.6 M concentrations of NaCl solutions. The protein quantification of a single-step purified active coagulant eluted using 0.6 M showed that the protein content of the M. stenopetala extract was 1016 µg/mL, whereas the concentration of the M. subcordata extract was 700 µg/mL. The dose for coagulation using crude extracts and purified proteins of M. subcordata on surface water was 4 and 2 mg/L, respectively, with 85% of coagulation activity. In the case of M. stenopetala, 14 mg/L dose of crude extracts, which was seven times higher than that of the purified protein, was needed to obtain the desired residual turbidity (5 NTU). The results also indicated that in the two-step purified coagulants, the sludge volume was reduced compared to crude extracts and alum. In conclusion, active coagulants from seeds of M. stenopetala could be scaled up and used in water treatment technology, whereas purifying the extracts from M. subcordata is not required as the crude extracts performed equally well.

Total phosphorus, phytate phosphorus contents and the correlation of phytates with amylose in selected edible beans in Sri Lanka

Phytate a major anti nutritional factors in legumes and it accounts for larger portion of the total phosphorus, while limiting the bioavailablity of certain divalent cations to the human body. Legumes of eleven varieties cultivated in Sri Lanka, Mung bean (MI5, MI6), Cowpea (Waruni, MICP1, Bombay, Dhawala, ANKCP1), Soybean (MISB1, Pb1) and Horse gram (ANKBlack, ANKBrown) were analyzed for phosphorus content and phytate content. Total phosphorus content was quantified by dry ashing followed by spectrophotometrical measurement of the blue colour intensity of acid soluble phosphate with sodium molybdate in the presence of ascorbic acid while phytate phosphorus using anion exchange chromatographic technique followed by spectrometrical measurement of the digested organic phosphorus and amylose content by Simple Iodine-Colourimetric method. Where the least value for phosphorus was observed 275.04 ±1.44 mg.100g-1 in ANKBlack (Horse gram) and the highest in MISB1 (Soyabean) with 654.94 ±0.05 mg.100g-1. The phytate phosphorus content (which is a ratio of phyate to total phosphorus) was highest in Dhawala (Cowpea). The phytate phosphorus (which is a ratio of phyate to total phosphorus) was highest in Dhawala with 67.42% and least in Bombay (Cowpea) with 24.87%. The amylose content of the legumes was least in Pb1 with 8.71 ±0.13 mg.100mg-1 and the highest in MI6 22.58 ±0.71 mg.100mg-1. The correlation between phyate and total phosphorus was significant (p <0.05) and positive (r = 0.62). Similarly the correlation coefficient for phytate phosphorus and total phosphorus was significant (p <0.05) and positive (r = 0.63). Amylose content of legumes was significantly correlated negatively (p <0.05) with the total phytates content (r = -0.82).

Purification, immobilization and characterization of thermostable α-amylase from a thermophilic bacterium Geobacillus sp. TF14

Abstract Objective In this study, α-amylase from a thermophilic bacterium Geobacillus sp. TF14 was purified and immobilized on two different supports. Methods Ion exchange and hydrophobic interaction chromatography techniques were employed for the purification. Results The enzyme was purified as 17.11 fold and determined as a single band of 54 kDa on SDS-PAGE. Purified enzyme showed two pH optimums of pH 5.00 and pH 9.00 and the enzyme is quite stable at these pHs over a period of 48 h. Purified enzyme showed maximal activity at 75°C and stability at this temperature over a period of 72 h. It was observed that Ca2+ activated the enzyme at about 70% at 5 mM final concentration. SDS, Triton X100, Triton X114 and Tween 20 caused around 50% loss of initial activity at a final concentration of 1% (w/v). Purified enzyme was immobilized on the surface of Dowex and chitin. Immobilization highly enhanced temperature optima and thermal stability. Dowex immobilized enzyme maintained most of its initial activity in the presence of SDS, Triton X100, Triton X114 and Tween 20 at a concentration of 1%. Conclusion It can be concluded that the purified enzyme may find application in many fields of starch based industries.

Pretreatment with ethanol as an alternative to improve steviol glycosides extraction and purification from a new variety of stevia

Leaves of a new variety of Stevia rebaudiana with a high content of rebaudioside A were pretreated with ethanol. The ethanolic extract showed high antioxidant potential and 39 compounds were identified, by UPLC/HRMS, among them one not yet mentioned in the literature for stevia leaves. From the in natura leaves and pretreated leaves, the conditions of aqueous extraction of steviol glycosides were investigated using response surface methodology. The aqueous extracts obtained were purified by ion exchange chromatography techniques and membrane separation methods. The recuperation of steviol glycosides was 4.02g for pretreated leaves and 2.20g for in natura leaves. The level of purity was, respectively, 87% and 84.8%. The results obtained demonstrate that pretreatment increases the yield and purity level of stevia sweeteners by the use of environmentally friendly methodologies and the final product presented acceptable sensory characteristics.

ISOLATION, PURIFICATION, AND OPTIMIZATION OF THERMOPHILIC AND ALKALIPHILC PROTEASE ORIGINATING FROM HOT WATER SPRING BACTERIA

Objective: The main objective of this study is to investigate the industrial applications of a thermophillic alkaline protease from a hot water spring bacterial isolate “A” and to study its production, optimization, and purification.Methods: The alkaline protease was produced using shake flask studies maintaining a pH of 9.0 and a temperature of 50°C. Optimization studies of the enzyme were carried out using variable pH, temperature, organic carbon, and nitrogen sources followed by purification of the enzyme using DEAE-cellulose ion exchange chromatography technique. Stability of the enzyme was analyzed in the presence of organic solvents and surfactants. The efficiency of the enzyme in the removal of proteinaceous stains in the presence of strong detergents under extreme conditions was assessed. The fibrinolytic activity of the enzyme in dissolving the blood clot was confirmed.Results: The isolated alkaline protease was purified to homogeneity with a 16-fold increase. Media optimization studies revealed that 1% glucose and 1 % casein-induced the production of alkaline protease. The purified enzyme retained stability in the presence of ethanol, methanol, and acetone and surfactants such as 0.5% (w/v) sodium dodecyl sulfate (SDS) and 0.5% (v/v) Triton-X-100. The isolated alkaline protease successfully removed the proteinaceous stains and showed significant results in the dissolution of blood clot.Conclusion: The above experimental results confirm that the isolated enzyme has both thermophilic and alkaliphilic protease properties. Thereby the enzyme finds promising industrial applications even in extreme conditions.

Purification, characterization, and bioassay of putative protease inhibitors from Hevea brasiliensis latex

Latex, a milky white liquid, is the main product from rubber tree (Hevea brasiliensis). Latex is the cytoplasm of complex cellular networks named laticifers in which it contains many different components, including important proteins. Various types of enzymes carrying functions associated with plant defense against pathogen and wounding have been detected in latex in which one of these enzymes is protease inhibitor (PI). Plant protease inhibitor has tremendous potential as an antifungal agent which can be developed as biofungicide. In this work, protease inhibitors from B-serum (lutoid) of rubber tree latex were isolated and purified using Ion Exchange Chromatography (IEC) technique. Of the total 70 fractions of proteins extracted from the columns, only 26 fractions showed measurable levels of protein. The concentration of obtained putative protease inhibitors (three fractions of IEC) ranged from 0.007 to 0.022 mL/g B-serum. Inhibitory activity against four protease enzymes (subtilisin A, trypsin, α-chymotrypsin, and papain) showed the characteristics of Hevea putative protease inhibitors from B-serum as serine and/or cysteine protease inhibitors with more than 15% inhibitory activity of target protease. Based on SDS-PAGE visualization, the molecular weight of dominant protein considered as Hevea putative protease inhibitors was 21.5 kDa. In vitro bioassay test of antifungal activity for Hevea putative protease inhibitors showed reduced mycelium growth of Ganoderma boninense, Sclerotium sp., and Rigidosporus lignosus.

Immobilization of Purified Phytase Enzyme from Tirmit (Lactarius volemus) on Coated Chitosan with Iron Nanoparticles and Investigation of Its Usability in Cereal Industry

In this study, phytase enzyme was purified and characterized from Tirmit (Lactarius volemus) mushroom and purified phytase enzyme was immobilized on the surface of modified chitosan with nano-Fe3O4 nanoparticles. Then, free and immobilized enzyme applications had been carried out in cereal crops. Phytase enzyme was purified 120.2-fold and characterized with 11.6 % yield in range of 40–80 % from Tirmit (L. volemus) by using, ammonium sulfate precipitation, DEAE-Sephadex ion exchange chromatography and Sephacryl S200 gel filtration chromatography techniques. The purification fold was calculated by determining the activity and amount of protein for each step. Then, purified phytase enzyme was immobilized on modified chitosan support material with Fe3O4 nanoparticles. For the immobilized and free enzyme, optimum pH was 5.5 and optimum temperature was 50 °C. First, the effects of some metal ions, such as CuCl2, HgCl2, FeCl2, MgCl2, ZnCl2 and CaCl2, were investigated on the bound and free purified phytase enzyme. Second, molecular weight of the enzyme was determined by SDS-PAGE electrophoresis method. Third, substrate specificity was determined for the purified phytase enzyme using some natural polymers, such as locust bean gum, gelatin, xylene, cellulose, phytate and starch. Finally, investigated whether the immobilized and free enzyme could be hydrolyzed the phytic acid in green lentils, red lentils, peas, pinto beans, beans, brass, corn, dried corn, oat, rye, wheat, broad bean, chickpea and peanuts or not.

PURIFICATION AND CHARACTERIZATION OF SALIVARY AMYLASE INHIBITOR EXTRACTED FROM BARLEY

Amylase inhibitors were purified by many sequential steps included concentration by gradual addition of ammonium sulfate at saturation ratios. ranged from 0 to 90% . The best ratio of saturation was found to be 70% as the specific activity and inhibition activity toward Human alpha-amylase(HAS) were the highest ( 8 U/mg and 6 U/ml respectively as compared to those of the rest ratios, the ratio of saturation with ammonium sulfate 60 % and then 50%, (5.8 ,5.5 )U/ml and( 7.7 ،7 )U/mg respectively for inhibition activity and specific activity and for 40% ,30%20% saturation the inhibition activity and specific activity were(5 ،4.8 ،4 ) u/ml (6.6 ،6 ،5.8) u/mg respectively .The precepitation step was followed by ionic exchange chromatography technique by DEAE-cellulose column( 3×11 )cm and the results showed that there was one peak with inhibition activity toward (HAS). Further purification steps were conducted using gel filtration on Sephacryl S-200 column (1.5 × 60)cm; the purification folds was5.59 times with outcome of 46.5%.The results of alpha-amylase inhibitors characterization showed that the molecular weight was about 23.44 and 22.9 kDa as determined by electrophoresis and gel filteration respectively.

A thermostable-endo-β-(1,4)-mannanase from Pediococcus acidilactici (M17): purification, characterization and its application in fruit juice clarification

A Turkish fermented sausage was bought from a local market in Erzurum and was used for isolation and characterization of lactic acid bacteria. As the result of 16S rRNA analysis, this bacterium was determined as Pediococcus acidilactici (Genbank number KF895384) at a rate of 99 %. The mannanase from P. acidilactici (M17) was purified using the (NH4)2SO4 precipitation, DEAE-cellulose ion exchange and Sephacryl S200 gel filtration chromatography techniques. The yield and purification fold of mannanase were 10.6 % and 106.1, respectively. The molecular mass of the enzyme (38 kDa) was determined by SDS-PAGE and gel filtration chromatography. In order to identify, the resistance of the purified mannanase to Mn2+, Co2+, Cu2+, Zn2+ and Ca2+ was examined, and all the metal ions activated the enzyme at a significant level. The V max and K m values for locust bean gum were calculated as 78.74 mmol/min mg and 0.592 mM, by Lineweaver–Burk. Purified mannanase was also used to clarify some fruit juices. The studied variables were, the incubation time (from 0 to 60 min), the enzyme concentration (17 EU/L), and the temperature (60 °C). It was observed that the purified mannanase had maximum yield on orange juice with 161.1 %. Also maximal clarification was obtained as 84.65 %, and the clarification for grape juice and sugar reduction was obtained as 19.26 mg/mL for apple juice. Consequently, it was concluded that the P. acidilactici (M17) mannanase could be successfully used in the fruit juice industry.

Refinement of high precision Ru isotope analysis using negative thermal ionization mass spectrometry

A refined method for the isolation, purification, and high precision measurement of Ru isotope compositions in natural samples by negative thermal ionization mass spectrometry (N-TIMS) is reported. After chemical purification of Ru using ion exchange chromatography and microdistillation techniques, the Ru isotopic composition is measured as RuO3− via N-TIMS. Data are corrected for oxide interferences using the simultaneously measured oxygen isotope composition, and subsequently for mass fractionation using an exponential law. Repeat analyses of an Alfa Aesar Ru standard solution demonstrate external reproducibility of 100Ru/101Ru to ±6.4ppm (2SD). This level of precision is more than two times better than prior techniques. Repeat analyses of gravimetrically prepared mixtures of a natural Alfa Aesar Ru standard and a 100Ru enriched spike show that isotopic differences of ≥13ppm can be resolved by single measurements of a material using this method. Repeat analyses of diverse terrestrial materials (chromitites and Os-Ir-Ru alloys) are characterized by compositions that are identical to the Alfa Aesar standard, and the external reproducibility for these materials is also identical to that of the chemically pure standard, demonstrating that chemical separation/purification methods introduce no bias to the analysis. These materials likely define the Ru isotopic composition of the Earth's mantle.

Efficient expression and purification of biologically active human cystatin proteins

Cystatins are reversible cysteine protease inhibitor proteins. They are known to play important roles in controlling cathepsins, neurodegenerative disease, and in immune system regulation. Production of recombinant cystatin proteins is important for biochemical and function characterization. In this study, we cloned and expressed human stefin A, stefin B and cystatin C in Escherichia coli. Human stefin A, stefin B and cystatin C were purified from soluble fraction. For cystatin C, we used various chaperone plasmids to make cystatin C soluble, as it is reported to localize in inclusion bodies. Trigger factor, GroES-GroEL, DnaK-DnaJ-GrpE chaperones lead to the presence of cystatin C in the soluble fraction. Immobilized metal affinity chromatography, glutathione sepharose and anion exchange chromatography techniques were employed for efficient purification of these proteins. Their biological activities were tested by inhibition assays against cathepsin L and H3 protease.

Immobilization of a thermostable phytase from Pinar melkior (Lactarius piperatus) onto magnetite chitosan nanoparticles

Phytase enzyme was purified from the Pinar melkior (Lactarius piperatus) mushroom using ammonium sulfate precipitation and DEAE-sephadex ion exchange chromatography techniques. First, the purified phytase enzyme was covalently bound to the surface of magnetite-CTS NPs in yield of 87%. Then, optimum pHs of the free and immobilized enzymes were determined as 5.0 and 4.0, respectively. Optimum temperature of free and immobilized enzymes was found as 60°C. Also, the effects of some metal ions on activity of free and immobilized phytase enzymes were investigated. Also, research was undertaken as to whether the purified free and immobilized phytase enzyme could hydrolyze the phytic acid in many cereal products or not. And, it was discovered that the immobilized phytase enzyme hydrolyzed the phytic acid at the highest rate (75.02% rate) in wheat. From the findings obtained, that immobilized enzyme was quite resistant to temperature, pH and metal ions.

Screening and identification of apolipoprotein A-I as a potential hepatoblastoma biomarker in children, excluding inflammatory factors.

The aim of the present study was to identify a child hepatoblastoma serum biomarker that is unaffected by inflammatory factors, with the ultimate aim of finding an effective method for the early diagnosis of hepatoblastoma. The magnetic bead-based weak cation exchange chromatography technique was used to process serum harvested from 30 children with hepatoblastoma, 20 children with systemic inflammatory response syndrome (SIRS) and 20 healthy children. Proteins differentially expressed in SIRS were excluded from consideration as biomarkers for hepatoblastoma. Proteins differentially expressed in hepatoblastoma and healthy controls were screened using surface-enhanced laser desorption/ionization-time of flight-mass spectrometry (SELDI-TOF-MS). Target proteins were purified by SDS-PAGE, and matrix-assisted laser desorption/ionization (MALDI)-TOF-MS was used to determine their amino acid sequences. Protein matches were searched in the SwissProt database. Quantitative polymerase chain reaction (qPCR) and ELISA were employed to confirm the expression of target proteins. Following screening to exclude inflammatory factors, SELDI-TOF-MS revealed a protein with a mass-to-charge ratio of 9,348 Da that was expressed at significantly lower levels in the serum of children with hepatoblastoma compared with healthy controls (P<0.01). Sequence analysis identified this protein as apolipoprotein A-1 (Apo A-I). qPCR and ELISA confirmed that the expression of Apo A-I mRNA and protein were significantly lower in children with hepatoblastoma compared with healthy controls (P<0.05). These results indicate that Apo A-I is a non-inflammatory protein marker for hepatoblastoma with the potential for use in early diagnosis of hepatoblastoma. In addition, the present study demonstrates the feasibility of proteomic screening for the identification of proteins that can serve as markers for a specific tumor.

Improved Detection of Sleeping Sickness Cases by LED Fluorescence Microscopy: Evidence from a Prospective Multi-Centric Study in the Democratic Republic of the Congo

Background: Confirmatory diagnosis of Trypanosoma brucei gambiense human African trypanosomiasis (HAT) is based on demonstration of parasites by microscopy. However, the sensitivity of routine microscopy methods is very low, and many cases are missed and left untreated. A clinical study was conducted in the Democratic Republic of the Congo to evaluate the accuracy of improved microscopy methods in diagnosis of HAT. These included examination by fluorescence microscopy (FM) of acridine orange (AO) stained smears of whole blood and smears made following a new procedure for concentrating trypanosomes by selective lysis of red blood cells (RBC). Methodology/Principal Findings: Venous blood was collected from 213 HAT cases, 101 HAT suspects and 95 controls and used to determine the accuracy of four microscopy methods: bright field microscopy of Giemsa-stained thick blood smears, FM of AO-stained thick blood smears, FM of AO-stained thick blood smears prepared after RBC lysis and concentration, and FM of AO-stained thin blood smears prepared after RBC lysis and concentration. The sensitivity of FM using thick blood smears stained with AO was 3 times higher than bright field microscopy using Giemsa-stained thick blood smears [19.7% (95% CI: 14.9% - 25.6%) versus 6.1% (95% CI: 3.6% - 10.2%)]. When the RBC lysis and concentration procedure was included, sensitivity of the test was further enhanced to 23.0% (95% CI: 17.9% - 29.1%) with thick blood smears and 34.3% (95% CI: 28.2% - 40.9%) with thin blood smears. Specificity of all four microscopy methods was 100% (95% CI: 96.1% - 100.0%). However, the miniature anion exchange chromatography technique (mAECT) and capillary tube centrifugation (CTC) method remained more sensitive. Conclusions: These new methods have practical advantages, including shorter staining time, ease of demonstration of parasites, and the possibility of archiving slides. They could, therefore, be alternative methods to improve case detection where concentration procedures such as mAECT or CTC are not performed.

A non-pediocin low molecular weight antimicrobial peptide produced by Pediococcus pentosaceus strain IE-3 shows increased activity under reducing environment.

BackgroundSpecies of the genus Pediococcus are known to produce antimicrobial peptides such as pediocin-like bacteriocins that contain YGNGVXC as a conserved motif at their N-terminus. Until now, the molecular weight of various bacteriocins produced by different strains of the genus Pediococcus have been found to vary between 2.7 to 4.6 kD. In the present study, we characterized an antimicrobial peptide produced by P. pentosaceus strain IE-3.ResultsAntimicrobial peptide was isolated and purified from the supernatant of P. pentosaceus strain IE-3 grown for 48 h using cation exchange chromatography and reversed-phase high-performance liquid chromatography (RP-HPLC) techniques. While MALDI-TOF MS experiments determined the precise molecular mass of the peptide to be 1701.00 Da, the de novo sequence (APVPFSCTRGCLTHLV) of the peptide revealed no similarity with reported pediocins and did not contain the YGNGVXC conserved motif. Unlike pediocin-like bacteriocins, the low molecular weight peptide (LMW) showed resistance to different proteases. Moreover, peptide treated with reducing agent like dithiothreitol (DTT) exhibited increased activity against both Gram-positive and Gram-negative test strains in comparison to native peptide. However, peptide treated with oxidizing agent such as hydrogen peroxide (H2O2) did not show any antimicrobial activity.ConclusionTo our knowledge this is the lowest molecular weight peptide produced by members of the genus Pediococcus. The low molecular weight peptide shared amino acid arrangement with N-terminal sequence of Class IIa, pediocin-like bacteriocins and showed increased activity under reducing conditions. Antimicrobial peptides active under reduced conditions are valuable for the preservation of processed foods like meat, dairy and canned foods where low redox potential prevails.

Study of the properties and inhibition of the polyamine oxidase enzyme partially purified from the fruit of green pepper by amyloride and medovi

The study included a detection of PAO in green pepper (Capsicum grossum) and its partial purification by using dialysis and anion exchange chromatography (using DEAE – cellulose) techniques . Two isoenzymes were obtained, with a specific activity of 3.9 and 2.6 unit / mg protein, and a purification fold of 21.4 and 14.3 respectively compared to crude enzyme. Copper ion in both isoenzymes was detected and found to be 0.1915 and 0.2947 mg / ml respectively. The maximum PAOI,II isoenzyme activity were obtained at 100 microlitre of enzyme, sodium phosphate buffer at pH 8.6,7.2, temperature at 40, 37°C, and 125 , 100 mM of spermidine as substrate respectively, and have a high specificity toward the polyamine, spermidine compared with other amine compounds. The inhibitory effect of the drugs amiloride and medovire on PAOI activity was studied and showed that increasing of inhibitor concentration led to a decrease in activity. The inhibition type was found to be competitive , and Ki were 0.88 and 1.1 mM respectively. Spermine was detected by HPLC technique from non-proteinous extract. Basic amino acids were isolated by using Amberlite 120 H+ and Amberlite 50NH4+ successively, the results indicated that the green pepper contained 96.1 mg / g of plant.

Recombinant Albumin-Partnering Technology: Development Of Balugrastim, a Novel Long-Acting Granulocyte Colony-Stimulating Factor

IntroductionVarious strategies have been used to extend the half-life of therapeutic proteins, including genetic fusion with carrier proteins. One such carrier protein is human serum albumin (HSA), a benign protein with minimal intrinsic biologic activity that is naturally present in the circulation at a high concentration. It has a long half-life (≈19 days in humans) and is highly soluble. Recombinant HSA produced from yeast retains the beneficial stabilizing properties of HSA while minimizing the potential disadvantages of a serum-derived product. Balugrastim, a novel, long-acting recombinant protein composed of HSA and human granulocyte colony-stimulating factor (G-CSF), was developed for once-per-cycle subcutaneous (SC) administration to provide a novel option for the prevention of severe neutropenia in patients with cancer receiving myelosuppressive chemotherapy. The rational design of balugrastim, differences in its protein chemistry compared with pegfilgrastim, and the clinical and practical implications are presented here. MethodsDuring the design phase of balugrastim, HSA was deemed an ideal candidate as a carrier protein because of its wide distribution in the body, long half-life, and low potential for affecting biological activity of G-CSF. A highly engineered proprietary yeast strain was chosen to achieve high levels of expression and quality. Balugrastim is manufactured using recombinant DNA technology in the yeast Saccharomyces cerevisiae in contrast to pegfilgrastim, which is a PEGylated form of a G-CSF expressed in the bacterium Escherichia coli and then modified by chemical conjugation to polyethylene glycol. Balugrastim was purified using a combination of ion exchange and affinity and chromatography techniques. For clinical testing, sensitive immunogenicity and serum concentration assays were developed for the product. ResultsThe manufacturing process produces balugrastim, a 759-amino-acid monomeric protein with a molecular mass of ≈85 kDa. It is a single continuous polypeptide chain in which residues 1–585 correspond to HSA and residues 586–759 correspond to the amino acid sequence of human G-CSF, connected via a peptide bond. The purified protein is >95% pure as determined by N-terminal sequencing. The result is a highly homogeneous product. The manufacturing process is straightforward, requiring no reformulations, additional chemical modifications, or secondary manufacturing, and is a scalable, modular production system. Balugrastim has a pharmacodynamic profile comparable to that of pegfilgrastim. In a clinical trial comparing balugrastim with pegfilgrastim in patients with breast cancer, the half-life of balugrastim 40 mg SC administered once per cycle was 37.7 hours, maximum plasma concentration was 875 ng/mL, and mean area under the concentration–time curve over 144 hours was 60321 h•ng/mL, providing sustained activity in the therapeutic window and stable blood levels (Pukac, MASCC/ISOO, 2012). Corresponding values for pegfilgrastim 6 mg SC were 47.1 hours, 164 ng/mL, and 11554 h•ng/mL, respectively. In this study, and in a randomized phase III trial in patients with breast cancer, balugrastim was noninferior to pegfilgrastim, with a safety profile similar to that of pegfilgrastim and low incidence of immunogenicity (Gladkov, ASCO, 2011). ConclusionsAlbumin partnering is an established technology used to generate innovative, half-life extended products. This technology formed the basis for the rational design for balugrastim, a novel once-per-cycle G-CSF for the prevention of severe neutropenia in patients with cancer receiving myelosuppressive chemotherapy. The technology provides balugrastim with several advantages, including a consistent, high-quality product with low immunogenic potential and an extended half-life that permits once-per-chemotherapy cycle administration. The low viscosity of balugrastim permits small needle size (29 gauge). Balugrastim, developed as an alternative to pegfilgrastim, has been shown to be noninferior to pegfilgrastim in clinical trials. Disclosures:Avisar:Teva Pharmaceuticals, Inc: Employment. Pukac:Teva Pharmaceuticals, Inc: Employment. Adar:Teva Pharmaceuticals, Inc: Employment. Barash:Teva Pharmaceuticals, Inc: Employment. Clark:Teva Pharmaceuticals, Inc: Employment. Liu:Teva Pharmaceuticals, Inc: Employment. Bock:Teva Pharmaceuticals, Inc: Employment. Shen:Teva Pharmaceuticals, Inc: Employment.

Isotope Separation of Nitrogen by Ion Exchange Chromatography in a Cascade System

The ion exchange chromatography technique in a cascade process was used to separate stable isotopes of nitrogen (14N/15N). For this purpose, three column systems (with different internal diameters, S1−14.5 cm; S2–9.5 cm; S3–5.2 cm) were used that contained the cationic resins DOWEX 50 WX8 (as H+ and equilibrated with H2O) and measured 1.7 m in height. Isotopic fractionation of nitrogen occurs during displacement band chromatography (DBC) of the NH3 (aq) solution with a 0.75 mol L−1 NaOH solution. The active sites of the resin were previously saturated to form NH4 + (RNH4) with a 0.75 mol L−1 solution of (NH4)2SO4 at a natural isotopic abundance (0.366 at. %) in 15N. The NH4 +/NH3 (aq) equilibrium was determined during the DBC, which enriched the rear portion of the band with 15N. In S1, enrichment of approximately 5 at. % of 15N in the last 10 cm was achieved. After four interactions (couplings) between S1–S2, an enrichment of approximately 50 at. % of 15N was obtained. During the interaction between S2–S3, after the second coupling, an isotopic enrichment of approximately 90 at. % of 15N was observed, which produced 70 g month−1 of (15NH4)2SO4.

Ion exchange chromatographic separation and isolation of oligosaccharides of intact low-molecular-weight heparin for the determination of their anticoagulant and anti-inflammatory properties

It is well known that enoxaparin, a widely used anticoagulant and low-molecular-weight heparin containing a large number of oligosaccharides, possesses anti-inflammatory activity. Whilst enoxaparin has shown promising results in various inflammatory disorders, some of its oligosaccharides have anti-inflammatory properties and others increase the risk of bleeding due to their anticoagulant effects. The aim of this study was to develop an effective ion exchange chromatographic (IC) technique which allows the separation, isolation and, consequently, the identification of different oligosaccharides of enoxaparin with or without anticoagulant activity. The developed method utilises a semi-preparative CarboPac PA100 (9 × 250mm) ion exchange column with sodium chloride gradient elution and UV detection at 232nm. The method successfully resolved enoxaparin into more than 30 different peaks. IC-derived oligosaccharides with high, moderate, low or no anticoagulant activity were identified using an anti-factor Xa assay. The anti-inflammatory activity of selected oligosaccharides was investigated using the Griess assay. Using this technique, the oligosaccharides of enoxaparin with low or no anticoagulant activity, whilst exhibiting significant anti-inflammatory activity, could be fractionated. This technique can provide a platform to identify the oligosaccharides which are devoid of significant anticoagulant activity and are responsible for the therapeutic effects of enoxaparin that have been observed in various inflammatory conditions.

Isolation and Characterization of Chelonia mydas Myoglobin

Green sea turtle – Chelonia mydas is a lung respiration animal that is able to dive and stay uder sea water for hours without needing to surface for oxygen. As oxygen supply in muscles is assured by myoglobin, we propose to study some characteristic of this muscle protein in green sea turtles. To achieve this objective, pure green sea turtle myoglobin has to be made available. Therefore, our first task is to purify this muscle protein from green sea turtles. Skeletal muscles from 3 green sea turtle hatchlings were studied microscopically and biochemically. Microscopy observation showed a general structure of striated muscle. Biochemical studies revealed that green sea turtle myoglobin could be more purely isolated to a certain degree by Sephadex G-75 gel filtration and purified by immunoaffinity gel chromatography rather than direct purification by DEAE-Sepharose ion exchange chromatography technique. SDS-PAGE analysis showed that green sea turtle myoglobin ran together with horse myoglobin as 17 kDa molecular weight proteins.