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Anion Exchange Chromatography (anion + exchange_chromatography)
Kinds of Anion Exchange Chromatography Selected AbstractsEffect of Conductivity, pH, and Elution Buffer Salinity on Glycomacropeptide Recovery from Whey Using Anion Exchange ChromatographyJOURNAL OF FOOD SCIENCE, Issue 4 2005Hatice N. Tek ABSTRACT: The objective of this study was to investigate the effect of whey conductivity, pH, and the salt concentration of the elution buffer on glycomacropeptide recovery and its extent of contamination using anion exchange chromatography. Glycomacropeptide was isolated from Mozzarella whey. Samples were analyzed for glycomacropeptide and contaminating whey proteins. Mass balances and percent recoveries were calculated from these data. Glycomacropeptide recovery increased substantially with decreasing conductivity and increasing pH of the whey feed stream. Increasing the pH, but not increasing the conductivity, increased contamination of the glycomacropeptide by primarily beta-lactoglobulin. Salt concentration of at least 0.1 M was required for complete elution of bound glycomacropeptide. These data define conditions needed for glycomacropeptide recovery by a process chromatography system that uses food-grade buffers, operates at industrially relevant flow rates, and achieves up to 98% recovery. [source] Anion exchange chromatography provides a robust, predictable process to ensure viral safety of biotechnology productsBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2009Daniel M. Strauss Abstract The mammalian cell-lines used to produce biopharmaceutical products are known to produce endogenous retrovirus-like particles and have the potential to foster adventitious viruses as well. To ensure product safety and regulatory compliance, recovery processes must be capable of removing or inactivating any viral impurities or contaminants which may be present. Anion exchange chromatography (AEX) is a common process in the recovery of monoclonal antibody products and has been shown to be effective for viral removal. To further characterize the robustness of viral clearance by AEX with respect to process variations, we have investigated the ability of an AEX process to remove three model viruses using various combinations of mAb products, feedstock conductivities and compositions, equilibration buffers, and pooling criteria. Our data indicate that AEX provides complete or near-complete removal of all three model viruses over a wide range of process conditions, including those typically used in manufacturing processes. Furthermore, this process provides effective viral clearance for different mAb products, using a variety of feedstocks, equilibration buffers, and different pooling criteria. Viral clearance is observed to decrease when feedstocks with sufficiently high conductivities are used, and the limit at which the decrease occurs is dependent on the salt composition of the feedstock. These data illustrate the robust nature of the AEX recovery process for removal of viruses, and they indicate that proper design of AEX processes can ensure viral safety of mAb products. Biotechnol. Bioeng. 2009;102: 168,175. © 2008 Wiley Periodicals, Inc. [source] Salt tolerant membrane adsorbers for robust impurity clearanceBIOTECHNOLOGY PROGRESS, Issue 6 2009William T. Riordan Abstract Clearance of impurities such as viruses, host cell protein (HCP), and DNA is a critical purification design consideration for manufacture of monoclonal antibody therapeutics. Anion exchange chromatography has frequently been utilized to accomplish this goal; however, anion exchange adsorbents based on the traditional quaternary amine (Q) ligand are sensitive to salt concentration, leading to reduced clearance levels of impurities at moderate salt concentrations (50,150 mM). In this report, membrane adsorbers incorporating four alternative salt tolerant anion exchange ligands were examined for impurity clearance: agmatine, tris-2-aminoethyl amine, polyhexamethylene biguanide (PHMB), and polyethyleneimine. Each of these ligands provided greater than 5 log reduction value (LRV) for viral clearance of phage ,X174 (pI , 6.7) at pH 7.5 and phage PR772 (pI , 4) at pH 4.2 in the presence of salt. Under these same conditions, the commercial Q membrane adsorber provided no clearance (zero LRV). Clearance of host-cell protein at pH 7.5 was the most challenging test case; only PHMB maintained 1.5 LRV in 150 mM salt. The salt tolerance of PHMB was attributed to its large positive net charge through the presence of multiple biguanide groups that participated in electrostatic and hydrogen bonding interactions with the impurity molecules. On the basis of the results of this study, membrane adsorbers that incorporate salt tolerant anion exchange ligands provide a robust approach to impurity clearance during the purification of monoclonal antibodies. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] Removal of endogenous retrovirus-like particles from CHO-cell derived products using Q sepharose fast flow chromatography,BIOTECHNOLOGY PROGRESS, Issue 4 2009Daniel M. Strauss Abstract Retrovirus-like particles (RVLPs) that are expressed during the production of monoclonal antibodies in Chinese hamster ovary (CHO) cell cultures must be removed during product recovery. Anion exchange chromatography (AEX) performed in product flow-through mode, a common component in the purification of monoclonal antibodies, has been shown to provide robust removal of a related retrovirus model, but it's ability to remove the actual RVLP impurities has not been directly investigated. We have determined the ability of a typical Q sepharose process to remove actual CHO RVLP impurities. Using small scale experiments with three model antibodies, we observe that this AEX process is capable of effectively removing both in-process and spiked RVLPs from different feedstocks containing different mAb products. In addition, we show that this AEX process also achieves a similarly high degree of RVLP removal during large scale manufacturing operations. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] Biomarker discovery in rat plasma for estrogen receptor-, actionELECTROPHORESIS, Issue 23 2005Tom G. Holt Dr. Abstract To support in vivo screening efforts for estrogen receptor (ER) subtype selective therapeutic agents, we initiated work to discover surrogate markers (biomarkers) in blood plasma that would change in response to ER subtype-specific action. We used a proteomic approach employing strong anion exchange chromatography (SAX), PAGE, and MS to identify potential plasma markers for selective ER-, action. The methodology was used to compare blood from vehicle-treated rats to blood from rats treated with either 17,-estradiol (an ER-,/ER-, agonist) or compound 1 (17,-ethynyl-[3,2-c]pyrazolo-19-nor-4-androstene-17,-ol, an ER-,-selective agonist). Blood samples were first fractionated by SAX to separate fractions containing dominant common plasma proteins from fractions enriched for less-abundant plasma proteins. 1-D PAGE analysis of fractions depleted of dominant plasma proteins revealed treatment-specific changes in protein profiles. Protein bands that changed reproducibly in response to ER-, action were excised from the gel, separated by capillary LC, and identified by microspray ESI-MS. Using this method, the plasma levels of two proteins, transthyretin and apolipoprotein E, were shown to decrease in response to ER-, agonism. The method lacked the sensitivity to identify the known, 1000-fold less-abundant, estrogenic marker prolactin (PRL). However, using a commercial RIA and immunoblots, we showed that PRL levels increase significantly in response to treatment with the ER-, selective agonist, compound 1. [source] Isolation of DNA from genetically modified oils by fast protein liquid chromatographyINTERNATIONAL JOURNAL OF FOOD SCIENCE & TECHNOLOGY, Issue 7 2010Li Huang Summary In this study, a novel method of fast protein liquid chromatography (FPLC) anion exchange chromatography was developed for isolation of DNA from processed genetically modified (GM) oils. Four kinds of different GM edible oil had been chosen as model sample. Salmon DNA was used as the control sample to determine the pH values and NaCl in mobile phase buffer. Applying pH 8 and NaCl gradient 0.5,2 m were chosen for the DNA isolation. The quality and purity of isolated DNA were tested with agarose gel electrophoresis, scanned with UV absorbance spectra and amplified by polymerase chain reaction (PCR). The result indicated that the quantity of DNA isolated by FPLC was suitable for further PCR analyses. Furthermore, it is more effective and less time-consuming in comparison with cetyltrimethylammonium bromide method and High Pure GMO Sample Preparation Kit method. [source] Purification and characterization of heparan sulfate from human primary osteoblastsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 5 2009Sadasivam Murali Abstract Heparan sulfate (HS) is a linear, highly variable, highly sulfated glycosaminoglycan sugar whose biological activity largely depends on internal sulfated domains that mediate specific binding to an extensive range of proteins. In this study we employed anion exchange chromatography, molecular sieving and enzymatic cleavage on HS fractions purified from three compartments of cultured osteoblasts,soluble conditioned media, cell surface, and extracellular matrix (ECM). We demonstrate that the composition of HS chains purified from the different compartments is structurally non-identical by a number of parameters, and that these differences have significant ramifications for their ligand-binding properties. The HS chains purified of conditioned medium had twice the binding affinity for FGF2 when compared with either cell surface or ECM HS. In contrast, similar binding of BMP2 to the three types of HS was observed. These results suggest that different biological compartments of cultured cells have structurally and functionally distinct HS species that help to modulate the flow of HS-dependent factors between the ECM and the cell surface. J. Cell. Biochem. 108: 1132,1142, 2009. © 2009 Wiley-Liss, Inc. [source] Bone-specific heparan sulfates induce osteoblast growth arrest and downregulation of retinoblastoma proteinJOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2006Kerry J. Manton The heparan sulfate (HSs) sugars of the extracellular matrix (ECM) play a key role during both development and wound repair in regulating the flow of growth and adhesive factors across their cell surface receptors. The aim of this study was to assess the structural and functional differences of HS chains extracted from the conditioned media (soluble), cell surface, and ECM of primary human osteoblast cultures, and to analyze their effects on osteoblast cell growth. HS chains from these compartments were characterized through a combination of enzymatic degradation, anion exchange chromatography, and molecular sieving. Although the chains were all approximately the same size, they varied systematically in their sulfate content, suggesting differences in their protein-binding domains. When added to pre-confluent hFOB1.19 osteoblast cultures, HS doses exceeding 500 ng/ml inhibited proliferation, without affecting viability, irrespective of their origin. Furthermore, HS doses of 500 ng/ml also downregulated retinoblastoma, Cyclin A and CDK1 protein expression, indicating that high doses of osteoblast HS negatively regulate cell cycle, resulting in growth arrest; when high doses of HS were withdrawn after a prolonged period, linear cell growth was reestablished. Thus, despite differences in sulfation, HS from either the soluble, cell surface, or matrix compartments of primary human osteoblast cultures are functionally similar with respect to their effects on growth. Binding assays revealed that the HS chains bound TGF,1, a known inhibitor of osteoprogenitor growth, at higher affinity than a suite of other bone-related, heparin-binding growth factors. Overcoming such sugar-mediated inhibition may prove important for wound repair. J. Cell. Physiol. 209: 219,229, 2006. © 2006 Wiley-Liss, Inc. [source] Purification and characterization of an organic solvent and detergent-tolerant novel protease produced by Bacillus sp.JOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 11 2008Abstract BACKGROUND: Purification and characterization of a novel protease produced by Bacillus sp. RKY3, has been investigated, with special emphasis on the stability of the enzyme in the presence of different oxidizing and reducing agents as well as organic solvents. The enzyme was purified in two steps through concentration of the crude enzyme by ammonium sulfate precipitation, followed by anion exchange chromatography. RESULTS: The purified protease had a molecular mass of approximately 38 kDa, which was highly active over a broad range of pH between 7.0 and 9.0 and was also stable over a wide pH range from 5.0 to 11.0. Although the optimum temperature for enzyme activity was found to be 60 °C, it was rapidly deactivated at temperatures above 60 °C. It also showed good stability at 50 °C, with a 70 min half-life. Ca2+ ions did not greatly enhance the activity or the stability of the enzyme. PMSF (1 mmol L,1) completely inhibited the protease activity, and thus the purified protease was considered to be serine protease. The purified protease was stable with oxidants (H2O2, 2%), reducing agents (sodium dodecyl sulfate, 2%), and organic solvents (25%) such as benzene, hexane, and toluene. CONCLUSION: The purified enzyme, protease, seems to possess potential applications in protease-based detergent and bleaching industries. The enzymatic activity against a wide variety of substrates suggests that the purified enzyme should be investigated for a range of commercial applications, especially for soy protein and gelatin hydrolysis in the food processing industry. Copyright © 2008 Society of Chemical Industry [source] Effect of Conductivity, pH, and Elution Buffer Salinity on Glycomacropeptide Recovery from Whey Using Anion Exchange ChromatographyJOURNAL OF FOOD SCIENCE, Issue 4 2005Hatice N. Tek ABSTRACT: The objective of this study was to investigate the effect of whey conductivity, pH, and the salt concentration of the elution buffer on glycomacropeptide recovery and its extent of contamination using anion exchange chromatography. Glycomacropeptide was isolated from Mozzarella whey. Samples were analyzed for glycomacropeptide and contaminating whey proteins. Mass balances and percent recoveries were calculated from these data. Glycomacropeptide recovery increased substantially with decreasing conductivity and increasing pH of the whey feed stream. Increasing the pH, but not increasing the conductivity, increased contamination of the glycomacropeptide by primarily beta-lactoglobulin. Salt concentration of at least 0.1 M was required for complete elution of bound glycomacropeptide. These data define conditions needed for glycomacropeptide recovery by a process chromatography system that uses food-grade buffers, operates at industrially relevant flow rates, and achieves up to 98% recovery. [source] STRUCTURE-ACTIVITY RELATIONSHIPS OF OLIGOAGAR ELICITORS TOWARD GRACILARIA CONFERTA (RHODOPHYTA)JOURNAL OF PHYCOLOGY, Issue 3 2001Florian Weinberger Agar oligosaccharides in the neoagarobiose series were prepared by partial enzyme hydrolysis, separated on Biogel P2 and P4, and analyzed by high-performance anion exchange chromatography with pulsed amperometric detection, yielding neoagarosaccharide fractions with a disaccharide repetition degree ranging from 1 (neoagarobiose) to more than 8 (neoagarohexadecaose). These fractions were analyzed for their biological activity toward the marine red alga Gracilaria conferta (Schousboe ex Montagne) J. et G. Feldmann in terms of increase of oxygen consumption, release of hydrogen peroxide, elimination of epiphytic bacteria, and induction of thallus tip bleaching. The structure,activity and dose,response relationships of neoagarosaccharides were very similar in the respiratory and oxidative burst responses and in their bactericidal properties, with neoagarosaccharides consisting of 6 to 8 disaccharide repeating units being the most active. All these responses were competitively inhibited by the reduced form of neoagarohexaose, neoagarohexaitol. In contrast, the tip-bleaching response was light dependent, required much higher concentrations of neoagarosaccharides, and was not inhibited by neoagarohexaitol, suggesting that it is an unspecific oxidative stress reaction. Putative structural effects on the recognition of endogenous agar-oligosaccharide elicitors by G. conferta are discussed. [source] Release and characterization of single side chains of white cabbage pectin and their complement-fixing activityMOLECULAR NUTRITION & FOOD RESEARCH (FORMERLY NAHRUNG/FOOD), Issue 6 2009Bjorge Westereng Abstract A mixture of single side chains from white cabbage pectin were obtained by anion exchange chromatography after applying mild chemical conditions promoting ,-elimination. These pectin fragments were characterized by their molecular weight distribution, sugar composition, 13C-NMR, and MALDI-TOF-MS analysis. These analyses revealed that the large oligosaccharides released by ,-eliminative treatment were composed of ,-1,5 linked arabinosyl residues with 2- and 3-linked ,-arabinosyl side chains, and, or ,-1,4 linked galactosyl side chains. Fractions were tested for complement-fixing activity in order to determine their interaction with the complement system. These results strongly indicated that there was a minimal unit size responsible for the complement-fixing activity. Neutral pectin fragments (,8 kDa) obtained from ,-elimination were inactive in the complement system, although they contained a sugar composition previously shown to be highly active. Larger pectin fragments (,17 kDa) retained some activity, but much lower than polymers containing rhamnogalacturonan type 1 (RGI) structures isolated from the same source. This implied that structural elements containing multiple side chains is necessary for efficient complement-fixing activity. [source] Purification and characterisation of two ACC oxidases expressed differentially during leaf ontogeny in white cloverPHYSIOLOGIA PLANTARUM, Issue 1 2000Deming Gong Two isoforms of ACC oxidase (ACO) (EC 1.4.3), expressed differentially during leaf ontogeny in white clover (Trifolium repens L.), have been identified and purified to homogeneity. One isoform, designated MGI, was purified from mature green leaf tissue while the second isoform, designated SEII, was purified from senescent leaf tissue. The isolation and purification of these isoforms were achieved using a combination of hydrophobic interaction chromatography, anion exchange chromatography, chromatofocusing and gel filtration column chromatography. The Mr of both MGI and SEII was determined to be 37.5 kDa by gel filtration, and 37 kDa (MGI), 35 kDa (SEII) by SDS-PAGE, indicating that both isoforms are active as monomers. During purification, both isoforms were recognised by a polyclonal antibody directed against a recombinant polypeptide derived from a white clover ACO gene expressed in mature green leaf tissue, TR-ACO2. In addition to molecular mass, differences between the two isoforms were observed in terms of pH optima, isoelectric point (pI), Km for ACC, optimal requirements for the co-substrate ascorbate, and NaHCO3 and Fe2+ as co-factors. The identification of distinct ACC oxidases from the same tissue at different developmental stages shows that the now widely observed transcriptional regulation of the ACO gene family in higher plants is also expressed in terms of differential regulation of enzyme isoforms. [source] Analysis of carbohydrates and amino acids in vegetable waste waters by ion chromatographyPHYTOCHEMICAL ANALYSIS, Issue 2 2003Michele Arienzo Abstract High-performance anion exchange chromatography coupled with pulsed amperometric detection was used for the quantitative determination of total and free sugars in olive oil mill waste waters (OMWW). Automated amino acid ion chromatography was employed to analyse total and free amino acids in the same OMWW. Sugars were analysed in samples pre-purified by means of a three-step purification procedure involving: (i) methanol precipitation of OMWW; (ii) dialysis of the obtained solid and liquid fractions; and (iii) chromatographic purification on RP18 phase followed by Amberlite resin. The amino acids were determined directly in samples obtained from the first two steps performed for sugar analysis. The analysis carried out with the reported methodologies allowed the quantitative determination of total sugars and amino acids and the differentiation between their free and bound forms. The sugars determined were arabinose, fructose, galactose, glucose, rhamnose, xylose, galacturonic and glucuronic acids, and the amino acids were Asp, Glu, Thr, Ser, Pro, Gly, Ala, Val, Met, Ile, Leu, Tyr, Phe, Lys, His, Arg and Cys. Asn, Gln, and Trp were not detected. The technological, biotechnological and environmental advantages arising from this analytical methodology applied to OMWW are briefly discussed. Copyright © 2003 John Wiley & Sons, Ltd. [source] The phosphoproteome of Fusarium graminearum at the onset of nitrogen starvationPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 1 2010Christof Rampitsch Abstract Fusarium graminearum grown under stress, such as nutrient deprivation, activates, among others, the trichothecene pathway that produces the mycotoxin deoxynivalenol and its derivatives. The kinase inhibitor staurosporine reduced the production of trichothecenes by 39% compared with control in vitro. On the other hand, phosphatase inhibitor okadaic acid increased the amount by 72% compared with the control in vitro. This suggests that phosphorylation events are involved in the signalling pathway, leading to the activation of the trichothecene pathway. Three approaches were used to study the phosphoproteome of F. graminearum under nitrogen-limiting conditions: 2-DE (2-DE: IEF×SDS-PAGE) in combination with MS protein identification; SDS-PAGE in combination with off-line IMAC and TiO2 enrichment and gel electrophoresis LC-MS analysis; and a gel-free approach using strong anion exchange chromatography, IMAC and LC-MS. A total of 348 phosphorylation sites localized in 301 peptides from 241 proteins were identified. By 2-DE, 20 phosphoproteins were identified, nine of which underwent changes during the time course examined. Using gel electrophoresis LC-MS 231 phosphopeptides were identified from three samples (ten gel slices each) at time points of nitrogen starvation t=0, 6, and 12,h. The gel-free analysis added 70 peptides from 65 proteins to the total. Proteins of unknown function and enzymes of known function comprised the largest groups overall. Ten protein kinases and seven transcription factors were identified. This is the first reported phosphoproteome of F. graminearum. [source] Large-scale phosphoproteome analysis of human liver tissue by enrichment and fractionation of phosphopeptides with strong anion exchange chromatographyPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 7 2008Guanghui Han Abstract The mixture of phosphopeptides enriched from proteome samples are very complex. To reduce the complexity it is necessary to fractionate the phosphopeptides. However, conventional enrichment methods typically only enrich phosphopeptides but not fractionate phosphopeptides. In this study, the application of strong anion exchange (SAX) chromatography for enrichment and fractionation of phosphopeptides was presented. It was found that phosphopeptides were highly enriched by SAX and majority of unmodified peptides did not bind onto SAX. Compared with Fe3+ immobilized metal affinity chromatography (Fe3+ -IMAC), almost double phosphopeptides were identified from the same sample when only one fraction was generated by SAX. SAX and Fe3+ -IMAC showed the complementarity in enrichment and identification of phosphopeptides. It was also demonstrated that SAX have the ability to fractionate phosphopeptides under gradient elution based on their different interaction with SAX adsorbent. SAX was further applied to enrich and fractionate phosphopeptides in tryptic digest of proteins extracted from human liver tissue adjacent to tumorous region for phosphoproteome profiling. This resulted in the highly confident identification of 274 phosphorylation sites from 305 unique phosphopeptides corresponding to 168 proteins at false discovery rate (FDR) of 0.96%. [source] Purification and properties of trypsin-like enzyme from the midgut of Morimus funereus (coleoptera, cerambycidae) LarvaeARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 4 2010Nikola Lon Abstract Trypsin-like enzyme (TLE) from the anterior midgut of Morimus funereus larvae was purified by anion exchange chromatography and gel filtration chromatography and characterized. Specific TLE activity was increased 322-fold by purification of the crude midgut extract. The purified enzyme had a pH optimum of 9.0 (optimum pH range 8.5,9.5) and temperature optimum of 45°C with the KM ratio of 0.065,mM for benzoyl-arginine- p -nitroanilide (BApNA). Among a number of inhibitors tested, the most efficient was benzamidine (KI value of 0.012,mM, Ic50 value of 0.204,mM) while inhibition of TLE activity by SBTI, TLCK, and PMSF was partial. Almost all divalent cations tested enhanced the enzyme activity, amongst them Co2+ and Mn2+ stimulated TLE activity for 2.5 times. The purified TLE (after gel-filtration on Superose 12 column) had a molecular mass of 37.5,kDa with an isoelectric point over 9.3. Sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed one band of 38,kDa, suggesting that the enzyme is a monomer. © 2010 Wiley Periodicals, Inc. [source] Identification of endo- and exo-polygalacturonase activity in Lygus hesperus (Knight) salivary glands,ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 2 2009Maria de la Paz Celorio-Mancera Abstract Polygalacturonase (PG) activity found in the salivary gland apparatus of the western tarnished plant bug (WTPB, Lygus hesperus Knight) has been thought to be the main chemical cause of the damage inflicted by this mirid when feeding on its plant hosts. Early viscosity and thermal stability studies of the PG activity in L. hesperus protein extracts were difficult to interpret. Thus, it has been suggested that one or more PG protein(s) with different hydrolytic modes of action are produced by this mirid. In order to understand the quantitative complexity of the WTPB salivary PG activity, PG purification from a protein extract from salivary glands excised from L. hesperus insects was performed using affinity and ion exchange chromatography. To elucidate the qualitative complexity of the purified PGs, the digestion products generated by the PGs were separated using high performance anion exchange chromatography with pulsed amperometric detection. At least five PG proteins were detected; these differing in terms of their glycosylation, mass-to-charge ratios, and/or molecular mass. The characterization of the products generated by these PGs showed that endo- and exo-acting PGs are produced by WTPB. Although none of the PGs was purified to homogeneity, the present work provides biochemical evidence of a multiplicity of PGs that degrade the pectin component of the plant tissue in different fashions. The implications of these findings affect the understanding of WTPB feeding damage and, potentially, help identify ways to control this important crop pest. Arch. Insect Biochem. Physiol. 2008. © 2008 Wiley-Liss, Inc. [source] Separation and quantification of inulin in selected artichoke (Cynara scolymus L.) cultivars and dandelion (Taraxacum officinale WEB. ex WIGG.) roots by high-performance anion exchange chromatography with pulsed amperometric detectionBIOMEDICAL CHROMATOGRAPHY, Issue 12 2006Katrin Schütz Abstract The profile of fructooligosaccharides and fructopolysaccharides in artichoke heads and dandelion roots was investigated. For this purpose, a suitable method for high-performance anion exchange chromatography with pulsed amperometic detection was developed. The separation of monomers, oligomers and polymers up to a chain length of 79 sugar residues was achieved in one single run. Glucose, fructose, sucrose and individual fructooligosaccharides (kestose, nystose, fructofuranosylnystose) were quantified in six different artichoke cultivars and in dandelion roots. The contents ranged from 12.9 g/kg DM to 71.7 g/kg DM for glucose, from 15.8 g/kg DM to 67.2 g/kg DM for fructose, and from 16.8 g/kg DM to 55.2 g/kg DM for sucrose in the artichoke heads. Kestose was the predominant fructooligosaccharide, followed by nystose and fructofuranosylnystose. In four cultivars fructofuranosylnystose was only detectable in traces and reached its maximum value of 3.6 g/kg DM in the cultivar Le Castel. Furthermore, an average degree of polymerization of 5.3 to 16.7 was calculated for the individual artichoke cultivars, which is noticeably lower than hitherto reported. In contrast, the contents of kestose, nystose and fructofuranosylnystose in dandelion root exceeded that of artichoke, reflecting the short chain characteristic of the inulin, which was confirmed by chromatographic analysis. Copyright © 2006 John Wiley & Sons, Ltd. [source] Heating-induced conformational change of a novel ,-(1,3)- D -glucan from Pleurotus geestanusBIOPOLYMERS, Issue 2 2010Mei Zhang Abstract Recently, we isolated and purified a neutral polysaccharide (PGN) from edible fungus Pleurotus geestanus. Its structure was characterized by a range of physical,chemical methods, including high performance anion exchange chromatography, uronic acid, and protein analyses, size exclusion chromatography with ultraviolet, refractive index and light scattering detectors, and nuclear magnetic resonance. Our results revealed that PGN is a novel ,-(1,3)- D -glucan with glucose attached to every other sugar residues at Position 6 in the backbone. It has a degree of branching of 1/2. Such structure is different from typical ,-(1,3)- D -glucans schizophyllan and lentinan in which DB is 1/3 and 2/5, respectively. Rheological study showed a very interesting melting behavior of PGN in water solution: heating PGN in water leads to two transitions, in the range of 8,12.5°C and 25,60°C, respectively. The melting behavior and conformational changes were characterized by rheometry, micro-differential scan calorimetry, atomic force microscopy, static and dynamic light scattering at different temperatures. The first heating-induced transition corresponds to the disintegration of polymer bundles into small helical clusters, resembling the heating-induced dissociation of SPG in water at 7°C; the second one might correspond to the dissociation of helical strands to individual chains. The ability of PGN to undergo a conformation/viscosity transition in water upon heating is very valuable to immobilize cells or enzymes or therapeutic DNA/RNA, which makes PGN a potentially useful biomaterial. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 121,131, 2010. This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com [source] Degree of blockiness of amide groups as indicator for difference in physical behavior of amidated pectinsBIOPOLYMERS, Issue 1 2006Stéphanie E. Guillotin Abstract Thickening and gelling properties of commercial amidated pectins depend on the degree of amidation and methyl-esterification, but also the distribution of these groups is of great importance. Methods have been developed during the last few years to determine the distribution of methyl esters over the pectic backbone. We applied the strategies developed for the analysis of high methyl-esterified pectins for studying the distribution of amide groups in amidated pectins. Low methyl-esterified amidated (LMA) pectins were digested before and after removal of methyl esters by an endo -polygalacturonase to determine the degree of blockiness of the substituents. The nature of the substituents (amide groups compared to methyl esters) did not modify the behavior of the enzyme. Oligomers released were separated by using high-performance anion exchange chromatography and pulsed amperometric detection (HPAEC,PAD) at pH 5. Fractions collected after on-line desalting were identified by using MALDI,TOF mass spectrometry. Oligomers were found to elute from the column as a function of their total charge. For the same overall charge and size, oligomers with methyl esters eluted before oligomers with amide groups. Both amide groups and methyl esters of the LMA pectins studied were found to be semirandomly distributed over the pectic backbone, but this may vary according to the amidation process used. © 2006 Wiley Periodicals, Inc. Biopolymers 82: 29,37, 2006 This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com [source] Selective precipitation-assisted recovery of immunoglobulins from bovine serum using controlled-fouling crossflow membrane microfiltrationBIOTECHNOLOGY & BIOENGINEERING, Issue 5 2008Adith Venkiteshwaran Abstract Efficient and economic recovery of immunoglobulins (Igs) from complex biological fluids such as serum, cell culture supernatant or fermentation cell lysate or supernatant, represents a substantial challenge in biotechnology. Methods such as protein A affinity chromatography and anion exchange chromatography are limited by cost and selectivity, respectively, while membrane chromatography is limited by low adsorptive area, flow distribution problems and scale-up difficulties. By combining the traditional salt-assisted precipitation process for selective removal of Igs from serum followed by constant-permeate flux membrane microfiltration for low fouling, we demonstrate an exciting new, efficient and economic hybrid method. The high selectivity of an ammonium sulfate-induced precipitation step was used to precipitate the Igs leaving the major undesirable impurity, the bovine serum albumin (BSA), in solution. Crossflow membrane microfiltration in diafiltration mode was then employed to retain the precipitate, while using axial flow rates to optimize removal of residual soluble BSA to the permeate. The selectivity between immunoglobulin G (IgG) and BSA obtained from the precipitation step was ,36, with 97% removal of the BSA with diafiltration in 5 diavolumes with resulting purity of the IgG of ,93% after the membrane microfiltration step. Complete resolubilization of the IgG was obtained without any aggregation at the concentrations of ammonium sulfate employed in this work. Further, membrane pore size and axial Reynolds number (recirculation rate) were shown to be important for minimizing fouling and loss of protein precipitate. Biotechnol. Bioeng. © 2008 Wiley Periodicals, Inc. [source] RNA interference of sialidase improves glycoprotein sialic acid content consistencyBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2006Frederyk A. Ngantung Abstract An important challenge facing therapeutic protein production in mammalian cell culture is the cleavage of terminal sialic acids on recombinant protein glycans by the glycosidase enzymes released by lysed cells into the supernatant. This undesired phenomenon results in a protein product which is rapidly cleared from the plasma by asialoglycoprotein receptors in the liver. In this study, RNA interference was utilized as a genetic approach to silence the activity of sialidase, a glycosidase responsible for cleaving terminal sialic acids on IFN-, produced by Chinese Hamster Ovary (CHO) cells. We first identified a 21-nt double stranded siRNA that reduced endogenous sialidase mRNA and protein activity levels. Potency of each siRNA sequences was compared using real time RT-PCR and a sialidase activity assay. We next integrated the siRNA sequence into CHO cells, allowing production and selection of stable cell lines. We isolated stable clones with sialidase activity reduced by over 60% as compared to the control cell line. Micellar electrokinetic chromatography (MEKC), thiobarbituric acid assay (TAA), and high performance anion exchange chromatography (HPAEC) coupled to amperometric detection were performed to analyze glycan site occupancy, sialic acid content, and distribution of asialo-/sialylated-glycan structures, respectively. Two of the stable clones successfully retained the full sialic acid content of the recombinant IFN-,, even upon cells' death. This was comparable to the case where a chemically synthesized sialidase inhibitor was used. These results demonstrated that RNA interference of sialidase can prevent the desialylation problem in glycoprotein production, resulting improved protein quality during the entire cell culture process. © 2006 Wiley Periodicals, Inc. [source] Large scale purification of linear plasmid DNA for efficient high throughput cloningBIOTECHNOLOGY JOURNAL, Issue 9 2010Dr. Marjolaine Noirclerc-Savoye Abstract In this report we describe a rapid, simple, and efficient method for large-scale purification of linear plasmid DNA to answer demand from high-throughput gene cloning. The process is based on the separation of the linear vector from small DNA fragments by anion exchange chromatography. Gene cloning experiments by restriction/ligation or the In-Fusion(tm) technique confirmed the high quality of the linearized vector as 100% of the genes were successfully cloned. [source] Enzymatic production of ,- D -glucose-1-phosphate from trehaloseBIOTECHNOLOGY JOURNAL, Issue 9 2010Jef Van der Borght Abstract ,- D -Glucose-1-phosphate (,Glc1P) is an efficient glucosyl donor for both enzymatic and chemical glycosylation reactions but is currently very costly and not available in large amounts. This article provides an efficient production method of ,Glc1P from trehalose and phosphate using the thermostable trehalose phosphorylase from Thermoanaerobacter brockii. At the process temperature of 60°C, Escherichia coli expression host cells are lysed and cell treatment prior to the reaction is, therefore, not required. In this way, the theoretical maximum yield of 26% could be easily achieved. Two different purification strategies have been compared, anion exchange chromatography or carbohydrate removal by treatment with trehalase and yeast, followed by chemical phosphate precipitation. In a next step, ,Glc1P was precipitated with ethanol but this did not induce crystallization, in contrast to what is observed with other glycosylphosphates. After conversion of the product to its cyclohexylammonium salt, however, crystals could be readily obtained. Although both purification methods were quantitative (>99% recovery), a large amount of product (50%) was lost during crystallization. Nevertheless, a production process for crystalline ,Glc1P is now available from the cheap substrates trehalose and inorganic phosphate. [source] Strategies for developing design spaces for viral clearance by anion exchange chromatography during monoclonal antibody productionBIOTECHNOLOGY PROGRESS, Issue 3 2010Daniel M. Strauss Abstract The quality-by-design (QbD) regulatory initiative promotes the development of process design spaces describing the multidimensional effects and interactions of process variables on critical quality attributes of therapeutic products. However, because of the complex nature of production processes, strategies must be devised to provide for design space development with reasonable allocation of resources while maintaining highly dependable results. Here, we discuss strategies for the determination of design spaces for viral clearance by anion exchange chromatography (AEX) during purification of monoclonal antibodies. We developed a risk assessment for AEX using a formalized method and applying previous knowledge of the effects of certain variables and the mechanism of action for virus removal by this process. We then use design-of-experiments (DOE) concepts to perform a highly fractionated factorial experiment and show that varying many process parameters simultaneously over wide ranges does not affect the ability of the AEX process to remove endogenous retrovirus-like particles from CHO-cell derived feedstocks. Finally, we performed a full factorial design and observed that a high degree of viral clearance was obtained for three different model viruses when the most significant process parameters were varied over ranges relevant to typical manufacturing processes. These experiments indicate the robust nature of viral clearance by the AEX process as well as the design space where removal of viral impurities and contaminants can be assured. In addition, the concepts and methodology presented here provides a general approach for the development of design spaces to assure that quality of biotherapeutic products is maintained. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] Production and Molecular Characterization of Clinical Phase I Anti-Melanoma Mouse IgG3 Monoclonal Antibody R24BIOTECHNOLOGY PROGRESS, Issue 5 2001Sven E. Kemminer R24 is a mouse IgG3 monoclonal antibody (mab) that reacts with the ganglioside GD3 expressed by cells of neuroectodermal origin. The anti-tumor activity of R24 has been demonstrated in initial phase I and pilot trials in patients suffering from metastatic melanoma. The purpose of this study was to investigate the biotechnological production and particularly the glycosylation of this clinically important antibody. Growth, metabolism, and IgG production of R24 secreting hybridoma cells were analyzed on 1 L bioreactor bench scale using repeated-batch mode. The amount of 57 mg of pure mab was obtained from 1.6 L crude supernatant by protein A chromatography. Western blot binding assays with sugar-specific lectins revealed glycosylation of the heavy chains, whereas no carbohydrates were detectable on the light chains. Because glycosylation is essential for antibody effector functions in vivo (such as complement fixation or binding to macrophage Fc receptors), mab R24 was subjected to both enzymatic deglycosylation using PNGase F and chemical deglycosylation by hydrazinolysis. Released glycans were structurally characterized by high pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD), matrix assisted laser desorption ionization time-of-flight (MALDI-TOF), and electrospray ionization quadrupole time-of-flight (ESI-QTOF) mass spectrometry. Six major biantennary chains of the complex glycosylation phenotype were found with variations in galactosylation and core fucosylation. The predominant N-linked structure, indicating the high degree of agalactosyl glycoforms, was the agalacto biantennary chain with a relative percentage of 57% (51% core-fucosylated, 6% nonfucosylated). The second most abundant oligosaccharide was the monogalacto biantennary chain amounting to 30% (26% core- and 4% nonfucosylated). The antibody contained 0.46 ,g sialic acid per mg protein, which splits into 0.243 ,g Neu5Gc and 0.217 ,g Neu5Ac, corresponding to a Neu5Ac:Neu5Gc ratio of 1:1.06. Furthermore, the antigen specificity of R24 was determined by immunodetection of GD3 on thin-layer chromatograms, and real time GD3-antibody binding interactions were measured with an optical biosensor (BIAcore). From the structural data obtained in this study it is concluded that glycosylation of the antibody may be important in the clinical outcome of targeted anti-cancer immunotherapy. [source] | |||||||||||||||||||||||||