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1.
The highly diastereoselective preparation of cis-N-Ts-iodoaziridines through reaction of diiodomethyllithium with N-Ts aldimines is described. Diiodomethyllithium is prepared by the deprotonation of diiodomethane with LiHMDS, in a THF/diethyl ether mixture, at -78 °Cin the dark. These conditions are essential for the stability of the LiCHI2 reagent generated. The subsequent dropwise addition of N-Ts aldimines to the preformed diiodomethyllithium solution affords an amino-diiodide intermediate, which is not isolated. Rapid warming of the reaction mixture to 0 °C promotes cyclization to afford iodoaziridines with exclusive cis-diastereoselectivity. The addition and cyclization stages of the reaction are mediated in one reaction flask by careful temperature control.Due to the sensitivity of the iodoaziridines to purification, assessment of suitable methods of purification is required. A protocol to assess the stability of sensitive compounds to stationary phases for column chromatography is described. This method is suitable to apply to new iodoaziridines, or other potentially sensitive novel compounds. Consequently this method may find application in range of synthetic projects. The procedure involves firstly the assessment of the reaction yield, prior to purification, by 1H NMR spectroscopy with comparison to an internal standard. Portions of impure product mixture are then exposed to slurries of various stationary phases appropriate for chromatography, in a solvent system suitable as the eluent in flash chromatography. After stirring for 30 min to mimic chromatography, followed by filtering, the samples are analyzed by 1H NMR spectroscopy. Calculated yields for each stationary phase are then compared to that initially obtained from the crude reaction mixture. The results obtained provide a quantitative assessment of the stability of the compound to the different stationary phases; hence the optimal can be selected. The choice of basic alumina, modified to activity IV, as a suitable stationary phase has allowed isolation of certain iodoaziridines in excellent yield and purity. 相似文献
2.
The preparation of plasmid DNA at large scale constitutes a pressing problem in bioseparation. This paper describes a first
investigation of displacement chromatography as a means to separate plasmid DNA (4.7 kb) from E. coli lipopolysaccharides
and protein (holo transferrin), respectively. Displacement chromatography has advantages in this regard, since the substance
mixture is resolved into rectangular zones of the individual components rather than into peaks. Thus a higher total concentration
can be maintained in the pooled product fractions. Hydroxyapatite (type I and II) and anion exchange stationary phases were
included in the experiments. In addition to a conventional anion exchange column packed with porous particles, the recently
introduced continuous bed UNOTM anion exchange column was investigated. No DNA purification was possible with either hydroxyapatite
material. Conventional particle based columns in general were not suited to the separation of any two substances varying considerably
in molecular mass, e.g. plasmid DNA and standard protein. Presumably, the direct competition for the binding sites, which
is essential in displacement chromatography, was restricted by the size dependency of the accessible stationary phase surface
area in this case. Better results were obtained with the continuous bed column, in which the adsorptive surface coincides
with the walls of the flow through pores. As a result the accessible surface does not vary as much with the size of the interacting
molecules as for the conventional stationary phase materials. Sharper transitions were also observed between substance zones
recovered from the UNOTM column. The steric mass action model was used to aid method development in case of the anion exchange
approach. While further research in obviously necessary, displacement chromatography on continuous bed columns has been shown
to be capable of separating plasmid DNA from typical impurities.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
3.
Bianchi F Careri M Corradini C Elviri L Mangia A Zagnoni I 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2005,825(2):193-200
Reversed phase ion-pair chromatography (RP-IPC) of seven heterocyclic aromatic amines encompassing quinoline (IQ, MeIQ), quinoxaline (MeIQx), pyridine (PhIP) and carboline derivatives (AalphaC, Harman, Norharman) was carried out with formate as counter ion in an aqueous eluent with acetonitrile as organic modifier. TSKgel ODS-80TS was used as the stationary phase. With the aim of acquiring a better insight into the mutual influence of ion-pair reagent and the organic modifier upon solute retention, the study was performed by using an experimental design approach able to evidencing the effect of the simultaneous variation of the two factors. A model for the chromatographic behavior of the amines is proposed that includes classical ion-pair mechanism involving formate in the case of MeIQx, PhIP, Harman and Norharman. A competitive ion-exchange mechanism was hypothesized to govern retention of quinoline compounds, whereas electrostatic interactions and hydrogen bond formation with the silanols of the stationary phase were judged to be responsible for the retention of AalphaC. Further, the chromatographic behavior of the analytes using the formic acid-ammonium formate buffer in the mobile phase was compared with that observed using acetic acid-ammonium acetate buffer. The method based on the use of RP IPC with tandem mass spectrometry when the eluent contained formate buffer at pH 2.8 exhibited higher detectability with respect to that achieved using the acetate buffer. 相似文献
4.
高速逆流双水相色谱法纯化卵白蛋白 总被引:7,自引:0,他引:7
生物大分子的液_固色谱纯化过程中固相载体会产生产物吸附、变性等不良影响。高速逆流色谱无需固相载体 ,且具有高分便率和高回收率的优点 ,其中有机相 水相体系在分离天然产物中应用广泛 ,而应用双水相体系分离生物大分子尚处于研究阶段。双水相高速逆流色谱体系的建立与仪器设备及操作工艺条件密切相关 ,因此利用多分离柱高速逆流色谱仪 ,研究了PEG1000-无机盐双水相体系对标准蛋白质混合物以及卵白蛋白的分离。pH值和PEG浓度对不同种类蛋白质的分配系数影响不同 ,实验发现在pH9.2的150% (W/W)PEG1000 170% (W/W)磷酸钾盐体系中 ,细胞色素C、溶菌酶和肌红蛋白的分配系数差异较大 ,且分布合理 ,因而采用该体系在 0 8mL min流速 ,85 0r min转速的条件下 ,成功分离了细胞色素C、溶菌酶和肌红蛋白的混合物。实验也发现在pH9 2的 16 0 % (W/W)PEG10 0 0 17 0 % (W/W)磷酸钾盐体系中 ,鸡蛋清样品中的主要蛋白质成分:卵转铁蛋白、卵白蛋白和溶菌酶的分配系数差异最大 ,因而采用该体系在 1 8mL min流速、85 0r mi转速的条件下,200min内从鸡蛋清样品中成功分离卵白蛋白,其电泳纯度为100%,收率为95%. 相似文献
5.
ε-聚赖氨酸生产菌株Streptomyces albulus PD-1可合成一种新型非蛋白质氨基酸均聚物聚二氨基丙酸,采用离子交换层析和反向色谱,对聚二氨基丙酸的分离纯化进行研究。离子交换层析柱选用DEAE-Sepharose Fast Flow填料,50 mmol/L磷酸盐缓冲液(p H 7.5)平衡上样,含0.5 mol/L Na Cl的磷酸盐缓冲液(p H 7.5)洗脱,收集洗脱液用分子筛Sephadex G-25除去磷酸盐缓冲液。然后用C18反相色谱进一步纯化,流动相为V(甲醇)/V(0.1%磷酸)=5/95。经过离子交换层析和反向色谱,纯化得到聚二氨基丙酸纯品,回收率为39.8%,样品纯度达98.4%,为后续的聚二氨基丙酸的深入研究奠定基础。 相似文献
6.
Nicholas Vecchiarello Steven M. Timmick Chaz Goodwine Laura E. Crowell Kerry R. Love J. Christopher Love Steven M. Cramer 《Biotechnology and bioengineering》2019,116(9):2178-2190
Integrated designs of chromatographic processes for purification of biopharmaceuticals provides potential gains in operational efficiency and reductions of costs and material requirements. We describe a combined method using screening and in silico algorithms for ranking chromatographic steps to rapidly design orthogonally selective integrated processes for purifying protein therapeutics from both process- and product-related impurities. IFN-α2b produced in Pichia pastoris containing a significant product variant challenge was used as a case study. The product and product-related variants were screened on a set of 14 multimodal, ion exchange, and hydrophobic charge induction chromatography resins under various pH and salt linear gradient conditions. Data generated from reversed-phase chromatography of the fractions collected were used to generate a retention database for IFN-α2b and its variants. These data, in combination with a previously constructed process-related impurity database for P. pastoris, were input into an in silico process development tool that generated and ranked all possible integrated chromatographic sequences for their ability to remove both process and product-related impurities. Top-ranking outputs guided the experimental refinement of two successful three step purification processes, one comprising all bind-elute steps and the other having two bind-elute steps and a flowthrough operation. This approach suggests a new platform-like approach for rapidly designing purification processes for a range of proteins where separations of both process- and product-related impurities are needed. 相似文献
7.
两种水溶性抗菌活性物质的分离提取 总被引:2,自引:0,他引:2
对水溶性、不解离的极性物质分离时,一般采用吸附层析和凝胶层析等途径。实验通过硅胶柱层析、葡聚糖凝胶柱层析以及硅胶GF254制备型薄板层析,从发酵液样品中分离出两种有抗菌活性的纯物质。薄层层析的展开剂为二氯甲烷-四氢呋喃-甲醇-水(25:30:2),分离出的组分中Rf=0.7和Rf=0.8两种物质有抗菌活性。硅胶柱层析洗脱过程为梯度洗脱,先用150ml上述展开剂洗脱,再用二氯甲烷-甲醇(20:80) 相似文献
8.
m-Hydroxybenzylalcohol dehydrogenase (EC 1.1.1.97), a secondary metabolism associated protein from stationary phase cultures of Penicillium urticae, was stabilized in crude extracts prior to purification. Stabilization studies resulted in the formulation of an optimal cell breakage and purification buffer. This buffer increased the enzyme's in vitro half-life at 30 degrees C from 14 to over 800 min which greatly aided purification and enhanced yields. Purification was achieved by salt fractionation, size-exclusion chromatography, affinity chromatography, and ion-exchange chromatography. The 1200-fold purified protein gave only one major band by sodium dodecyl sulphate - polyacrylamide gel electrophoresis. 相似文献
9.
High affinity, low molecular weight anionic displacers were successfully employed for the purification of antisense oligonucleotides. Several important structural characteristics were identified that contribute to the affinity of low molecular weight displacers to a hydrophilized polystyrene divinyl benzene anion exchanger. Sulfonic acid groups were found to possess higher affinity than carboxylic acid and phosphate functionalities, and nonspecific interactions (particularly hydrophobic interactions) were shown to play a major role in the retention process on this stationary phase material. Using this information, two high affinity, low molecular weight displacers were identified. These molecules are relatively inexpensive organic dyes that possess multiple sulfonic acid moieties, as well as aromatic functionalities, which increase nonspecific interactions with the stationary phase. These high affinity displacers, which can be readily detected, were then employed to displace several strongly retained antisense oligonucleotides that could not be displaced by previously established low molecular weight displacers. The displacement process resulted in very high purities of the antisense oligonucleotides. The results presented in this paper are significant in that they demonstrate that low molecular weight displacers for ion-exchange chromatography can possess equal to or greater affinities than their higher molecular weight counterparts, when nonspecific interactions with the stationary phase are exploited. In addition, the results illustrate the high resolutions possible with displacement chromatography and demonstrate an attractive technology for the process scale purification of oligonucleotides. 相似文献
10.
Vaccine manufacturing strategies that lower capital and production costs could improve vaccine access by reducing the cost per dose and encouraging localized manufacturing. Continuous processing is increasingly utilized to drive lower costs in biological manufacturing by requiring fewer capital and operating resources. Aqueous two-phase systems (ATPS) are a liquid–liquid extraction technique that enables continuous processing for viral vectors. To date, no economic comparison between viral vector purifications using traditional methods and ATPS has been published. In this work, economic simulations of traditional chromatography-based virus purification were compared to ATPS-based virus purification for the same product output in both batch and continuous modes. First, the modeling strategy was validated by re-creating a viral subunit manufacturing economic simulation. Then, ATPS capital and operating costs were compared to that of a traditional chromatography purification at multiple scales. At all scales, ATPS purification required less than 10% of the capital expenditure compared to chromatography-based purification. At an 11 kg per year production scale, the ATPS production costs were 50% less than purification with chromatography. Other chromatography configurations were explored, and may provide a production cost benefit to ATPS, but the purity and recovery were not experimentally verified. Batch and continuous ATPS were similar in capital and production costs. However, manual price adjustments suggest that continuous ATPS plant-building costs could be less than half that of batch ATPS at the 11 kg per year production scale. These simulations show the significant reduction in manufacturing costs that ATPS-based purification could deliver to the vaccine industry. 相似文献
11.
Wang C Geng X Wang D Tian B 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2004,806(2):185-190
Purification of the prion protein (PrP) is a major concern for biological or biophysical analysis as are the structural specificities of this protein in relation to infectivity. A simple and efficient method for purification of recombinant bovine normal prion protein containing residues 104-242, PrP(104-242) expressed in Escherichia coli by high performance hydrophobic interaction chromatography (HPHIC) was presented in this work. The solution containing denatured and reduced protein in 8.0 mol/L urea extracted from the inclusion body was directly injected into the HPHIC column, aggregates were prevented by the interaction between the denatured PrP(104-242) molecules and the stationary phase during the chromatographic process, the soluble form of PrP(104-242) in aqueous solution was obtained after desorbed from the column. Several factors, including pH value, types of stationary phase and salt, and gradient mode, influencing the purification results were investigated. Optimal conditions were obtained for the purification of PrP(104-242) by HPHIC. This procedure yield PrP(104-242) of a purity of 96% with a recovery of 87%, respectively, for a single step purification of 40 min. 相似文献
12.
Purification and scale-up of a recombinant heavy chain fragment C of botulinum neurotoxin serotype E in Pichia pastoris GS115 总被引:1,自引:0,他引:1
Dux MP Barent R Sinha J Gouthro M Swanson T Barthuli A Inan M Ross JT Smith LA Smith TJ Webb R Loveless B Henderson I Meagher MM 《Protein expression and purification》2006,45(2):359-367
A recombinant C-terminus heavy chain fragment from botulinum neurotoxin serotype E (BoNT/E) is proposed as a vaccine against the serotype E neurotoxin. This fragment, rBoNTE(Hc), was produced intracellular in Pichia pastoris GS115 by a three-step fermentation process, i.e., glycerol batch phase and a glycerol fed-batch phase to achieve high cell densities, followed by a methanol fed-batch induction phase. The rBoNTE(Hc) protein was purified from the soluble fraction of cell lysates using three ion-exchange chromatography steps (SP Sepharose Fast Flow, Q Sepharose Fast Flow, Sp Sepharose High Performance) and polished with a hydrophobic charge induction chromatography step (MEP HyperCel). Method development at the bench scale was achieved using 7-380 mL columns and the process was performed at the pilot scale using 0.5-3.1 L columns in preparation for technology transfer to cGMP manufacturing. The purification process resulted in greater than 98% pure rBoNTE(Hc) based on HPLC and yielded up to 1.01g of rBoNTE(Hc)/kg cells at the bench scale and 580mg vaccine/kg cells at the pilot scale. N-terminal sequencing showed that the purified rBoNTE(Hc) N-terminus is intact and was found to protect mice against a challenge of 1000 mouse intraperitoneal LD50's of BoNT/E. 相似文献
13.
Yoji Nakagawa Kenichi Kishida Yoshifumi Kodani Tohey Matsuyama 《Microbiology and immunology》1997,41(1):27-32
Through the adoption of a chiral stationary phase in high-performance liquid chromatography and a simple derivatization method for hydroxy fatty acids, it became easy to separate and identify the optical isomers of 2- and 3-hydroxy fatty acids composing several kinds of microbial lipids. The 2- and 3-hydroxy fatty acids were converted with dinitrophenyl isocyanate to their 3, 5-dinitrophenyl urethane derivatives (DU-derivatives), which were analyzable by HPLC using a chiral column. By varying the composition of an eluent, separation of the DU-derivatives of hydroxy fatty acids differing in optical configuration, chain length and position of hydroxyl group was achieved. The general elution orders of these DU-derivatives were determined with authentic 2- and 3-hydroxy fatty acids. Small amounts (~300 μg) of ornithine-containing lipids isolated from the Serratia marcescens strains were examined by this method to identify 3-hydroxy fatty acids of the lipids as D isomers. 相似文献
14.
Enantiomeric separation of a variety of drugs and related compounds was achieved on an (S)-naphthylethylcarbamoylated-β-cyclodextrin (S-NEC-CD) chiral stationary phase (CSP) using sub- and supercritical fluid chromatography (SFC). Compounds previously resolved on native or derivatized cyclodextrin CSPs in liquid chromatography (LC) using reversed phase or polar organic mobile phase modes could be resolved in SFC using a simple carbon dioxide/methanol eluent. Resolution of cromakalim, which is not possible on the S-NEC-CD column in LC, was readily accomplished in SFC. The importance of modifier, temperature, and pressure was assessed in relation to retention, selectivity, and resolution. The nature of the modifier and the modifier concentration were found to be crucial parameters. © 1996 Wiley-Liss, Inc. Contribution of the National Institute of Standards and Technology. Not subject to copyright. 相似文献
15.
采用大孔吸附树脂层析结合硅胶柱层析,对环孢菌素A的分离纯化进行研究,确定了最佳层析条件,建立了工业化制备环孢菌素A的工艺。大孔吸附树脂层析选用D101树脂作为吸附介质,提取液丙酮含量控制在50%,最大吸附量为35 mg/g湿树脂,洗脱剂选用丙酮;硅胶柱层析选用42~64μm硅胶作为层析介质,最优层析条件为柱床高径比10∶1,流动相配比V(石油醚)∶V(丙酮)=70∶30,流速80 mL/m in,环孢菌素A上样质量浓度100 g/L,硅胶层析平均收率为84.2%,环孢菌素A纯度可达到97%以上,整个工艺总收率为65%~70%。 相似文献
16.
Beckley K. Nfor Tangir Ahamed Martijn W.H. Pinkse Luuk A.M. van der Wielen Peter D.E.M. Verhaert Gijs W.K. van Dedem Michel H.M. Eppink Emile J.A.X. van de Sandt Marcel Ottens 《Biotechnology and bioengineering》2012,109(12):3070-3083
A multi‐dimensional fractionation and characterization scheme was developed for fast acquisition of the relevant molecular properties for protein separation from crude biological feedstocks by ion‐exchange chromatography (IEX), hydrophobic interaction chromatography (HIC), and size‐exclusion chromatography. In this approach, the linear IEX isotherm parameters were estimated from multiple linear salt‐gradient IEX data, while the nonlinear IEX parameters as well as the HIC isotherm parameters were obtained by the inverse method under column overloading conditions. Collected chromatographic fractions were analyzed by gel electrophoresis for estimation of molecular mass, followed by mass spectrometry for protein identification. The usefulness of the generated molecular properties data for rational decision‐making during downstream process development was equally demonstrated. Monoclonal antibody purification from crude hybridoma cell culture supernatant was used as case study. The obtained chromatographic parameters only apply to the employed stationary phases and operating conditions, hence prior high throughput screening of different chromatographic resins and mobile phase conditions is still a prerequisite. Nevertheless, it provides a quick, knowledge‐based approach for rationally synthesizing purification cascades prior to more detailed process optimization and evaluation. Biotechnol. Bioeng. 2012; 109: 3070–3083. © 2012 Wiley Periodicals, Inc. 相似文献
17.
Rosangela Marchelli Roberto Corradini Terenzio Bertuzzi Gianni Galaverna Arnaldo Dossena Francesco Gasparrini Beatrice Galli Claudio Villani Domenico Misiti 《Chirality》1996,8(6):452-461
The copper(II) complexes of two new diastereomeric ligands, N2-(R)- and N2-(S)-2′-hydroxypropyl-(S)-phenylalaninamide [(R, S)-1 and (S, S)-1], have been used as additives to the eluent in high-performance liquid chromatography (HPLC) reversed phase for the chiral separation of DNS-amino acids. The aim was that of comparing the separation process obtained by the chiral eluent with that obtained by an analogous bonded stationary phase containing (S)-phenylalaninamide, previously studied [CSP-(S)-Phe-NH2]. The affinity of the ternary complexes for the C18 column was determined by adsorption experiments in HPLC. It was shown that the two systems (chiral eluent, chiral stationary phase) work according to different mechanisms. Ternary complex formation in solution was studied by fluorescence spectroscopy. It was shown that chiral separation with the Cu(II) complexes added to the eluent was determined by the relative affinities of the ternary complexes for the column-stationary phase rather than by their stabilities in solution. With CSP-(S)-Phe-NH2 the separation is accounted for by the relative stabilities of the ternary complexes, which depends mainly on the “allowed” geometry of the complex and on the steric repulsion of the amino acid side chain with the spacer. © 1996 Wiley-Liss, Inc. 相似文献
18.
Purification of P0 Myelin Glycoprotein by a Cu2+-Immobilized Metal Affinity Chromatography 总被引:2,自引:0,他引:2
P0 is an abundant myelin glycoprotein of peripheral nerves of vertebrates. Various point mutations of this protein are responsible for hereditary neuropathies. In this paper we described purification of P0 glycoprotein using SDS and a metal chelate affinity chromatography. Purified myelin fraction from bovine spinal roots in 0.5% SDS, 0.5 M NaCl, 50 mM Tris-HCl, pH 7.4 is filtered and applied directly to the Cu2+-immobilized affinity chromatography column, equilibrated with the same buffer. After eluting a void volume (or pass through) fraction, P0 protein was eluted by the same buffer but without salt. To remove contamination from the eluent, further purification is continued on a Concanavalin-A coupled agarose column. We purify within two days, 30 mg of P0 protein of apparent molecular weight 27 kDa. The method can be used to purify recombinant or mutated P0 protein found in severe pathologies. 相似文献
19.
Plasma protein fractionation with advanced membrane adsorbents 总被引:1,自引:0,他引:1
High capacity membrane adsorbents have been used as a stationary phase for the preparative chromatographic purification of human serum albumin. A two-step ion exchange fractionation scheme yields albumin with 98% purity from clarified, microfiltrated, and desalted human plasma. Experiments with laboratory and pilot scale membrane modules are compared to literature data obtained with conventional Fast Flow Sepharose in a similar purification protocol. Increased productivity in combination with excellent reproducibility and stability was found using the membrane adsorbents. Scale-up of the process based on standard microfiltration equipment was successful but resulted in reduced capacity and productivity due to deteriorated flow characteristics of the module. This was attributed to the effects of substantial axial dispersion in the pilot scale module. Methods to reduce this limitation were identified. The concept of membrane adsorption chromatography for the fast purification of proteins is illustrated and engineering aspects important for the process design are discussed. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 181-189, 1997. 相似文献
20.
蛋白质层析用离子交换和疏水作用层析介质的发展概况 总被引:2,自引:0,他引:2
层析是蛋白质纯化的关键技术之一 ,作为层析技术的核心———层析介质一直以来是层析技术研究的一个热点。近年来 ,越来越多的新型层析介质被开发出来 ,如粒度均匀的交联多糖、人工合成的大孔聚合物、触角型吸附剂、软胶包裹在硬胶表面等介质。主要介绍应用较为广泛的IEC和HIC介质的组成、特性及其在蛋白质纯化中的应用 ,还研究了与HIC技术相关的两种新技术 :亲硫层析和疏水电荷诱导层析 (HCIC) ,重点介绍了HCIC的介质及其应用 ,同时也讨论了在蛋白质纯化中应用的三相纯化策略 (富集、中间纯化和精制 )。结合我国的实际情况 ,就当前蛋白质纯化的离子交换和疏水层析介质面临的挑战和未来的发展进行讨论并提出了建议 相似文献