Optimal operating mode for enantioseparation of SB-553261 racemate based on simulated moving bed technology

The performance of the simulated moving bed (SMB) technology and its modification, the Varicol process, was optimized using an experimentally verified model for the enantioseparation of SB-553261 racemate. Single and multiobjective optimizations have been carried out for both existing as well as design stage and their efficiencies were compared. The optimization problem involves a relatively large number of decision variables, both continuous variables such as flow rates, switching time and length of the columns, as well as discrete variables like number and distribution of columns. A state-of-the-art new optimization technique based on a genetic algorithm (nondominated sorting genetic algorithm with jumping genes) was utilized which allows handling of these complex optimization problems. The optimization results showed that significant improvement could be made to the chiral drug separation process using both the SMB and the Varicol process. It was found that the performance of a Varicol process is superior to that of a SMB process in terms of treating more feed using less desorbent or increasing productivity while at the same time achieving better product quality. Optimum results were explained using equilibrium theory by locating them in the pure separation region.     

Simulated moving bed chromatography with supercritical fluids for the resolution of bi-naphthol enantiomers and phytol isomers

The combination of the simulated moving bed (SMB) technique with supercritical fluid chromatography (SFC) leads to a process with unique features. Besides the known advantages of the SMB process, the use of supercritical carbon dioxide as the mobile phase offers the advantages of reduction in organic solvents and an easy eluent/solute separation. Because of the low viscosity and high diffusion coefficients of supercritical fluids, a high efficiency is possible. The steps of process development for SMB SFC are presented using the separations of the bi-naphthol enantiomers and phytol isomers as examples. The development of a packed column SFC method at an analytical scale is shown for the separation of the bi-naphthol enantiomers on a chiral stationary phase and CO(2) with a modifier as the mobile phase. The influence of the modifier, modifier content, and column configuration on productivity of the SMB SFC process was investigated by simulation. The first set of experiments was performed in the SMB separation of phytol isomers at low concentration to test the feasibility of the SMB SFC high purity separation of the binary mixtures. In the second set of experiments, the productivity of the process was increased by increasing the feed concentration up to 54 grams feed per liter stationary phase (SP) and hour (g(feed)/l(SP) h).     

A novel design of simulated moving bed (SMB) chromatography for separation of ketoprofen enantiomer

A simulated moving bed (SMB) chromatography system is a powerful tool for preparative scale separation, which can be applied to the separation of chiral compound. We have designed our own lab-scale SMB chromatography using 5 HPLC pumps, 6 stainless steel columns and 4 multi-position valves, to separate a racemic mixture of ketoprofen in to its enantiomers. Our design has the characteristics of the low cost for assembly for the SMB chromatography and easy repair of the unit, which differs from the designs suggested by other investigators. It is possible for the flow path through each column to be independently changed by computer control, using 4 multi-position rotary valves and 5 HPLC solvent delivery pumps. In order to prove the operability of our SMB system, attempts were made to separate the (S)-ketoprofen enantiomer from a ketoprofen racemic mixture. The operating parameters of the SMB chromatography were calculated for ketoprofen separation from a batch chromatography experiment as well as by the triangle theory. With a feed concentration of 1 mg/mL, (S)-ketoprofen was obtained with a purity of 96% under the calculated operating conditions.     

Chiral separation of flurbiprofen enantiomers by preparative and simulated moving bed chromatography

This study presents the chiral resolution of flurbiprofen enantiomers by preparative liquid chromatography using the simulated moving bed (SMB) technology. Flurbiprofen enantiomers are widely used as nonsteroidal anti‐inflammatory drugs, and although demonstrate different therapeutic actions, they are still marketed as a racemic mixture. The results presented here clearly show the importance of the selection of the proper solvent composition for the preparative separation of flurbiprofen enantiomers. Chiral SMB separation is carried out using a laboratory‐scale unit (the FlexSMB‐LSRE®) with six columns, packed with the Chiralpak AD® stationary phase (20 μm). Results presented include the experimental measurement of equilibrium and kinetic data for two very different solvent compositions, a traditional high hydrocarbon content [10%ethanol/90%n‐hexane/0.01% trifluoroacetic acid (TFA)] and a strong polar organic composition (100%ethanol/0.01%TFA). Experimental data, obtained using the two mobile phase compositions, are used to predict and optimize the SMB operation. After selecting 10%ethanol/90%n‐hexane/0.01%TFA as the most appropriate solvent composition, three feed concentrations of racemic flurbiprofen were considered. Using 40 g/l of racemic flurbiprofen feed solution, the purities for both outlet streams were above 99.4%, the productivity was 13.1 g_feed/(L_bed h), and a solvent consumption of 0.41 L_solvent/g_feed was achieved. Chirality, 2011. © 2011 Wiley‐Liss, Inc.     

Preparative chromatographic separation: Simulated moving bed and modified chromatography methods

Chromatography has been the method of choice for the separation of complex biological mixtures for analytical purposes, particularly for the last fifty years. Its use has recently been extended to preparative separation where the productivity relative to the amount of resin and solvent used is a matter of concern. To overcome the inherent thermodynamic inefficiency of batch chromatography, as exemplified by the partial temporal usage of the resin and dilution of the product with the solvent, chromatography has been continually modified by separation engineers. Column switching and recycling represent some of the process modifications that have brought high productivity to chromatography. Recently, the simulated moving bed (SMB) method, which claims a high separation efficiency based on counter-current moving bed chromatography, has become the mainstay of preparative separation, especially in chiral separation. Accordingly, this paper reviews the current status of SMB, along with several chromatographic modification, which may be helpful in routine laboratory and industrial chromatographic practices.     

Continuous preparative liquid chromatography in the downstrem processing of biotechnological products

Continuous counter‐current chromatographic processes have been successfully used in the petrochemical and sugar industry over the last 30 years. Only recently has simulated moving bed (SMB)‐technology attracted widespread interest in the pharmaceutical industry, mainly as a very efficient system for chromatographic enantioseparation. The application of this technique to the downstream processing of biotechnological products requires some specific changes to meet the special demands of bioproduct isolation. Production processes are set up on an multi‐ton scale, for example, for the purification of fructose with both yield and purity higher than 90%. Examples for other mono‐ and oligosaccharides are reported. In the purification of fatty acids or fat soluble vitamins, SMB technology under supercritical fluid conditions gives additional benefits and increases the productivity by a factor of four when a pressure gradient is applied. Another field of operation is the isolation of drug compounds from natural sources where different batch‐ and SMB‐chromatographic steps could be successfully combined. First examples are reported for cyclosporine A and paclitaxel isolation. Finally, step‐gradient elution modes can be used continuously, as demonstrated for the isolation of monoclonal antibodies.     

2-(2,3-Dihydro-1H-indol-3-yl)ethanol: synthesis, separation of enantiomers, and assignment of absolute stereochemistry by X-ray structure analysis

The first direct resolution of racemic 2-(2,3-dihydro-lH-indol-3-yl)ethanol-prepared by catalytic hydrogenation of 2-(lH-indol-3-yl)ethanol-has been accomplished by chiral simulated moving bed (SMB) chromatography. The single enantiomers were isolated as their dihydrogen phosphate salts. Single-crystal X-ray analyses were successful, revealing that the (+)-enantiomer of 2-(2,3-dihydro-lH-indol-3-yl)ethanol has the (S) configuration. Chirality 16:126-130, 2004.     

The effects of vitronectin on specific interactions between urokinase-type plasminogen activator and its receptor: ab initio molecular orbital calculations

Binding of urokinase-type plasminogen activator (uPA) to its receptor (uPAR) on the surface of a cancer cell is considered to be a trigger for starting cancer invasions. In addition, the somatomedin B (SMB) domain of vitronectin binds simultaneously to uPAR to construct a ternary complex of uPAR–uPA–SMB. Here we present stable structures of the solvated complexes of uPAR–uPA and uPAR–uPA–SMB obtained by classical molecular mechanics simulations, and the specific interactions between uPAR, uPA and SMB are investigated by ab initio fragment molecular orbital calculations. The result indicates that the SMB binding enhances the binding affinity between uPAR and uPA, although there is no direct contact between SMB and uPA. In particular, the specific interaction between uPAR and the Lys36 residue of uPA is significantly affected by the SMB binding. The positively charged Lys23, Lys46 and Lys61 residues of uPA have strong attractive interactions to uPAR in both the uPAR–uPA and uPAR–uPA–SMB complexes, demonstrating the importance of these residues in the specific binding between uPAR and uPA. The current results on the specific interactions are informative for proposing potent antagonists, which block the uPA and SMB bindings to uPAR.     

Solution structure of recombinant somatomedin B domain from vitronectin produced in Pichia pastoris

The cysteine-rich somatomedin B domain (SMB) of the matrix protein vitronectin is involved in several important biological processes. First, it stabilizes the active conformation of the plasminogen activator inhibitor (PAI-1); second, it provides the recognition motif for cell adhesion via the cognate integrins (alpha(v)beta(3), alpha(v)beta(5), and alpha(IIb)beta(3)); and third, it binds the complex between urokinase-type plasminogen activator (uPA) and its glycolipid-anchored receptor (uPAR). Previous structural studies on SMB have used recombinant protein expressed in Escherichia coli or SMB released from plasma-derived vitronectin by CNBr cleavage. However, different disulfide patterns and three-dimensional structures for SMB were reported. In the present study, we have expressed recombinant human SMB by two different eukaryotic expression systems, Pichia pastoris and Drosophila melanogaster S2-cells, both yielding structurally and functionally homogeneous protein preparations. Importantly, the entire population of our purified, recombinant SMB has a solvent exposure, both as a free domain and in complex with PAI-1, which is indistinguishable from that of plasma-derived SMB as assessed by amide hydrogen ((1)H/(2)H) exchange. This solvent exposure was only reproduced by one of three synthetic SMB products with predefined disulfide connectivities corresponding to those published previously. Furthermore, this connectivity was also the only one to yield a folded and functional domain. The NMR structure was determined for free SMB produced by Pichia and is largely consistent with that solved by X-ray crystallography for SMB in complex with PAI-1.     

Development of a three-zone simulated moving bed process based on partial-discard strategy for continuous separation of valine from isoleucine with high purity,high yield,and high product concentration

The issue of separating valine from isoleucine has been a major concern in the biotechnological process for production of valine. To address this issue, an optimal three-zone simulated moving bed (SMB) process for continuous separation of valine was developed in this study. It was first found that an Amberchrom-CG161C resin was highly suitable for the adsorbent of such SMB process. The adsorption isotherm and mass-transfer parameters of valine and isoleucine on the Amberchrom-CG161C adsorbent were then determined through multiple frontal experiments. The determined parameters were used in the next stage of optimizing the SMB for valine separation, which was performed on the basis of genetic algorithm. For the optimized SMB process, a partial-discard strategy was applied to the raffinate port in order to make a further improvement in the valine product concentration. Finally, the optimized SMB based on the partial-discard strategy was tested experimentally using the self-assembled SMB equipment. The experimental results showed that the developed process in this study was highly effective in continuous separation of valine from isoleucine while ensuring the attainment of high product concentration. The experimental data for the SMB effluent histories and the SMB column profiles were also in close agreement with the model predictions.     

A note on the effects of calf stimuli on the response of Zebu cows to Synchro-mate-B

The effect of Synchro-mate-B (SMB) was measured on the estrous response and the establishment of cyclicity using different calf stimuli. Multiparous Zebu cows, were divided in three groups. In the first, 32 animals were treated with SMB leaving the calves present (SMB+CP). In the second, 33 cows were treated with SMB and calves partially removed (fence-line contact) for 48h (SMB+CPR). In the third group (n=33), cows received SMB and calves were removed for 48h (SMB+CCR) with no visual or olfactory contact. A control group (CG, n=33) involved neither SMB or calf separation. Blood samples for progesterone assessment were obtained at 11 and 4d prior to SMB treatment and on days 7 and 11 after the average return to estrous for each group. All animals were observed continuously for mounting activity during 72h after SMB implant removal. A significant difference (P<0.05) in estrous response was found between SMB-treated and non-treated animals, regardless of calf management (56 versus 8%, respectively). Cows with SMB+CCR and SMB+CPR came into estrous sooner (P<0.05) (26.5+/-2.6 and 18.1+/-4.94h, respectively), than those that remained with their calf present (40.4+/-12.8h). Cows with SMB+CCR displayed longer (P<0.05) periods of mounting behavior (13.0+/-4.4h) in comparison with SMB+CPR and SMB+CP (7.4+/-1.8 and 8.1+/-4.0h), respectively. Furthermore, 84% (P<0.05) of the cows in the SMB+CCR had high concentrations of progesterone after mounting behavior was displayed, in comparison with 68 and 54% in the other two groups, respectively. No difference was found (P>0.05) in the number of mounts per hour in estrous. It was concluded that: (1) SMB increases the number of cows that display estrous; (2) temporary weaning shortens the period from SMB implant withdrawal to mounting activity, and (3) SMB+CCR, increases the length of sexual receptivity and the number of cows that continue to cycle.     

Optimal design of a tandem simulated moving bed process for separation of paclitaxel, 13-dehydroxybaccatin III,and 10-deacetylpaclitaxel

Paclitaxel, 13-dehydroxybaccatin III (13-DHBIII), and 10-deacetylpaclitaxel (10-DAP), which came from the plant cell culture, have been regarded as highly valuable because of their efficacy in the anticancer treatments. Due to these values, the necessity for separating the three components in an economical way has been a matter of grave concern in industry. In this study, a tandem simulated moving bed (SMB) process that consisted of two four-zone SMB units in series was applied to such a ternary separation. First, a series of pulse injection experiments were performed for estimation of the adsorption isotherm and mass-transfer parameters. The estimated parameters were utilized in the tandem SMB optimization tool that was prepared based on the standing wave design principle. During the optimization of interest, the throughput of the tandem SMB was maximized while meeting the requirements on product purities and pressure drop. The results proved that the most economical strategy of utilizing the tandem SMB was to recover paclitaxel in the first SMB unit and then separate the remaining two components (13-DHBIII and 10-DAP) in the second SMB unit. Furthermore, such a strategy was also found to result in better tandem SMB performances over the entire region of a pressure drop limit.     

Is the microwave irradiation a suitable method for measuring soil microbial biomass?

Soil microbial biomass (SMB), the living part of soil organic matter, is used to quantify the total biomass of microorganisms present in the soil. The importance of studies about SMB has emphasized the need to identify methods which can measure the size of SMB. Among the methods currently available, chloroform-fumigation extraction and incubation are the most commonly used for estimation of SMB. However, several studies have proposed the microwave (MW) irradiation as a quick, simple and safe alternate method. There are different opinions about suitability of this method for measuring SMB. There is a question to do “Is the microwave irradiation a suitable method for measuring soil microbial biomass?” Most of the published papers comparing MW and chloroform-fumigation showed strong relationship between both methods. Therefore, we consider MW a suitable method for measuring SMB; however, it is necessary to calibrate the MW methods in different soils with a range of properties, such as clay content, to find an appropriate conversion factor in order to generate correct values for SMB with MW method.     

Comparison of Amberchrom-CG161C and Dowex99 as the adsorbent of a four-zone simulated moving bed process for removal of acetic acid from biomass hydrolyzate

One of the important steps in the application of biomass to producing sugars, which can be converted into bio-ethanol and other valuable chemicals by fermentation, is to hydrolyze the biomass components by sulfuric acid. It was reported that such a hydrolysis entailed the generation of acetic acid, which has been recognized as a key impurity to be surely removed from the biomass hydrolyzate for ensuring high fermentability of the hydrolyzed sugars. Regarding such a removal task, there has been a previous application of a simulated moving bed (SMB) process based on the Dowex99 adsorbent, whose performance, however, was limited by low selectivity between acetic acid and sugars. To overcome such a limitation, another adsorbent alternative to Dowex99 was searched in this study. It was found that Amberchrom-CG161C allowed higher selectivity between acetic acid and sugars than Dowex99. To investigate the relative superiority of Amberchrom-CG161C over Dowex99 as the adsorbent of an SMB process for removing acetic acid from the biomass hydrolyzate, the two SMB processes based on Amberchrom-CG161C and Dowex99 were optimized using the SMB optimization tool based on standing wave design (SWD) method. The optimization results revealed that the Amberchrom-CG161C SMB outperformed the Dowex99 SMB by a wide margin.     

Box-Behnken响应面法优化司替霉素B产生菌发酵条件

【背景】粗糙链霉菌(Streptomyces scabrisporus) HBERC-53204是本中心自主分离的一株链霉菌,经鉴定,其产生一种活性化合物司替霉素B (steffimycin B,SMB),对多种动植物重要病原菌具有良好生物活性。【目的】提高SMB发酵水平,拓宽放线菌活性天然产物在农牧业领域的研究及应用。【方法】以本实验室筛选出的一株产SMB的粗糙链霉菌HBERC-53204为研究对象,运用单因素试验筛选培养基的主效碳源、氮源、无机盐及各营养成分最适浓度,并基于单因素试验结果,通过Plackett-Burman(PB)试验设计筛选出显著影响因素,再结合最陡爬坡试验、Box-Behnken (BB)响应面法拟合显著因子与产量的非线性方程求解,进一步优化菌株产SMB的最佳发酵培养基配方。【结果】优化后最佳培养基配方为：葡萄糖36.22 g/L,蛋白胨8.00 g/L,酵母粉8.51 g/L,酸水解酪蛋白1.50 g/L,MgSO₄ 0.68 g/L,KNO₃ 1.00 g/L。经摇瓶验证,优化后SMB效价达到477.26 mg/L...     

The in vivo genotoxic effects of sodium metabisulfite in bone marrow cells of rats

This study is designed to investigate the genotoxic effect of sodium metabisulphite (SMB), which is used as an antimicrobial substance in foods on bone marrow cells of rats. Four different concentrations of SMB (250, 500, 750 and 1000 mg/kg body weight) were given rats (Rattus norvegicus var. albinos) for 6, 12 and 24 h treatment period by intraperitoneal (IP) and gavage (GV) administrations. In this study, we found that intraperitoneal implement of SMB generally more effectively increases the percentage of abnormal cells and CA/cell in all concentrations and treatment period. In addition, mitotic index (MI) data of intraperitoneal injection are lower than gavage. It can be concluded that potential genotoxic effects of SMB by IP injection are higher than GV injection. The text was submitted by the authors in English.     

Motif-Independent Prediction of a Secondary Metabolism Gene Cluster Using Comparative Genomics: Application to Sequenced Genomes of Aspergillus and Ten Other Filamentous Fungal Species

Despite their biological importance, a significant number of genes for secondary metabolite biosynthesis (SMB) remain undetected due largely to the fact that they are highly diverse and are not expressed under a variety of cultivation conditions. Several software tools including SMURF and antiSMASH have been developed to predict fungal SMB gene clusters by finding core genes encoding polyketide synthase, nonribosomal peptide synthetase and dimethylallyltryptophan synthase as well as several others typically present in the cluster. In this work, we have devised a novel comparative genomics method to identify SMB gene clusters that is independent of motif information of the known SMB genes. The method detects SMB gene clusters by searching for a similar order of genes and their presence in nonsyntenic blocks. With this method, we were able to identify many known SMB gene clusters with the core genes in the genomic sequences of 10 filamentous fungi. Furthermore, we have also detected SMB gene clusters without core genes, including the kojic acid biosynthesis gene cluster of Aspergillus oryzae. By varying the detection parameters of the method, a significant difference in the sequence characteristics was detected between the genes residing inside the clusters and those outside the clusters.     

Optimal design and experimental validation of a three-zone simulated moving bed process based on the Amberchrom-CG161C adsorbent for continuous removal of acetic acid from biomass hydrolyzate

In the production process of bio-ethanol from biomass, acetic acid is recognized as the key impurity to be removed from the sugar components that are generated by hydrolyzing biomass. In regard to this issue, it has recently been confirmed that the Amberchrom-CG161C resin was highly qualified as the adsorbent of a simulated moving bed (SMB) process for continuous separation of acetic acid from the biomass hydrolyzate, i.e., sugars. However, the previous study on the Amberchrom-CG161C SMB with the aforementioned separation goal has been limited to only a theoretical work, including some batch-chromatography tests. The experimental validation of such an Amberchrom-CG161C SMB process, including its optimal design, was attempted in this article. This task began by assembling the experimental unit of the SMB process with three zones. Its operating conditions were then optimized by using genetic algorithm. Under the optimized operating conditions, the relevant three-zone SMB experiment was conducted. The assay of all the resultant product samples verified that the SMB separation of interest was performed successfully as designed. The experimental data were also found to agree closely with the model predictions. Finally, a partial-discard strategy was applied to maintain the sugar product concentration as high as possible.     

A method for defining binding sites involved in protein-protein interactions: analysis of the binding of plasminogen activator inhibitor 1 to the somatomedin domain of vitronectin

Plasminogen activator inhibitor type-1 (PAI-1) is bound to vitronectin (VN) in plasma and in the extracellular matrix. We previously employed a domain-swapping approach to show that the high-affinity binding site for PAI-1 in VN is contained within residues 12-30 in the amino-terminal somatomedin B (SMB) domain. In this study, we attempt to further delineate the location of this site by employing a novel approach that is based on the use of monoclonal antibodies (Mabs) together with site-directed mutagenesis. Six separate Mabs were identified that bound to the SMB domain and competed with PAI-1 for binding to VN. The relative affinity of each of the Mabs, and of PAI-1 itself, for binding to individual variants of SMB (prepared by alanine scanning mutagenesis), was then determined and compared in competitive binding experiments. Three separate, partially overlapping Mab epitopes within SMB were defined by these studies, and the PAI-1 binding site was localized to the region between residues 24 and 37. When considered together with the domain swapping data, these studies suggest that the PAI-1 binding site is contained within a common seven-residue region (i.e., residues 24-30) in the SMB domain.     

Plasminogen activator inhibitor-1 regulates cell adhesion by binding to the somatomedin B domain of vitronectin

Plasminogen activator inhibitor-1 (PAI-1) binds to the somatomedin B (SMB) domain of vitronectin. It inhibits the adhesion of U937 cells to vitronectin by competing with the urokinase receptor (uPAR; CD87) on these cells for binding to the same domain. Although the inhibitor also blocks integrin-mediated cell adhesion, the molecular basis of this effect is unclear. In this study, the effect of the inhibitor on the adhesion of a variety of cells (e.g., U937, MCF7, HT-1080, and HeLa) to vitronectin was assessed, and the importance of the SMB domain in these interactions was determined. Although PAI-1 blocked the adhesion of all of these cells to vitronectin-coated wells, it did not block adhesion to a variant of vitronectin which lacked the SMB domain. Interestingly, HT-1080 and U937 cells attached avidly to microtiter wells coated with purified recombinant SMB (which does not contain the RGD sequence), and this adhesion was again blocked by the inhibitor. These results affirm that PAI-1 can inhibit both uPAR- and integrin-mediated cell adhesion, and demonstrate that the SMB domain of vitronectin is required for these effects. They also show that multiple cell types can employ uPAR as an adhesion receptor. The use of purified recombinant SMB should help to further define this novel adhesive pathway, and to delineate its relationship with integrin-mediated adhesive events.