The application of simulated moving bed chromatography for the separation between 2,6- and 2,7-dimethylnaphthalene

The possibility of applying SMB chromatography for the separation of 2,6-DMN and 2,7-DMN was investigated by numerical simulation based on the single column chromatography experimental data. It was found that 2,6-DMN could be separated from 2,7-DMN in the ODS-modified silica gel by using methanol and water as the mobile phase. A systematic method of obtaining model parameters for the SMB simulation from single column chromatography experiments was presented. The adsorption isotherms of 2,6-DMN and 2,7-DMN were obtained by the pulse method. The mass transfer kinetics was very fast, indicating that the mobile phase and the stationary phase are very close to equilibrium for both 2,6-DMN and 2,7-DMN. SMB simulation was performed by using the model parameters. Pure 2,6-DMN and 2,7-DMN can be obtained by selecting suitable operating conditions, indicating that SMB chromatography can be employed to separate 2,6-DMN and 2,7-DMN. The influence of operating parameters (feed flow rates, raffinate flow rate, and switch time) was also investigated to recommend the optimum operating conditions.     

Hybrid Process for o‐ and p‐Xylene Production in Aromatics Plants

A new hybrid process for the production of o‐ and p‐xylene is proposed to replace the traditional plant of aromatics in refineries. The proposed process comprises a simulated moving bed (SMB) unit and two crystallizers. The SMB technology as the first unit of the suggested process is applied for the separation of xylene isomers and was investigated by simulation of an industrial size unit, using experimentally measured adsorption equilibrium data on MIL‐53(Al)‐shaped material. The separation of p‐xylene from o‐xylene with m‐xylene as desorbent is the key characteristic of this method. An industrial‐scale SMB unit could provide extract and raffinate streams with very high purities.     

Separation of mixed solutes using reciprocating size exclusion chromatography: Computer simulation based upon experimental parameters

Mixed solutes of different molecular size were separated by reciprocating size exclusion chromatography and the results were compared with conventional size exclusion chromatography with repeated feeds using computer simulation. Operational conditions, such as overlapping between elution curves and diffusivities of solutes, were varied in simulation. Simulation showed the separation efficiency was higher in RSEC than SEC with repeated feeds in the region of high diffusivity of either solutes at low degree of overlapping. Further study to improve RSEC by employing temperature swing is being made.     

Modeling of the α-lactalbumin and β-lactoglobulin protein separation

This work used the General Rate Model (GRM) to evaluate the experimental data of α-lactalbumin (α-la) and β-lactoglobulin (β-lg) mass transfer using size exclusion chromatography (SEC). The chromatographic simulation has become necessary in large scale production processes. Mathematical models have been used for the optimization and control of different operating conditions of the process, as well as providing calculations for the process scale-up. For the SEC experiments, the aqueous biphasic system was composed of polyethylene glycol 1500 g/mol, potassium phosphate and whey protein isolate. The polymeric phase was enriched with α-la and the saline phase with β-lg. The experiments were conducted using a glass column packed with the Sephadex G-25^® gel. Both proteins were quantified by reverse phase liquid chromatography. The experimental data were fitted by non-linear regression, using the successive quadratic programming algorithm. The mass transfer model utilized represented adequately the SEC experimental results.     

Size-exclusion simulated moving bed chromatographic protein refolding

Size-exclusion chromatographic refolding (SECR) has successfully proven its capability to refold a variety of proteins using a range of gel filtration column materials. Several approaches have also been undertaken to improve the refolding yield of these systems, mostly under batch operation. Although, these approaches may lead to an increase on refolding yield, it is not expected that they will lead to significant increases in other important process indicators, such as volumetric productivity, specific eluent consumption and product concentration in the product stream; as these indicators are strongly dependent on the mode of operation. To overcome the shortcomings of batch chromatography, the size-exclusion refolding reactor may be operated in a continuous mode, with the aid of Simulated Moving Bed (SMB) technology. Albeit, SMB technology has inherent advantages over batch chromatography, these have been proven mainly in the context of conventional purifications and are still to be addressed in the context of chromatographic refolding reactors. In this work we report the on-column refolding of an industrially relevant protein, produced in inclusion bodies, by batch size-exclusion chromatography (SEC) and simulated moving bed size-exclusion chromatography (SMBSEC). The presented study encompasses: (1) a statistical design of experiments (DOE) to study the combined effect of the mobile phase pH (9.0–11.20) and the feed concentration of denatured and reduced protein (C_f,D&R=2.50–7.50 mg ml^?1) on the refolding yield of the model protein; (2) a mechanistic analysis of the SMBSECR data, using a detailed model that accounts for both separation and refolding; and (3) a detailed comparison of the SMBSECR against the SECR, based both on quantitative and qualitative criteria. Our work showed that: (1) refolding yields of 50% are attainable by tuning pH and C_f,D&R, and that the positive effect of pH is strongly dependent on the C_f,D&R; (2) the modeling tool captured well the SMBSECR behavior, based solely on the effect that C_f,D&R has on the reaction rates; and (3) the volumetric productivity of the SMBSECR is about 53 times higher than that of the SECR, the specific solvent consumption is approximately 1/10th of that of the SECR, and the concentration of the product (i.e., native protein), leaving the SMBSECR, is roughly 4.5-fold higher than the one leaving the SECR. Accordingly, the comparison revealed the significant advantages that SMB technology has to offer to the design of chromatographic refolding reactors.     

Chiral Separation of R,S‐α‐Tetralol by Simulated Moving Bed

Abstract

This work deals with chiral separation by Simulated Moving Bed (SMB) chromatography. A simulation package is used to predict the effect of the operating variables on the process performance. The “separation volume” methodology is used to study effect of the presence of mass transfer resistances presented by different particle sizes on operating conditions of the SMB unit with emphasis to the net flow in zone I and IV in terms of interstitial velocity ratio (γ₁ and γ₄). The experimental operation of the a pilot plant SMB unit LICOSEP 12–26 (Novasep, France) is carried out for the separation of enantiomers of R,S‐α‐Tetralol using the chiral adsorbent CHIRALPAK AD with particle size 20 µm. To obtain real insight to operation of the SB unit Licosep 12–26 the mathematical model accounts for all dead volumes inside of the SMB unit and time delay in shifting of injection and withdrawal points. Experimental and theoretical results based on the SMB model are compared in terms of process performance parameters and internal concentration profiles.     

Micro-simulated moving bed (μSMB) systems: A numerical study

Continuous chromatographic separation processes like simulated moving bed (SMB) systems have been employed in petrochemicals, sugar, and more recently, in pharmaceutical industries by virtue of their superior separation efficiency. Miniaturization of chromatography-based analytical techniques (e.g., high performance liquid chromatography, micro-HPLC or μHPLC) has already been successfully demonstrated in the last few years owing to the rapid development in MEMS technology. With such a rapid progress in technology, it is definitely possible to realize the miniaturization of a powerful continuous chromatographic process such as SMB. Micro-SMB (μSMB) systems could not only inherit the merits of μHPLC, but also provide efficient separation of compounds such as isomers and enantiomers that are otherwise very difficult to isolate. In this paper, new simulations of the performance of a μSMB system for the separation of a mixture of phenol and o-cresol, using a robust numerical algorithm developed that mimics the dynamic operation of the μSMB system, are presented. A systematic parametric sensitivity analysis that addresses the effects of various process parameters on the performance of the μSMB system is also presented. High purities and yields of both phenol and o-cresol is achieved in the μSMB by judicious choice of process parameters.     

重组瑞替普酶包涵体制备及其体外复性

将携带重组瑞替普酶(reteplase, rt-PA)的质粒成功转化到大肠杆菌BL21(DE3)后,诱导表达获得包涵体,考察了诱导剂浓度、培养温度和培养时间等条件对目标蛋白表达量的影响。在此基础上,对高效表达的rt-PA包涵体体外复性过程进行了详细研究。首先利用单因素实验考察了复性液pH、GSH浓度、GSH/GSSG比例、蛋白浓度等各种复性条件对复性效果的影响;并结合正交实验设计,进一步研究了高蛋白浓度下复性后rt-PA酶活变化情况。以0.2 mmol·L^-1 IPTG诱导,在33℃下培养6 h,每升发酵液约可获得1.7 g粗制包涵体。适宜的复性条件为蛋白浓度50 mg·ml^-1,pH 10.0,GSH浓度1 mmol·L^-1,GSH/GSSG比例8,复性收率为87.2%。影响高蛋白浓度下rt-PA复性的关键因素为复性液初始pH及GSH浓度,在800 mg·ml^-1蛋白浓度下复性后rt-PA比活可达7.54×10⁴ IU·mg^-1,荧光光谱分析结果表明复性后rt-PA恢复了其天然态结构。     

Continuous separation of glucose and fructose at high concentration using two-section simulated moving bed process

A two-section simulated moving bed (SMB) modified from a three-section SMB [Barker et al., 1975] was applied to the separation of an aqueous mixture of glucose and fructose at high concentration up to 500 kg/m³. Dowex 50W-X12 resin of Ca⁺⁺ form was used as an adsorbent and water as an isocratic eluent. The equilibrium isotherms in terms of a quadratic expression and a plug flow model with mass transfer effect were used to predict both the products and on-concentrations in the two-section SMB process. The two-section SMB process suggested in this work was successful in obtaining high fructose corn syrup (fructose with 55-90% w/w) at the high concentration of 500 kg/m³.     

Effect of the SA content of a novel thermo‐sensitive P(NIPAM‐co‐SA) copolymer on denatured lysozyme refolding in vitro

A novel hydrogel of P(NIPAM‐co‐SA) copolymer was synthesized by inverse suspension polymerization by adding sodium acrylate (SA) to improve the phase transition properties of poly(N‐isopropylacrylamide) (PNIPAM). The morphologies, size distribution and thermosensitive characteristics of gel particles were studied and the maximal swelling ratio and LCST (Lower Critical Solution Temperature) of gel particles increased obviously with the addition of SA comonomer. When the protein concentration was 250 μg/mL, the optimized refolding conditions of denatured lysozyme with P(NIPAM‐co‐SA) hydrogel were that operating at the temperature of 35°C and a urea concentration of 2M, in which the mass ratio of P(NIPAM‐co‐SA) hydrogel with 4% SA copolymerized to lysozyme was 10 : 1. Under the optimized conditions, the activity recovery of lysozyme increased to 76.5% assisted by P(NIPAM‐co‐SA) gel particles compared with 55.6% by simple dilution. When refolding finished, the gel particles could be removed and recovered easily and the activity recovery of lysozyme was still as high as 61.5% after reused for 5 batches. With the addition of different amounts of SA comonomer, the hydrophobicity of the copolymer could be varied. Then the copolymerized hydrogel inhibits protein molecules aggregation more effectively through the moderate hydrophobic interactions between copolymers and protein molecules in the course of lysozyme refolding compared with the presence of PNIPAM polymer. All results above demonstrate that the P(NIPAM‐co‐SA) is a cost effective additive with tunable hydrophobicity for application in the refolding of recombinant proteins expressed as inclusion bodies in vitro. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

流加操作与稀释添加耦合辅助高浓度变性蛋白质复性动力学

研究了流加操作与稀释添加耦合辅助高浓度变性还原溶菌酶的复性,建立了相关的过程动力学模型.利用三态复性的过程模型对实验数据进行了拟合,获得了较好的拟合效果.利用模型分析了复性过程的动力学特性,结果表明,两者耦合可在较低的盐酸胍浓度(1 mol•L^-1)存在下使较高浓度的（5 mg•ml^-1）变性溶菌酶获得80%以上的复性收率;并且发现添加剂乙酰胺的辅助复性作用与盐酸胍相同,降低盐酸胍浓度,适当提高乙酰胺浓度即可使聚集体生成速率常数最小,酶的复性收率最大;但甘油与盐酸胍具有协同作用,只有在适当的盐酸胍浓度下添加甘油才可获得理想的复性效果.     

Surfactant-modified bentonite as adsorbent for the removal of humic acid from wastewaters

The effectiveness of surfactant-modified bentonite (SMB) in removing humic acid (HA) from wastewaters was evaluated. Hexadecyl trimethylammonium (HDTMA) chloride was used to modify the surface of the clay mineral. The SMB exhibits very high adsorption potential for HA and at pH 3.0 more than 99% removal was achieved from an initial concentration of 25 μmol/L. The experimental kinetic data were analyzed using the pseudo-first-order kinetic model. Adsorption occurs through film diffusion at low as well as at higher concentrations and temperatures. The adsorption of HA using SMB was an exothermic process. HA adsorption was found to decrease with increase of ionic strength due to the formation of outer-sphere surface complexes on SMB. The equilibrium isotherms were determined and data were analysed using the Langmuir isotherm model. The maximum adsorption capacity, Q° was 73.52 μmol/g with binding constant, b = 0.155 L/μmol at 30 °C and pH 3.0. The adsorbent was suitable for repeated use (more than 3 cycles) without any noticeable loss of capacity.     

利用体积排阻色谱法进行蛋白质折叠

以溶菌酶为模拟体系对体积排阻色谱法进行蛋白质折叠过程实验研究 .圆二色性光谱法分析结果证实了复性溶菌酶与天然溶菌酶的二级结构一致性 ;复性溶菌酶与天然溶菌酶色谱保留体积的差异揭示出折叠过程中无活性蛋白质聚集体的存在及其向复性蛋白质转化的机制 ;不同初始浓度的复性实验证实了蛋白质聚集体的存在及其与变性蛋白质初始浓度的关系 ;采用短色谱柱的折叠分离实验结果表明蛋白质折叠是一个快速过程 ;不同尿素浓度下的折叠分离实验结果表明尿素在SEC法中具有非常重要的作用 .与稀释复性法的对比实验表明 :体积排阻色谱法具有稀释倍数小、复性产品活性收率高、复性蛋白质浓度高等优点 .     

Effects of desorbent flowrate on simulated moving bed process

This article presents the effects of the desorbent flowrate (or m₁) on simulated moving bed (SMB) process performance at constant m₂, m₃ and m₄, where the m-values (m _j ) are defined as the net flowrate ratios of the zone, j, to the solid flowrate. A complete m-plane analysis of SMB systems (ComPASS) has been developed that illustrates how the desorbent flowrate is determined within the complete m-plane (i.e., fluid-solid flowrate ratio plane) to enhance the SMB process performance. The effects of desorbent flowrate are presented numerically for linear/equilibrium and nonlinear/nonequilibrium SMB processes. The results of the studies using ComPASS show that for linear/equilibrium SMB process, it is desirable to determine the minimum desorbent flowrate (m₁) within the complete separation region. For the nonlinear/nonequilibrium SMB process, the desorbent flowrate can be determined at the minimum value which maximizes the raffinate purity.     

液固层析辅助蛋白质从变性态恢复到活性态

层析是化工中分离纯化一些价格昂贵的精细化工产品的常用手段,而液固层析因其温和、快捷、高效等优点已经成为蛋白质纯化过程中必不可少的工具.近年来层析技术的一个拓展是用于辅助蛋白质从变性态恢复到活性态,即所谓的复性,取得了令人瞩目的进展.液固层析辅助蛋白质复性可在高蛋白浓度下操作,适用于大规模生产,同时还能使目标蛋白得到部分纯化.对近年来发展的利用液固层析辅助蛋白质复性的方法做了归纳,包括凝胶过滤层析、离子交换层析、疏水相互作用层析、亲和层析和固定化的折叠催化剂及人工分子伴侣辅助蛋白质复性的方法,并对各自的优缺点和适用范围进行了比较分析.     

Thermosensitive poly(N‐isopropylacrylamide) hydrogel for refolding of recombinant bovine prethrombin‐2 from E. coli inclusion bodies

The poly(N‐isopropylacrylamide) (PNIPA) hydrogel, which is a kind of temperature‐sensitive polymer, was synthesized by inverse suspension polymerization. The microscopy and scan electron microscopy (SEM) of PNIPA hydrogel were studied. The microscope photograph showed that the particles were in the range of 0.2–0.5mm in diameter, with numerous conjoint pores about 1–2μm spreading all over the surface of the beads. The swelling properties of PNIPA gel beads indicated that the lower critical solution temperature (LCST) of the gel was 33°C. The PNIPA prepared was applied to the renaturation of bovine prethrombin‐2 (pThr‐2) from inclusion bodies produced in E. coli. It was observed that PNIPA was quite efficient in assisting protein renaturation at high protein concentration. When mixing with 105mg/mL PNIPA hydrogel during the refolding, the total activity of the thrombin was about 6222U/mL, compared with only 2800U/mL by simple dilution refolding. The kinetics of pThr‐2 refolding with the absence or the presence of PNIPA was also studied respectively. The time required for the refolding with PNIPA gel was a little bit longer than that by the dilution method owing to the diffusion resistance of the protein into the network of the gel and the hydrophobic interaction between the protein and the polymer. The mechanism of the enhancement for the PNIPA gel to the refolding was further discussed. The porosity of the PNIPA hydrogel allows penetration of the unfolded protein into the inside of the polymer with a hydrophobic side chain, which can facilitate the formation of intermediate via hydrophobic interaction with the unfolded protein and the folding intermediate that are liable to re‐aggregation. About 1.2mg of purified active thrombin could be recovered from 1 L of cells, which greatly facilitated the scale‐up to the quantities of protein necessary for further functional and structural studies. A novel protein renaturation method mediated by PNIPA hydrogel beads, which highly increases the refolding efficiency with easy handling, recycling, and low cost, was proposed. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 96: 1734–1740, 2005     

Improved performance of simulated moving bed process using column‐modified feed

A “FeedCol” strategy was developed to improve separation performance in simulated moving bed (SMB) processes. In the FeedCol operation, a short chromatographic column was simply added to the SMB unit and feed was supplied by a pulse input through the column to the SMB process. Because the feed was made in the shape of partially separated chromatographic peaks through the column, the purities in the raffinate and extract products were improved efficiently in the SMB process. All the performance parameters for a binary mixture with low selectivity (α = 1.1) were better for the FeedCol operation than for the conventional SMB operation (2‐2‐2‐2). Because the feed injection through the feed column was synchronized with the SMB process during the switching period, two new operating variables were introduced: injection length and injection time. Their effects on the suggested strategy were evaluated in terms of performance parameters through a detailed simulation study. © 2010 American Institute of Chemical Engineers AIChE J, 2011     

Optimal design of a four‐zone simulated moving bed process for separation of homoharringtonine and harringtonine

A simulated moving bed (SMB) technology was applied to the separation of homoharringtonine (HHT) and harringtonine (HT), which were known to have the potentiality of being used as anti‐cancer agents. First, a series of pulse injection experiments were performed for estimation of the adsorption isotherm and mass‐transfer parameters of HHT and HT. The estimated parameters were utilised in the SMB optimisation tool based on the standing wave design method. From the optimisation tool prepared, the SMB operating parameters (zone flow rates and step time) that led to the highest throughput were obtained under the constraints of product purities (=99.0%) and pressure drop (≤1000 psi). Such an optimisation work was then extended to determine an optimal size of the adsorbent particle for the SMB of interest. The results showed that a particle size of 29 µm was the optimal one for maximising the SMB throughput under the conditions that the column configuration was 2–2–2–2 and the length of each column was 25 cm. If the SMB had the particle size other than 29 µm, its throughput was limited by either the maximum operating pressure or the mass‐transfer efficiency. Finally, an efficient procedure of removing a mobile‐phase additive (ammonium formate) from the product stream of the aforementioned SMB system was developed using a liquid–liquid extraction (LLE) technique. From the results of this study, it was confirmed that the SMB process coupled with a LLE procedure could be highly effective in separating HHT and HT with high throughout and high purity.     

Dispersion-enhanced chromatography refolding of denatured protein

Protein folding following the expression of insoluble gene products in bacterial cell factories such as Escherichia coli is a significant technical challenge. Preparative refolding is often conducted chromatographically using automated equipment, allowing simple exchange of buffer conditions and integrated removal of contaminants. While an understanding of the role of mixing on dilution protein refolding has recently emerged, the role of dispersion in chromatographic refolding has not been reported. Here we report that protein refolding yield during chromatography is influenced strongly by column dispersion. Specifically, modelling studies predicted that a significant yield increase could be obtained by introducing a highly dispersive gap at the top of the refolding column (i.e. between the inlet distributor and the top of the packed chromatographic bed). Experimentally, the introduction of a 15 mm gap increased yield from 47% to 64% at . This counter-intuitive strategy of dispersion-enhanced chromatography refolding (DCR) is potentially generic for other proteins, and suggests the need for gel-filtration refolding columns and resins that achieve adequate buffer exchange yet maximise, rather than minimise, peak broadening.     

Design of Simulated Moving Bed Plants by Using Noncompetitive Langmuir Isotherms

The simulated moving bed process is increasingly used for the separation of binary mixtures. To ensure proper operation, the volumetric flow rates and the time interval must be exactly adjusted. This study presents a general method for determining the control variables for a dispersion‐free SMB process. For noncompetitive Langmuir isotherms, explicit equations are derived for the case of complete separation. The proposed method allows both a good estimation of the time trajectories of the concentrations at the drains and the design of new applications for optimization and control of SMB plants.