Less common applications of simulated moving bed chromatography in the pharmaceutical industry

Simulated moving bed (SMB) chromatography is often perceived in the pharmaceutical industry as chromatographic method for separating binary mixtures, like racemates. However, SMB can also be used for unbalanced separations, i.e. binary mixtures of varying compositions and multi-component mixtures. These less common application modes of isocratic SMB chromatography are exemplified for four different compounds (racemates and diastereomers) and discussed in view of the so-called 'triangle theory' from an industrial perspective.     

Two-fraction and three-fraction continuous simulated moving bed separation of nucleosides

A new experimental set-up and a new simulated moving bed (SMB) operation are presented in this work. A desktop SMB unit developed as a modification of the commercial AKTA explorer working platform has been utilized for the separation of different mixtures of nucleosides. Both two fraction and three fraction SMB separations have been carried out, the latter made possible by the adoption of a new SMB configuration and operating mode (three fraction SMB, 3F-SMB, operation). Experiments demonstrate the feasibility of the 3F-SMB operation, and confirm the trends predicted based on considerations about retention of the components to be separated along the unit.     

Simulated moving bed chromatography for the separation of enantiomers

Simulated moving bed (SMB) chromatography, a continuous multi-column chromatographic process, has become one of the preferred techniques for the separation of the enantiomers of a chiral compound. Several active pharmaceutical ingredients, including blockbuster drugs, are manufactured using the SMB technology. Compared to single column preparative chromatography, SMB separations achieve higher productivity and purity, while reducing the solvent consumption. The SMB technology has found applications both at small and large scales. Design methods have been developed for robust operation and scale-up, using data obtained from analytical experiments. In the last few years, rapid developments have been made in the areas of design, improved process schemes, optimization and robust control. This review addresses these developments, as well as both the fundamentals of the SMB science and technology and some practical issues concerning the operation of SMB units. Particular emphasis is placed on the consolidation of the “triangle theory”, a design tool that is used both in the academia and industry for the design of SMB processes.     

丙二酰胺型大环多胺键合固定相在毛细管开管电色谱中的应用

毛细管电色谱 ( Capillary electrochromatography,简称 CEC)是近年发展起来的一种高效、快速的新型微柱分离方法 [1～ 4] .由于它在毛细管柱内填充液相色谱固定相或者在毛细管内壁键合固定相 ,且采用电渗流作为驱动力 ,因而兼有高效液相色谱和毛细管电泳的分离特点 ,已应用于复杂的药物分析 [2 ] .填充柱毛细管柱具有工艺要求高、容易产生气泡、焦耳热和价格昂贵等缺点 .开管柱电色谱( Open- tubular CEC,简称 OTCEC)是将固定相键合或涂覆在毛细管的内壁 ,避免了上述缺陷 .因此已引起高度重视 [3,4] .大环多胺的结构与冠醚类似 ,是一…     

High-efficiency capillary isoelectric focusing of protein complexes from Escherichia coli cytosolic extracts

High-efficiency capillary isoelectric focusing (cIEF) separations of protein complexes obtained from soluble protein fractions are demonstrated. Size-exclusion chromatography was used as a first dimension separation to fractionate putative protein complexes with apparent molecular masses of up to 1,500,000 from an Escherichia coli cytosolic fraction. Non-denaturing cIEF separations using highly hydrophilic polymer-coated capillaries constituted the second dimension. The conditions developed produced reproducible and high-efficiency separations, corresponding to approximately 2 x 10(6) theoretical plates and peak capacities of approximately 10(3) for pH 3-10 cIEF separations in 65 cm long capillaries. Combination of the two non-denaturing separation dimensions permitted isolation and analysis of individual protein complexes from complicated biological samples. Studies indicated that many E. coli complexes were stable on the time scale of the cIEF separations, but were degraded upon more extended periods of storage on ice, necessitating rapid sample processing and fast analysis techniques.     

Continuous voltage gradients and their application to true moving bed electrophoresis

Gradient elution has been practiced in chromatographic separations for many years. The application of discontinuous "step" gradients in simulated moving bed (SMB) chromatography has been very successful in increasing both processing rates and column productivity, resulting in a reduction in the number of SMB columns required. With the advent of the field gradient focusing techniques, electrophoresis has gained the ability to apply a continuous electric field gradient to a true moving bed (TMB) electrophoretic separation. Application of a spatial gradient allows a large degree of control of the product concentrations inside the separation unit as well as a large increase in product throughput. A model of moving bed electrophoretic separations has been developed that demonstrates the potential advantages of applying a continuous gradient to the moving bed process. These advantages include the reduction of detrimental peak tailing and the ability to decrease the concentrations of the compounds being separated in comparison with commonly used step gradient elution.     

Step gradients in 3-zone simulated moving bed chromatography. Application to the purification of antibodies and bone morphogenetic protein-2

The simulated moving bed (SMB) technology is a proven tool for efficient separation of binary mixtures. However, relying on isocratic conditions limits the applicability of the classical SMB approach when considering the field of bioseparations. Here, the use of gradients opens up new possibilities. A gradient in a SMB process can be established by using different solvent strengths in the incoming feed and desorbent streams, resulting in two internal plateaus of elution strength. Thus, compared to the conventional process, the overall amount of solvent needed can be reduced, productivity can be increased and more concentrated product streams can be obtained. In this contribution, two case studies will be presented. At first, the separation of bovine IgG from lysozyme will be analyzed as a model system. Antibodies are a common target substance in bio-chromatography, as therapeutic monoclonal antibodies are among the most promising biopharmaceuticals. Using adsorption data obtained from single-column experiments, an appropriate SMB process was designed and implemented. The second target component is the active dimeric form of the bone morphogenetic protein-2 (BMP-2). This protein was isolated from a renaturation solution, which also contained its inactive monomeric form as well as other undefined proteins from the bacterial production strain. A 3-zone open-loop gradient-SMB approach was used successfully for both separations.     

Enantiomers separation by simulated moving bed chromatography. Non-instantaneous equilibrium at the solid-fluid interface

The simulated moving bed (SMB) technology, first conceived for large bulk-scale separations in the petrochemical industry, has found increasingly new applications in the pharmaceutical industry. Among these, the separation of fine chemicals has been the subject of considerable study and research. This work presents the modeling, simulation and design of the operation of a SMB plant in order to separate a binary chiral mixture. The usual assumption of instantaneous equilibrium at the solid-fluid interface is questioned and a first-order kinetics of adsorption is taken into account. The cases of linear, Langmuir and modified Langmuir equilibria are studied. The equivalent true moving bed (TMB) model was used assuming axial dispersion for the fluid flow and plug flow for the solid-phase flow. Intraparticle diffusion was described by a linear driving force (LDF) approximation. Simulation results indicate that, under certain conditions, equilibrium is not actually reached at the adsorbent surface. This leads to different unit performances, in terms of product purities and recoveries, as compared to those predicted assuming instantaneous equilibrium. Moreover, SMB units may be improperly designed by the usual methods (flow-rate ratio separation regions) if non-equilibrium effects are overlooked.     

Etched chemically modified capillaries: novel separation media for electrophoretic analyses

The fabrication, properties, and applications of etched chemically modified capillaries for electrophoretic analysis are reviewed. It is shown that the etching process creates a surface that is fundamentally different than a bare fused silica capillary. The new surface matrix produces unique electroosmotic flow properties and is more compatible with basic and biological compounds. After chemical modification of the surface, the bonded organic moiety (stationary phase) contributes to the control of migration of solutes in the capillary. Both electrophoretic and chromatographic processes take place in the etched chemically modified capillaries leading to a variety of experimental variables (pH, buffer type, presence and amount of organic modifier, and temperature) that can be used to optimize separations. A number of examples of separations on these capillaries are presented as well as data on column ruggedness and reproducibility.     

Simulated moving bed technology: old and new

The Simulated Moving Bed (SMB) concept has been applied to the separation of different mixtures as a continuous counter current separation process, avoiding several problems related with solid motion. The aim of this work is to present some relevant examples of SMB separations corresponding to the two major ages in the use of the SMB concept, here named “old” and “new” applications. The “old” applications of SMB technology in the petrochemical industry are still important, with large and highly productive units; and the “new” applications of the second “age” of SMB concept are from the fine chemical, pharmaceutical and biochemistry areas, associated with the demand of high purity products during the last 10 years. Different examples are presented for different ages: a UOP Parex ^® process for the “old”, modelled with the equivalent True Moving Bed (TMB) approach; and a chiral resolution for the “new”, modelled by the real SMB model. Some of the latest developments are also mentioned: the non conventional techniques as the Varicol ^® process, PowerFeed, Modicon, M3C or Enriched Extract-SMB (EE-SMB), MultiFeed (MF), Outlet Streams Swing (OSS) or Pseudo-SMB, involving considerable changes in the SMB concept itself. The use of the last optimization/modelling packages for the development of design techniques, either at the conception stage as well as for performance improvements of existing units is emphasized.     

Investigation of the stability of polyelectrolyte multilayer coatings in open-tubular capillary electrochromatography using laser scanning confocal microscopy

A simple polyelectrolyte multilayer (PEM) coating procedure was used for the development of stable modified capillaries. PEM coatings were constructed in fused-silica capillaries using alternating rinses of cationic and anionic polyelectrolytes. The multilayer coatings investigated in this study consisted of two and twenty layer pairs, or bilayers. A bilayer is one layer of a cationic polymer and one layer of an anionic polymer. Poly(diallyldimethylammonium chloride) was used as the cationic polymer, and the polymeric surfactant poly(sodium N-undecanoyl-L-leucylvalinate) was used as the anionic polymer. Previous studies for both chiral and achiral separations have shown that PEM-coated capillaries have excellent reproducibilities, remarkable endurance, and strong stabilities against extreme pH values when used in open-tubular capillary electrochromatography (OT-CEC). In this study, the stability of the coatings was further investigated after exposure to 0.1 M and 1.0 M NaOH. Structural changes of these coatings were monitored using laser scanning confocal microscopy (LSCM) after flushing the capillaries with NaOH. This technique allowed observation of the degradation of the coatings. Observations are discussed in terms of separations using OT-CEC. Electropherograms obtained from the chiral separation of 1,1'-binaphthyl-2,2'-dihydrogenphosphate in OT-CEC showed a decrease in selectivity and an increase in electroosmotic mobility after long exposure to NaOH. The ability to recover the capillaries by exposure to NaOH was also demonstrated. Measurements of electroosmotic mobility and selectivity showed that 2-bilayer and 20-bilayer PEM coatings could be completely removed from the capillary surface after approximately 3.5 and 9.5 h, respectively, of continuous exposure to 1 M NaOH.     

CE-ESI-MS of biological anions in plastic capillaries at high pH

In this study, the potential of poly(methylmethacrylate) (PMMA, Plexiglas) and polyether ether ketone (PEEK) tubing for CE-ESI-MS separations of anions at high pH values was examined. A set of model compounds of biological interest was used to investigate the main operational parameters for CE-ESI-MS, such as the sheath-flow interface design, the polarity of the ionization voltage, the use of ammonia-based separation electrolytes, and the sheath liquid composition. Optimum separations and detection sensitivities in negative ESI mode were obtained using a running electrolyte of 75 mM of ammonia at pH 11 and a sheath liquid of 60:40 v/v or 75:25 v/v isopropanol/water with 0.5% v/v of ammonia. At these experimental conditions, PMMA and PEEK capillaries show good hydrolytic stabilities and lower EOF values than fused-silica columns. Better separation resolutions were obtained with PMMA capillary, but this plastic rapidly swelled and bled because of its limited chemical resistance to the sheath liquid. PMMA columns equipped with a fused-silica tip were used for a safer exposure to the sheath liquid, but the inner surface of the fused-silica tips had limited stability at pH 11. On the other hand, good separations and reproducibility on migration times and peak areas were obtained using PEEK capillaries without capillary column deterioration.     

Fritless capillary electrochromatography

The preparation of packed capillaries with stable frits of good quality can be a hurdle to obtain efficient separations in capillary electrochromatography (CEC). Especially with particles smaller than 3 microm, frit preparation is cumbersome. Highly efficient separations using packed capillaries without frits are presented. Under appropriate CEC conditions the particles were retained by electrophoretic attraction towards the anode by a tapered capillary inlet, without the need of a frit at the outlet end. Such fritless capillaries, packed with 1.5 microm nonporous reversed-phase particles, allowed separations with efficiencies of more than 500,000 plates/m. Once the capillaries were conditioned properly, more than 100 separations could be performed with good repeatability. With respect to separation efficiency, fritless capillaries packed with 3 microm particles were comparable with standard CEC capillaries with frits. Examples of separations of steroids, a pesticide and its by-products, and cardiac glycosides under various CEC conditions are shown.     

Evaluation of 1.5 microM reversed phase nonporous silica in packed capillary electrochromatography and application in pharmaceutical analysis.

Reversed-phase nonporous silica (RP-NPS) of 1.5 microm dp is employed to demonstrate rapid and efficient separations in packed capillary electrochromatography (CEC). Two methods for packing capillaries and two techniques to manufacture frits used to hold the packing in place are evaluated for their effect upon separation performance using polyaromatic hydrocarbons (PAHs) and polar neutral pharmaceutical compounds. Attention is given to conditioning of the packed capillaries for high efficiency separations without necessity for sodium dodecyl sulfate (SDS). Separation conditions for the nonporous materials were modified from those previously determined on porous reversed-phase silica. Feasibility for method development and validation of a parent pharmaceutical compound and related impurities in the range of 0.1-120% of a 5 mg/mL concentration was assessed and reported. An approach to improving detection sensitivity through use of large-bore capillaries is briefly discussed.     

Purification of an ascomycin derivative with simulated moving bed chromatography. A case study

A purification process was developed for the separation of a semi-synthetic ascomycin derivative from its by-products. The process consists of a silica gel filtration and crystallization step prior to two simulated moving bed (SMB) separations, where in the first part the polar by-products and in the second part the apolar by-products were removed. The desired purity was achieved in a final crystallization step. Key elements of the whole process were the design of the first crystallization to obtain a product feasible for SMB chromatography and the specification of operating parameters for the two corresponding SMB steps. Starting from a crude product with an assay of only 44.9% an overall yield for the whole process of 81.0% was achieved with a final purity of >98%.     

Hydride-Based Separation Materials for High Performance Liquid Chromatography and Open Tubular Capillary Electrochromatography

 Silica hydride is a recent development in chromatographic support materials for high performance liquid chromatography (HPLC) where hydride groups replace 95% of the silanols on the surface. This conversion changes many of the fundamental properties of the material as well as the bonded stationary phases that are the result of further chemical modification of the hydride surface. Some unique chromatographic properties of hydride-based phases are described as well as some general application areas where these bonded materials may be used in preference to or have advantages not available from typical stationary phases. The fabrication, properties and applications of etched chemically modified capillaries for electrophoretic analysis are also reviewed. It is shown that the etching process creates a surface that is fundamentally different than a bare fused silica capillary. The new surface matrix produces unique electroosmotic flow properties and is more compatible with basic and biological compounds. After chemical modification of the surface, the bonded organic moiety (stationary phase) contributes to the control of migration of solutes in the capillary. Both electrophoretic and chromatographic processes take place in the etched chemically modified capillaries leading to a variety of experimental variables that can be used to optimize separations. A number of examples of separations on these capillaries are described.     

Contactless Conductivity Detection in Capillary Electrophoresis Employing Capillaries with Very Low Inner Diameters

The behaviour of a contactless conductivity detector was studied in its application to capillary electrophoresis employing quartz capillaries with inner diameters (i.d.) of 10 to 75 µm. The detector output signal was measured using KCl as the test electrolyte, within a KCl concentration range from 0 to 100 mmol/L, corresponding to specific conductivities from 0 to 1295 mS/m. When using capillaries with high inner diameters, then the signal‐to‐noise ratio is high in electrolytes of low conductivity. On the other hand, the use of capillaries with low i.d. values is useful in separations employing solutions of high conductivities. The advantages of capillaries with small i.d. values, combined with contactless conductivity detection, are demonstrated on separations of mixtures of inorganic ions and on separations of neutral mono‐ and disaccharides.     

Experimental implementation of automatic 'cycle to cycle' control of a chiral simulated moving bed separation

In the absence of a suitable controller, currently simulated moving beds (SMBs) are operated suboptimally to cope with system uncertainties and to guarantee robustness of operation. Recently, we have developed a 'cycle to cycle' optimizing controller that not only makes use of minimal system information, i.e. only the Henry constants and average bed voidage, but also optimizes the process performance and taps the full economic potential of the SMB technology. The experimental implementation of the 'cycle to cycle' optimizing controller had been carried out for achiral separation. For chiral separation however, application of any online controller has not been possible because an appropriate online monitoring system has not been available. This work reports and discusses the first experimental implementation of the 'cycle to cycle' optimizing control for chiral separations. A mixture of guaifenesin enantiomers is separated on Chiralcel OD columns with ethanol as mobile phase in a eight-column four sections laboratory SMB unit. The results show that the controller, although using minimal information about the retention of the two enantiomers, is able to meet product and process specifications, can optimize the process performance, and is capable of rejecting disturbances that may occur during the operation of the SMB plant.     

Solvent gradient operation of simulated moving beds. 2. Langmuir isotherms

Simulated Moving Bed separations of enantiomers or fine chemicals are usually carried out in the isocratic mode, i.e. by applying the same operating conditions (temperature, pressure, mobile phase composition, pH) in the whole SMB unit. However, it has been recently recognized that by properly modulating operating conditions in the SMB sections. i.e. Sections 1-4 normally, separation performance in terms of productivity and solvent consumption can be significantly improved. In this work, we study solvent gradient SMB (SG-SMB) operation, where the concentration of a modifier in the main solvent constituting the mobile phase is adjusted along the SMB unit, so as to have weaker retention of the species to be separated in the first two sections, and stronger retention in Sections 3 and 4. Overload chromatographic conditions are considered, where the adsorption behavior is characterized by a nonlinear competitive adsorption isotherm, e.g. a binary Langmuir isotherm. Design criteria to achieve complete separation are developed in the frame of the equilibrium theory of chromatography. The theoretical findings are discussed in view of typical effects of the modifier concentration on retention times and solubility of the species to be separated, and an overall assessment of the SG-SMB technology is attempted.     

Internal electrolyte temperatures for polymer and fused-silica capillaries used in capillary electrophoresis

Polymers are important as materials for manufacturing microfluidic devices for electrodriven separations, in which Joule heating is an unavoidable phenomenon. Heating effects were investigated in polymer capillaries using a CE setup. This study is the first step toward the longer-term objective of the study of heating effects occurring in polymeric microfluidic devices. The thermal conductivity of polymers is much smaller than that of fused silica (FS), resulting in less efficient dissipation of heat in polymeric capillaries. This study used conductance measurements as a temperature probe to determine the mean electrolyte temperatures in CE capillaries of different materials. Values for mean electrolyte temperatures in capillaries made of New Generation FluoroPolymer (NGFP), poly-(methylmethacrylate) (PMMA), and poly(ether ether ketone) (PEEK) capillaries were compared with those obtained for FS capillaries. Extrapolation of plots of conductance versus power per unit length (P/L) to zero power was used to obtain conductance values free of Joule heating effects. The ratio of the measured conductance values at different power levels to the conductance at zero power was used to determine the mean temperature of the electrolyte. For each type of capillary material, it was found that the average increase in the mean temperature of the electrolyte (DeltaT(Mean)) was directly proportional to P/L and inversely proportional to the thermal conductivity (lambda) of the capillary material. At 7.5 W/m, values for DeltaT(Mean) for NGFP, PMMA, and PEEK were determined to be 36.6, 33.8, and 30.7 degrees C, respectively. Under identical conditions, DeltaT(Mean) for FS capillaries was 20.4 degrees C.