Continuous Chromatographic Separation Research Articles

A novel method for continuous chromatographic separation of monoclonal antibody charge variants by combining displacement mode chromatography and step elution.

Charge heterogeneity of monoclonal antibodies is considered a critical quality attribute and hence needs to be monitored and controlled by the manufacturer. Typically, this is accomplished via separation of charge variants on cation exchange chromatography (CEX) using a pH or conductivity based linear gradient elution. Although an effective approach, this is challenging particularly during continuous processing as creation of linear gradient during continuous processing adds to process complexity and can lead to deviations in product quality upon slightest changes in gradient formation. Moreover, the long length of elution gradient along with the required peak fractionation makes process integration difficult. In this study, we propose a novel approach for separation of charge variants during continuous CEX chromatography by utilizing a combination of displacement mode chromatography and salt-based step elution. It has been demonstrated that while the displacement mode of chromatography enables control of acidic variants ≤26% in the CEX eluate, salt-based step gradient elution manages basic charge variant ≤25% in the CEX eluate. The proposed approach has been successfully demonstrated using feed materials with varying compositions. On comparing the designed strategy with 2-column concurrent (CC) chromatography, the resin specific productivity increased by 95% and resin utilization increased by 183% with recovery of main species >99%. Further, in order to showcase the amenability of the designed CEX method in continuous operation, the method was examined in our in-house continuous mAb platform.

Methods for the Continuous Chromatographic Separation of Substances

Methods for the Continuous Chromatographic Separation of Substances

Possibilities of Optimizing the Purity of Mannose in Continuous Chromatographic Separation

AbstractThe development of separation technology based on continuous simulated moving bed chromatography in sugar technology is described. This is a potential economic alternative method both in terms of separation efficiency and process performance, and in terms of the high purity of the resulting products. This work focuses in detail on the isolation of mannose from natural polysaccharides, such as galactomannans; however, the developed procedures and equipment can be further applied to the separation of various liquid mixtures of carbohydrates. The main aim was to find optimal operating conditions for hydrolytic and separation steps of the whole technology. The obtained results provide a basic data set for further development of the industrial application.

Dihydroxyacetone Production: From Glycerol Catalytic Oxidation with Commercial Catalysts to Chromatographic Separation

A process for catalytic production of dihydroxyacetone through glycerol oxidation and dihydroxyacetone purification by chromatographic processes is herein presented. Glycerol oxidation over commercial Pt/AC and Pt–Bi/AC catalysts was performed on a batch reactor operating under base-free conditions, using water as a solvent and oxygen as the oxidant agent. The highest dihydroxyacetone yield obtained was 36%, using the Pt5%-Bi1.5%/AC catalyst, whereas the monometallic Pt/AC catalyst was selective to glyceric acid. It is herein reported for the first time dihydroxyacetone separation from unreacted glycerol and reaction byproducts (organic acids). Two commercial polystyrene-divinylbenzene ion-exchange resins were used as a stationary phase, one in the hydrogen form and the other in the calcium form, and water was used as the mobile phase. Adsorption equilibrium data for the species present in the reaction mixture were determined in both resins through single-component adsorption breakthrough experiments. A continuous chromatographic separation process was designed based on the simulated moving bed (SMB) technology and simulated using the gPROMS model builder software. A two SMB cascade was then considered, performing the pseudobinary mixture separation on the first SMB. The extract stream from the first SMB containing dihydroxyacetone and glycerol was fed to the second SMB, obtaining dihydroxyacetone with a purity of 97%. Considering the overall unit, a dihydroxyacetone productivity of 64 kgDHA (LAds day)−1 was obtained with an eluent consumption of 1218 LDes kgDHA–1.

Experimental application of individual column state and parameter estimation in SMB processes to an amino acid separation

Abstract The simulated moving bed (SMB) process is a highly efficient continuous chromatographic separation process. Due to its hybrid process dynamics that lead to discontinuities and sharp fronts on the state trajectories, optimal SMB process operation is challenging. Process performance can be improved by applying model-based optimizing control methods. For this, online information about states and individual column parameters are required. The strategy for simultaneous state and parameter estimation used here exploits the switching nature of the SMB process. The successful experimental application of the strategy is demonstrated for the continuous separation of two amino acids on an SMB pilot plant where extra-column equipment effects need to be considered.

Expanding Simulated Moving Bed Chromatography into Ternary Separations in Analogy to Dividing Wall Column Distillation

Chromatographic separation techniques are widely applied for complex separations. For binary or pseudo-binary separation problems, it is well known that continuous chromatographic separation proces...

Effects of the mobile phase on the chromatographic separation of l-lysine and 5-aminovaleric acid

5-Aminovaleric acid (5AVA), an attractive bio-based carbon-5 building block for polymer synthesis, is produced from l-lysine. However, their structural similarity makes separating 5AVA from l-lysine challenging. The simulated moving bed (SMB), continuous chromatographic separation process provides a solution with high productivity and low eluent consumption. Here, the effects of the mobile phase on the separation of 5AVA and l-lysine on a C18 column are reported. Pre-column derivatization using diethylethoxymethylene malonate enhanced the detection sensitivity and separation performance, which was strongly affected by the phosphate buffer pH. A 70/30 (v/v) mobile phase of 20 mM phosphate buffer at pH 6.8 and acetonitrile showed the best separation performance in isocratic mode. The selectivity at adsorption equilibrium was determined from pulse experiments, and the optimal operating conditions of the SMB process were determined from the equilibrium constants of both components. The feasibility of separating 5AVA and l-lysine by the SMB process was confirmed with purity and recovery of greater than 99%. The SMB process using pre-column derivatization can provide the competiveness of the bio-based chemical production because separation processes typically contribute to more than 70% of the total production cost.

Competitive adsorption of vanillin and syringaldehyde on a macro-mesopore polymeric resin: modeling.

Vanillin and syringaldehyde are widely used as flavoring and fragrance agents in the food products. The potential of a macro-mesoporous adsorption resin was assessed for separation of these binary mixtures. This work focuses on modeling of the competitive adsorption behaviors and exploration of the adsorption mechanism. The characterization results showed the resin had a large BET surface area and specific pore structure with hydrophobic properties. By analysis of the physicochemical properties of the solutes and the resin, the separation mechanism was mainly contributed by hydrophobic effect. Subsequently, the competitive Langmuir isotherm model was used to fit the competitive adsorption isotherms. The pore diffusion coefficient was obtained by macropore diffusion model. Afterwards, a mathematical model was established to predict the breakthrough curves of the binary mixture at various operating conditions. The data and model presented are valuable for design and simulation of the continuous chromatographic separation process.

Anthraquinone and naphthopyrone glycosides from Cassia obtusifolia seeds mediate hepatoprotection via Nrf2-mediated HO-1 activation and MAPK modulation.

Cassia obtusifolia L. seed is one of the most popular traditional Chinese medicine for mutagenicity, genotoxicity, hepatotoxicity, and acute inflammatory diseases. We evaluated the hepatoprotective activity of anthraquinone and naphthopyrone glycosides isolated from the butanol fraction of C. obtusifolia seeds and explored their effects on cell signaling pathways. Continuous chromatographic separation led to the isolation of 1-desmethylaurantio-obtusin 2-O-β-D-glucopyranoside (1), rubrofusarin 6-O-β-D-apiofuranosyl-(1 → 6)-O-β-D-glucopyranoside (2) and rubrofusarin 6-O-β-gentiobioside (3). All glycosides were non-toxic at concentrations up to 80µM. The increased intracellular reactive oxygen species (ROS) and decreased glutathione levels observed after tert-butylhydroperoxide (t-BHP) intoxication were ameliorated by all three glycosides, with compound 3 being the most active. Pretreatment with the three glycosides increased nuclear factor erythroid-2-related factor 2 (Nrf2)-mediated heme oxidase-1 (HO-1) expression. All the glycosides enhanced the phosphorylation of c-JunN-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK), and the dephosphorylation of p38. The protective effects of the anthraquinone and naphthopyrone glycosides against t-BHP-induced oxidative damage in human liver-derived HepG2 cells were due to the prevention of ROS generation and up-regulated activity of HO-1 via Nrf2 activation and modulation of the JNK/ERK/MAPK signaling pathway. The data indicate the potential of these compounds as hepatoprotective agents in pharmaceuticals and/or nutraceuticals.

Effect of separation material particle size on pressure drop and process efficiency in continuous chromatographic separation of glucose and fructose

Abstract Effect of the particle size (250–320 µm) of gel type strong cation exchange resins in Ca2+ form on the pressure drop across the resin bed and on the efficiency of chromatographic glucose–fructose separation was investigated experimentally. Pressure drop caused by the resin bed was found to depend strongly on the particle size. Strong dependency of the pressure drop on the solution viscosity was also observed. No elastic deformation of the resin particles was observed as in each case the dependence of the pressure drop on flow rate was linear. Both material (water consumption) and energy aspects were addressed in the investigation of the effect of particle size on the efficiency of continuous glucose–fructose separation. An eight-column four-zone simulated moving bed (SMB) process was used. The water consumption was found to decrease by 53% by change of particle size from 320 µm to 250 µm. This could be achieved without compromising specific productivities or product yields and purities by proper adjustment of the operating parameters. Lower water consumption results in more concentrated product fractions which decreases the energy consumption of the product concentration step (evaporation). Change in resin particle size from 320 µm to 250 µm decreased the energy consumption in evaporation by 18%, but resulted in 50% increase in the energy consumption of the pumps due to higher pressure drop. However, the total (pumps and evaporation) energy consumption decreased as the product concentration step is significantly more energy intensive unit operation.

Production of Monosugars from Lignocellulosic Biomass in Molten Salt Hydrates: Process Design and Techno-Economic Analysis.

ZnCl2 hydrate, the main molten salt used in biomass conversion, combined with low concentration HCl is an excellent solvent for the dissolution and hydrolysis of the carbohydrates present in lignocellulosic biomass. The most recalcitrant carbohydrate, cellulose, is dissolved in a residence time less than 1 h under mild conditions without significant degradation. This technology is referred to as BIOeCON-solvent technology. Separation of the sugars from the solution is the main challenge. The earlier conclusion regarding the potential of zeolite beta for selective adsorption has been used as the basis of a scale-up study. The technology of choice is continuous chromatographic separation (e.g., simulated moving bed, SMB). The sugar monomers are separated from the sugar oligomers, allowing the production of monosugars at high yield, using water as an eluent. Results of a pilot plant study are presented showing a stable operation at high selectivity. Several process designs are discussed, and the techno-economic performance of the BIOeCON-solvent technology is demonstrated by comparison with the state-of-the-art technology of NREL (National Renewable Energy Laboratory), which is based on enzymatic conversion of cellulose. It is concluded that the BIOeCON-solvent technology is technically and economically viable and is competitive to the NREL process. Because the BIOeCON-solvent process is in an early stage of development and far from fully optimized, it has the potential to outperform the existing processes.

Advanced Reprocessing – The Potential for Continuous Chromatographic Separations

This concept paper, discusses the challenges and opportunities for an extractive chromatographic process for the separation of fission products and minor actinides from uranium and plutonium isotopes in irradiated nuclear fuel. The paper highlights the constraints of the PUREX process, a process that is universally accepted for reprocessing of spent nuclear fuel now and for GEN IV reactor systems. It also identifies the challenges that a new separation process would have to overcome to dislodge its acceptance by both the operators and regulators. Although the concept of using a chromatography technique for this separation is challenging, recent developments of continuous chromatography such as simulated moving bed (SMB) and/or continuous annular chromatography (CAC) provides a degree of encouragement. Equally the development of new stationary phases in particular inorganic exchangers, many of which have not been examined for this application enhances confidence that an alternative to the PUREX process is possible.

Open-loop optimal control of batch chromatographic separation processes using direct collocation

This contribution presents a novel model-based methodology for open-loop optimal control of batch high-pressure liquid chromatographic (HPLC) separation processes. The framework allows for simultaneous optimization of target component recovery yield and production rate with respect to a parameterization of the input elution trajectory and fractionating interval endpoints. The proposed methodology implies formulating and solving a large-scale dynamic optimization problem (DOP) constrained by partial differential equations (PDEs) governing the multi-component system dynamics. It is based on a simultaneous method where both the control and state variables are fully discretized in the temporal domain, using direct local collocation on finite elements, and the state variables are discretized in the spatial domain, using an adaptive finite volume weighted essentially non-oscillatory (WENO) scheme. The direct transcription of the DOP described by Modelica, and its extension Optimica, code into a sparse nonlinear programming problem (NLP) is thoroughly presented. The NLP was subsequently solved using CasADi's (Computer algebra system with Automatic Differentiation) interface to the primal-dual interior point method IPOPT. The advantages of the open-loop optimal control strategy are highlighted through the solution of a challenging ternary complex mixture separation problem of human insulin analogs, with the intermediately eluting component as the target, for a hydrophobic interaction chromatography system. Moreover, the high intercorrelation between the shape of the optimal elution trajectories and the fractionation interval endpoints is thoroughly investigated. It is also demonstrated that the direct transcription methodology enabled accurate and efficient computation of optimal cyclic-steady-state solutions, which govern that state and control variables conform to periodicity constraints imposed on column re-generation and re-equilibration. By these means, the generic methods and tools developed here are applicable to continuous chromatographic separation technologies, including the continuous simulated moving bed (SMB) and the multicolumn counter-current solvent gradient purification (MCSGP) process.

Evaluation of center-cut separations applying simulated moving bed chromatography with 8 zones

Different multi-column options to perform continuous chromatographic separations of ternary mixtures have been proposed in order to overcome limitations of batch chromatography. One attractive option is given by simulated moving bed chromatography (SMB) with 8 zones, a process that offers uninterrupted production, and, potentially, improved economy. As in other established ternary separation processes, the separation sequence is crucial for the performance of the process. This problem is addressed here by computing and comparing optimal performances of the two possibilities assuming linear adsorption isotherms. The conclusions are presented in a decision tree which can be used to guide the selection of system configuration and operation.

Continuous downstream processing for high value biological products: A Review.

There is growing interest in the possibility of developing truly continuous processes for the large-scale production of high value biological products. Continuous processing has the potential to provide significant reductions in cost and facility size while improving product quality and facilitating the design of flexible multi-product manufacturing facilities. This paper reviews the current state-of-the-art in separations technology suitable for continuous downstream bioprocessing, focusing on unit operations that would be most appropriate for the production of secreted proteins like monoclonal antibodies. This includes cell separation/recycle from the perfusion bioreactor, initial product recovery (capture), product purification (polishing), and formulation. Of particular importance are the available options, and alternatives, for continuous chromatographic separations. Although there are still significant challenges in developing integrated continuous bioprocesses, recent technological advances have provided process developers with a number of attractive options for development of truly continuous bioprocessing operations.

Mathematical modeling of the competitive sorption dynamics of acetone–butanol–ethanol on KA-I resin in a fixed-bed column

The recovery and purification of biobutanol based on the adsorption method were performed in dynamic conditions. Computational and theoretical modeling is an important tool in the characterization, development, and validation of fixed-bed columns. Relevant breakthrough curves provide valuable information for designing fixed-bed adsorption processes for field applications. In the present study, a general rate model (GRM), implementing convection/diffusion approach theory and a competitive isotherm model, was used to predict the competitive sorption dynamics of acetone–butanol–ethanol (ABE) on a KA-I resin in a fixed-bed column under different operating conditions, i.e., inlet feed flow rate, initial adsorbate concentration, and bed height. The model simulation was quantified by the absolute average deviation (AAD). The calculated AAD values, ranging from 0.05 to 0.1, indicated that the GRM gives a general prediction for experimental data. The axial dispersion, external mass transfer, and pore diffusion coefficients were calculated by a series of empirical correlations. Biot number was used to identify the rate controlling step for the adsorption process of ABE on the resin. And the pore diffusion coefficient was found to be major governing factor for adsorption of ABE. The data and modeling presented are valuable for designing the continuous chromatographic separation process and simulation of ABE.

Chromatographic Separation of Isomaltooligosaccharides on Ion-Exchange Resins: Effect of the Cationic Form

Prebiotic isomaltooligosaccharides (IMOs) are composed of α-d-glucose units linked by glucosidic α (1O6) bonds and they remain intact until reaching the large bowels, where they are metabolized by bifidobacteria and lactobacillus. These saccharides can also be synthesized enzymatically, wherein the reaction media is composed of monosaccharides (glucose and fructose), disaccharides (sucrose and maltose), IMOs (the product of interest) and dextrans. Dextrans are readily separated by solvent precipitation; however, continuous chromatographic separation (simulated moving bed or SMB) should be an interesting option for the enrichment and purification of IMOs from mono- and disaccharides. The objective of the present work was to measure fundamental adsorption data of IMOs, mono- and disaccharides in preparative columns packed with ion-exchange resin of different cationic forms (K+, Ca+, H+ and Na+), so as to verify the influence of the cation on the selectivity and capacity. Ion exchange in H+ form showed the b...

Supercritical fluid simulated moving bed chromatography for the separation of ethyl linoleate and ethyl oleate

Supercritical fluid-simulated moving bed (SF-SMB) chromatography is an attractive technology thanks to the outstanding performance of continuous countercurrent adsorption processes in combination with the tunability of eluent strength of supercritical CO2. This work focuses on the process development steps of simulated moving bed chromatography for the separation of ethyl linoleate and ethyl oleate on silica gel using supercritical carbon dioxide under linear conditions. Adsorption isotherms of the components of interest were determined at analytical scale and applied together with the equilibrium theory for the design of a two-step SF-SMB process for the separation of the fatty acid ethyl esters. Results of the experimental validation of the continuous chromatographic separation of ethyl linoleate and ethyl oleate are shown and discussed in detail.

Varicol process optimization strategy based on the solution of a MINLP

Varicol is a continuous chromatographic separation process which is based on the simulated moving bed principle with an asynchronous commutation of the inlet and outlet streams. The objective of this work is to develop a systematic procedure to optimize the operating conditions of the Varicol process, including the commutation sequence and the internal flow rates. To this end, the continuous dynamics are represented using a conventional mass-balance partial differential equation model, whereas the discrete dynamics are described by a timed transition Petri net. A Mixed-Integer Non-Linear Problem (MINLP) is formulated to maximize the process productivity under constraints on the desired purities of both the extract and the raffinate components. The MINLP is solved by constructing a set of candidate integer parameters and solving the resulting nonlinear problem for a subset of candidates selected on the basis of an approximate evaluation using the (true moving bed) triangle theory. Numerical results show the feasibility of this approach.

Chromatographic Separation and Purification of Mitotane Racemate in a Varicol Multicolumn Continuous Process

AbstractThe multicolumn continuous chromatographic separation process that increases throughput, purity, and yield compared to batch chromatography is considered as an important preparative technique to purify pharmaceutical drugs. Mitotane is a chiral drug marketed in the racemic form but pharmacological effects due to molecule chirality indicate that one of the enantiomers is more potent. Preparative separation of the mitotane enantiomers was performed by a continuous Varicol unit operated on a scale of 30 g/day. Amylose tris(3,5‐dimethylphenylcarbamate) functioned as the stationary phase and acetonitrile‐isopropanol mixtures as mobile phases. The enantiomeric purities obtained were 97.0 % for S‐(–)‐mitotane and 96.8 % for R‐(+)‐mitotane in the raffinate and extract streams, respectively. The unit provided productivities of 1.14 kg raffinate per day and kg adsorbent and 0.68 kg extract per day and kg adsorbent.