Cell-Free Multi-Enzyme Synthesis and Purification of Uridine Diphosphate Galactose.

High costs and low availability of UDP-galactose hampers the enzymatic synthesis of valuable oligosaccharides such as human milk oligosaccharides. Here, we report the development of a platform for the scalable, biocatalytic synthesis and purification of UDP-galactose. UDP-galactose was produced with a titer of 48 mM (27.2 g/L) in a small-scale batch process (200 µL) within 24 h using 0.02 genzyme /gproduct . Through in-situ ATP regeneration, the amount of ATP (0.6 mM) supplemented was around 240-fold lower than the stoichiometric equivalent required to achieve the final product yield. Chromatographic purification using porous graphic carbon adsorbent yielded UDP-galactose with a purity of 92 %. The synthesis was transferred to 1 L preparative scale production in a stirred tank bioreactor. To further reduce the synthesis costs here, the supernatant of cell lysates was used bypassing expensive purification of enzymes. Here, 23.4 g/L UDP-galactose were produced within 23 h with a synthesis yield of 71 % and a biocatalyst load of 0.05 gtotal_protein /gproduct . The costs for substrates per gram of UDP-galactose synthesized were around 0.26 €/g.

Continuous synthesis and purification by direct coupling of a flow reactor with simulated moving-bed chromatography.

Continuous synthesis meets continuous purification to produce pure products from crude reaction mixtures. In the nucleophilic aromatic substitution of 2,4-difluoronitrobenzene with morpholine the desired monosubstituted product can be continuously separated from the byproducts in a purity of over 99 % by coupling a flow reactor to a simulated moving bed (SMB) chromatography module.

Analysis and experimental demonstration of temperature step gradients in preparative liquid chromatography.

The amount of substance adsorbed on solid surface depends on temperature. Therefore, the migration velocities of the solutes in a chromatographic column can be altered by introducing temperature gradients. Such gradients designed to change retention behaviours can be exploited to improve the separation performances in preparative chromatography. To describe key process features, we used analytical solutions of the equilibrium model with instant stepwise shift of temperature. To achieve a more realistic description, the equilibrium dispersion model was additionally applied to treat finite column efficiencies. The effect of temperature gradients was illustrated experimentally using two identical columns sequentially connected. Temperature of the second column was modulated by thermostats. Wide pulse injections of a single component led to instructive elution profiles in a preliminary investigation. The observations were found to be in qualitative agreement with predictions of the equilibrium dispersion model. Subsequently, the separation of a ternary model mixture was investigated considering a simple two-step temperature gradient. To support the quantitative analysis and to identify suitable switching and cycle times, the temperature dependencies of the Henry constants were determined by short pulse injections. A meaningful variation of the parameters of the temperature gradient is required for adjusting the cycle times, which is the time difference between two consecutive injections that needs to shorten. Decreasing this time is connected with a desirable increase in process productivity. The results achieved revealed that relatively simple to implement stepwise temperature gradients offer an option to improve and fine-tune the performance of repetitive batch chromatography.

Method to predict the bandwidth of elution profile under the linear gradient elution in reversed-phase HPLC.

Solute migration in a chromatographic column is an important consideration when designing batch or continuous chromatographic separation processes. Most design methods for the chromatographic processes are based on the equilibrium theory which concerns only the migration velocity of the solute. However, in real cases, it is important to predict the zone spreading which occurs by axial dispersion and mass transfer resistance. To predict the actual solute profiles in the column or effluent stream, numerical methods to solve nonlinear partial differential equations have been used. However, these methods involve much time and expense. In this work, two different rate factors are considered to predict the characteristics of the solute profiles. The first is solute migration velocity and the second is the zone spreading rate. The zone spreading rate can be estimated by the apparent axial dispersion coefficient which is obtained from the height of the equivalent theoretical plate in particular. Four benzene derivatives (benzene, toluene, p-xylene, and acetophenone) were used as model solutes, and two mobile phase systems, water/methanol and water/ACN, were used in RP-HPLC. The bandwidths and retention times of the solutes were predicted under several linear gradient conditions. The predicted and experimental bandwidths and retention times showed good agreement.

Separation of D-psicose and D-fructose using simulated moving bed chromatography.

Simulated moving bed (SMB) processes have been widely used in the sugar industries with ion-exchange resin as a stationary phase. D-psicose, a rare monosaccharide known as a valuable pharmaceutical substrate, was synthesized by the enzymatic conversion from D-fructose. The SMB process was adopted to separate D-psicose from D-fructose. Before the SMB experiment, the reaction mixture including D-psicose and D-fructose was treated by a deashing process to remove contaminants, such as buffers, proteins, and other organic materials. Four columns packed with Dowex 50WX4-Ca2+ (200-400 mesh) ion-exchange resins were used in the four-zone SMB. Single-step frontal analysis was performed to estimate the isotherm parameters of each monosaccharide. The operating conditions of the SMB process were determined based on the Equilibrium Theory. According to the simulation of the SMB process, the purity and yield of extract product (D-psicose) achieved were 99.04 and 97.46%, respectively and those of raffinate product (D-fructose) were 99.06 and 99.53%, respectively. Under the optimized operating condition, complete separation (extract purity = 99.36%, raffinate purity = 99.67%) was achieved experimentally.

Continuous synthesis of artemisinin-derived medicines.

Described is a continuous, divergent synthesis system which is coupled to continuous purification and is capable of producing four anti-malarial APIs. The system is comprised of three linked reaction modules for photooxidation/cyclization, reduction, and derivatization. A fourth module couples the crude reaction stream with continuous purification to yield pure API.

Design of pseudo-simulated moving bed process with multi-objective optimization for the separation of a ternary mixture: linear isotherms.

Pseudo-SMB, often called "J-O process", is a modified SMB process to completely separate a ternary mixture with two discrete steps per one cycle. For improved separation, two new design parameters, the position of step 1 (chi(S1)) and the number of port switches during step 2 (n(SMB)), were introduced. A multi-objective optimization method was used to optimize the operating conditions of the pseudo-SMB process with four average zone flow-rate ratios for one cycle. Nadolol isomers were selected for the model solutes and the global objective for the design of the pseudo-SMB was to collect 99% of the intermediate retained solute. The separation was optimized for 8-column pseudo-SMB system with three column lengths (2.5, 5.0, and 10 cm) and three feed composition ratios (1/1/1, 1/2/1, and 2/1/2). The simulation results showed that productivity was increased 4.3 times (n(SMB)=20, chi(S1)=0.5, 1/1/1) and desorbent to feed ratio D/F was decreased 45% (n(SMB)=16, chi(S1)=0.5, 1/1/1) compared to normal operation (n(SMB)=8, chi(S1)=0.5, 1/1/1). Productivity and D/F were significantly improved when short columns were used in the pseudo-SMB process. The pseudo-SMB was compared with recycle chromatography and SMB cascades for the same total amount of adsorbent. Recycle chromatography and 8-column SMB cascades using 20 cm and 40 cm of total column lengths were not able to separate the intermediate component with the target purity and the same feed rate of the pseudo-SMB process.

Optimized design of recycle chromatography to isolate intermediate retained solutes in ternary mixtures: Langmuir isotherm systems.

Batch chromatography with a recycle stream is a popular and simple technique to separate a single target component in a complex mixture with moderate operating conditions. Design of recycle chromatography depends on the retention behaviors of the mixture components. In this work, four nucleosides were considered as solutes. Feed concentration and recycle methods were optimized to isolate only the intermediate retained solute in ternary and pseudo-ternary mixtures. Two recycle methods introduced in our previous work for linear isotherms, the desorbent and feed recycle methods, were compared in terms of productivity and desorbent to feed ratio, D/F, with various feed concentrations for competitive Langmuir isotherm systems. The simulation results show that the target (intermediate retained solute) was separated with over 99.76% purity and 99.88% yield. Productivity of the feed recycle method was increased by up to 162% and D/F was decreased by up to 59% compared to the desorbent recycle method. For the separation of nucleosides, recycle chromatography was compared to eight column simulated moving bed (SMB) cascades with a recycle stream and D/F of the SMB cascades was 58% lower than D/F of recycle chromatography at the same productivity. However, recycle chromatography is much simpler.

Prediction of elution bandwidth for purine compounds by a retention model in reversed-phase HPLC with linear-gradient elution.

In reversed-phase liquid chromatography, the retention mechanism of solute has been studied under linearly programmed gradient mobile-phase conditions. The separation of a mixture of four purine compounds (purine, theobromine, theophylline, and caffeine) was considered as a practical case in two binary mobile phase systems, water/methanol and water/acetonitrile. The retention model which describes how the retention factor is related to the mobile-phase composition has been developed in various mathematical forms to predict the retention time in both linear and gradient elutions. For a pulse injection of sample, two important factors, the retention time and the bandwidth of solute, might be computable to predict the elution profiles estimated by the distribution function, such as the Gaussian distribution function. In this work, a prediction method based on the analogue of the retention model was proposed to calculate the bandwidth in linear gradient elutions. Band broadening was caused by the different migration velocities of the front and rear ends of the solute band in a chromatographic column. Therefore, the migration behaviors of the front and rear ends of the solute band were explained with the same retention model which had been used to predict the retention time of solute. For the well retained solutes, theophylline and caffeine, the predicted bandwidth and experimentally obtained bandwidth showed good agreement in both isocratic and gradient elutions.