Experimental and model-based approach to evaluate solvent effects on the solubility of the pharmaceutical artemisinin.

The separation and purification of plant-based Active Pharmaceutical Ingredients (API) from extracts is a crucial part in pharmaceutical process development. For the purification of the antimalarial drug component artemisinin (ARTE) from an Artemisia anna L. toluene extract, antisolvent crystallization is considered. Solubilities of ARTE in binary solvent mixtures of toluene and two potential antisolvents, n-heptane and ethanol, were determined at temperatures from 278.15 K to 313.15 K. The experimental work was supported by the application of various models, utilizing varying amounts of experimental input data. The goal was the identification of models that are able to predict solubilities in binary solvent mixtures sufficiently accurate and, thus, can help to reduce the experimental effort for future solvent screenings. In this study, we applied the PC-SAFT model both with and without fitting the binary interaction parameter kij between ARTE and the respective solvent, as well as the empirical Jouyban-Acree model. From the experiments, n-heptane demonstrated to be a promising antisolvent, while ethanol acted more as a cosolvent. All models tested were capable of distinguishing between effective and ineffective antisolvents. The purely predictive PC-SAFT model applied with kij = 0 exhibited the largest deviation from the experimental data. This was followed by the PC-SAFT model including fitted kij values, based on at least four experimental data points. The Jouyban-Acree model fitted the data most accurately. Its parametrization required a minimum of ten experimental data points.

Real-Time Crystal Growth Monitoring of Boric Acid from Sodium or Lithium Sulfate Containing Aqueous Solutions by Atomic Force Microscopy.

The crystal growth of boric acid from an aqueous solution in the absence and presence of sodium and lithium sulfate was studied by real-time monitoring. For this purpose, atomic force microscopy in situ has been used. The results show that the growth mechanism of boric acid from its pure and impure solutions is spiral growth driven by screw dislocation and that the velocity of advancement of steps on the crystal surface, and the relative growth rate (ratio of the growth rate in presence and absence of a salt) is reduced in the presence of salts. The reduction of the relative growth rate could be explained by the inhibition of advancement of steps of the (001) face mainly in the growth direction [100] caused by the adsorption of salts on the actives sites and the inhibition of the formation of sources of steps such as dislocations. The adsorption of the salts on the crystal surface is anisotropic and independent of the supersaturation and preferentially on the active sites of the (100) edge. Moreover, this information is of significance for the improvement of the quality of boric acid recovered from brines and minerals and the synthesis of nanostructures and microstructures of boron-based materials.

Shortcut Model for Batch Preferential Crystallization Coupled with Racemization for Conglomerate-Forming Chiral Systems.

Kinetically controlled preferential crystallization (PC) is a well-established elegant concept to separate mixtures of enantiomers of conglomerate-forming systems. Based on a smaller number of laboratory investigations, the key parameters of an available shortcut model (SCM) can be estimated, allowing for a rapid and reliable process design. This paper addresses a severe limitation of the method, namely, the limitation of the yield to 50%. In order to exploit the valuable counter enantiomer, the crystallization process is studied, coupled with a racemization reaction and a recycling step. It will be shown that the process integration can be performed in various ways. To quantify the different options in a unified manner and to provide a more general design concept, the SCM of PC is extended to include a kinetic model for the enzymatically catalyzed reaction. For illustration, model parameters are used, which characterize the resolution of the enantiomers of asparagine monohydrate and the racemization rate using an amino acid racemase. The theoretical study highlights the importance of exploiting the best stop time for batch operations in order to achieve the highest process productivity.

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.

A Contribution to the Solid State Forms of Bis(demethoxy)curcumin: Co-Crystal Screening and Characterization.

Bis(demethoxy)curcumin (BDMC) is one of the main active components found in turmeric. Major drawbacks for its usage are its low aqueous solubility, and the challenging separation from other curcuminoids present in turmeric. Co-crystallization can be applied to alter the physicochemical properties of BDMC in a desired manner. A co-crystal screening of BDMC with four hydroxybenzenes was carried out using four different methods of co-crystal production: crystallization from solution by slow solvent evaporation (SSE), and rapid solvent removal (RSR), liquid-assisted grinding (LAG), and crystallization from the melt phase. Two co-crystal phases of BDMC were obtained with pyrogallol (PYR), and hydroxyquinol (HYQ). PYR-BDMC co-crystals can be obtained only from the melt, while HYQ-BDMC co-crystals could also be produced by LAG. Both co-crystals possess an equimolar composition and reveal an incongruent melting behavior. Infrared spectroscopy demonstrated the presence of BDMC in the diketo form in the PYR co-crystals, while it is in a more stable keto-enol form in the HYQ co-crystals. Solubility measurements in ethanol and an ethanol-water mixture revealed an increase of solubility in the latter, but a slightly negative effect on ethanol solubility. These results are useful for a prospective development of crystallization-based separation processes of chemical similar substances through co-crystallization.

Immobilization of an amino acid racemase for application in crystallization-based chiral resolutions of asparagine monohydrate.

Integration of racemization and a resolution process is an attractive way to overcome yield limitations in the production of pure chiral molecules. Preferential crystallization and other crystallization-based techniques usually produce low enantiomeric excess in solution, which is a constraint for coupling with racemization. We developed an enzymatic fixed bed reactor that can potentially overcome these unfavorable conditions and improve the overall yield of preferential crystallization. Enzyme immobilization strategies were investigated on covalent-binding supports. The amino acid racemase immobilized in Purolite ECR 8309F with a load of 35 mg-enzyme/g-support showed highest specific activity (approx. 500 U/g-support) and no loss in activity in reusability tests. Effects of substrate inhibition observed for the free enzyme were overcome after immobilization. A packed bed reactor with the immobilized racemase showed good performance in steady state operation processing low enantiomeric excess inlet. Kinetic parameters from batch reactor experiments can be successfully used for prediction of packed bed reactor performance. Full conversions could be achieved for residence times above 1.1 min. The results suggest the potential of the prepared racemase reactor to be combined with preferential crystallization to improve resolution of asparagine enantiomers.

Speeding up Viedma Deracemization through Water-catalyzed and Reactant Self-catalyzed Racemization.

Viedma deracemization is based on solution phase racemization, dissolution of racemic or scalemic conglomerates and crystal growth through autocatalytic cluster formation. With rate limiting racemization, its acceleration by appropriate catalysts may result in speeding up deracemization. A conglomerate-forming chiral compound may principally racemize directly, or via reverse of its formation reaction. For a hydrazine derivative, we investigated available racemization pathways in presence of pyrrolidine or thiourea amine as base catalysts: via Mannich or aza-Michael reaction steps and their reverse, or by enolization. Racemization by enolization was computationally found to dominate, both under water-free conditions and in presence of water, involving a multitude of different pathways. Faster racemization in presence of water resulted indeed in more rapid deracemization, when the base was pyrrolidine. Under water-free conditions, the role of water as enolization catalyst is assumed by chiral hydrazine itself - in autocatalytic racemization and in which both reactant and product are catalysts.

Separation Processes to Provide Pure Enantiomers and Plant Ingredients.

Enantiomer separation and the isolation of natural products from plants pose challenging separation problems resulting from the similarity of molecules and the number of compounds present in synthesis or extract mixtures. Furthermore, limited theory is available to predict productivities for possible alternative separation techniques. The application and performance of chromatography- and crystallization-based processes are demonstrated for various case studies devoted to isolating valuable target compounds from complex initial mixtures. In all cases, the first emphasis is set to determine the process-specific phase equilibria to identify feasible process options. For all examples considered, yields and productivities are evaluated and compared for different scenarios. Guidelines to approach and solve similar separation tasks are given.

Resolution of Racemic Guaifenesin Applying a Coupled Preferential Crystallization-Selective Dissolution Process: Rational Process Development.

Preferential crystallization is a cost efficient method to provide pure enantiomers from a racemic mixture of a conglomerate forming system. Exploiting small amounts of pure crystals of both enantiomers, several batch or continuous processes were developed, capable of providing both species. However, an intermediate production step has to be used when pure enantiomers are not available. In such cases, partially selective synthesis, chromatography, or crystallization processes utilizing chiral auxiliaries have to be used to provide the initial seed material. Recently, it was shown that a coupled Preferential Crystallization-selective Dissolution process (CPCD) in two coupled crystallizers can be applied if at least one pure enantiomer is available to produce both antipodes within one batch. The corresponding process is carried out in one reactor (crystallization tank) by seeding a racemic supersaturated solution with the available enantiomer at a certain temperature. The second reactor (dissolution tank) contains a saturated racemic suspension at a higher temperature. Both reactors are coupled via the fluid phase, allowing for a selective dissolution of the preferentially crystallizing enantiomer from the solid racemic feed provided in the dissolution vessel. The dissolution and crystallization processes continue until the solid racemic material is completely resolved and becomes enantiopure. At this point, both enantiomers can be harvested in their pure crystalline form. For a specific pharmaceutically relevant case study, a rational process design and the applied empirical optimization procedure will be described. The achieved productivities after optimization show the great potential of this approach also for industrial applications. Also, a strategy to control this process based on inline turbidity measurement will be presented.

Shortcut Model for Describing Isothermal Batch Preferential Crystallization of Conglomerates and Estimating the Productivity.

Preferential crystallization (PC) is a powerful method to separate the enantiomers of chiral molecules that crystallize as conglomerates. The kinetically controlled separation method works in a typically narrow metastable zone. Currently, there are no simple models available that allow estimating the productivity of PC and, thus, the comparison with rivalling resolution techniques. In this Article, we suggest a simple shortcut model (SCM) capable of describing the main features of batch-wise operated PC using three ordinary differential equations originating from the mass balance of the target enantiomer and solvent in the liquid and solid phases. Compared to population balance models, the basis of the SCM is the assumption that the crystals for each enantiomer have the same size, which increases continuously from prespecified initial values. The goal of the model is to describe the initial period of the batch, during which the purity is within the specification required. It is accepted that after reaching this border, the precision of predictions can drop. This Article also illustrates a simple strategy how to parametrize the model based on a few experimental runs of PC. At first, for demonstration purposes, theoretical transients generated using the more rigorous PBE model is analyzed using SCM considering the separation of the enantiomers of dl-threonine. Subsequently, results of an experimental study with the enantiomers of asparagine monohydrate are presented to validate the shortcut model, which is seen as a new valuable tool to quantify more rapidly the productivity of PC and to further promote this elegant technique capable to resolve enantiomers of conglomerate forming chiral systems.

Literally Green Chemical Synthesis of Artemisinin from Plant Extracts.

Active pharmaceutical ingredients are either extracted from biological sources-where they are synthesized in complex, dynamic environments-or prepared in stepwise chemical syntheses by reacting pure reagents and catalysts under controlled conditions. A combination of these two approaches, where plant extracts containing reagents and catalysts are utilized in intensified chemical syntheses, creates expedient and sustainable processes. We illustrate this principle by reacting crude plant extract, oxygen, acid, and light to produce artemisinin, a key active pharmaceutical ingredient of the most powerful antimalarial drugs. The traditionally discarded extract of Artemisia annua plants contains dihydroartemisinic acid-the final biosynthetic precursor-as well as chlorophyll, which acts as a photosensitizer. Efficient irradiation with visible light in a continuous-flow setup produces artemisinin in high yield, and the artificial biosynthetic process outperforms syntheses with pure reagents.

Analysis of linear two-dimensional general rate model for chromatographic columns of cylindrical geometry.

This work is concerned with the analytical solutions and moment analysis of a linear two-dimensional general rate model (2D-GRM) describing the transport of a solute through a chromatographic column of cylindrical geometry. Analytical solutions are derived through successive implementation of finite Hankel and Laplace transformations for two different sets of boundary conditions. The process is further analyzed by deriving analytical temporal moments from the Laplace domain solutions. Radial gradients are typically neglected in liquid chromatography studies which are particularly important in the case of non-perfect injections. Several test problems of single-solute transport are considered. The derived analytical results are validated against the numerical solutions of a high resolution finite volume scheme. The derived analytical results can play an important role in further development of liquid chromatography.

Solid-Phase and Oscillating Solution Crystallization Behavior of (+)- and (-)-N-Methylephedrine.

This work involves the study of the solid-phase and solution crystallization behavior of the N-methylephedrine enantiomers. A systematic investigation of the melt phase diagram of the enantiomeric N-methylephedrine system was performed considering polymorphism. Two monotropically related modifications of the enantiomer were found. Solubilities and the ternary solubility phase diagrams of N-methylephedrine enantiomers in 2 solvents [isopropanol:water, 1:3 (Vol) and (2R, 3R)-diethyl tartrate] were determined in the temperature ranges between 15°C and 25°C, and 25°C and 40°C, respectively. Preferential nucleation and crystallization experiments at higher supersaturation leading to an unusual oscillatory crystallization behavior as well as a successful preferential crystallization experiment at lower supersaturation are presented and discussed.

Center-cut separation of intermediately adsorbing target component by 8-zone simulated moving bed chromatography with internal recycle.

An 8-zone simulated moving bed chromatography with internal recycle (8ZSMB-IR) has been designed for center-cut separation, that is, for isolating an intermediately adsorbed component out of a multicomponent mixture. The system consists of two integrated subunits and operates in a fully continuous manner. In the first subunit the feed mixture is split into two fractions containing either a single component or a binary mixture. The binary mixture is recycled through the internal raffinate or extract port into the second subunit, where the target product is isolated. Additionally, the solvent is also recycled internally. For a case study, the separation of a ternary mixture of cycloketones as a model system under weakly non-linear isotherm conditions has been investigated. A few novel configurations of the 8ZSMB-IR unit including the arrangement of the internal recycle of extract, raffinate and solvent streams between two subunits have been examined with respect to various performance indicators for the process realization. The unit performed best with the developed configuration when the internal raffinate stream was recycled and the solvent recycling loop was closed between the last and the first zone of the first subunit. That configuration has further been analyzed experimentally and numerically. On the basis of the results a strategy for determining reliable operating conditions for the 8ZSMB-IR process has been developed. The procedure exploited a model of the process dynamics, which was implemented to refine the isotherm coefficients and to quantify the mixing effect of the liquid stream inside the recycling loops. The upgraded model with the adjusted parameters has been validated based on experimental data and successfully applied for optimizing the operating conditions of the separation.

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.

Physical-Chemical Properties of the Chiral Fungicide Fenamidone and Strategies for Enantioselective Crystallization.

Thermodynamic and kinetic parameters are of prime importance for designing crystallization processes. In this article, Preferential Crystallization, as a special approach to carry out enantioselective crystallization, is described to resolve the enantiomers of the chiral fungicide fenamidone. In preliminary investigations the melting behavior and solid-liquid equilibria in the presence of solvents were quantified. The analyses revealed a stable solid phase behavior of fenamidone in the applied solvents. Based on the results obtained, a two-step crystallization route was designed and realized capable of providing highly pure enantiomers. An initial Preferential Crystallization of the racemate was performed prior to crystallizing the target enantiomer preferentially out of the enriched mother liquor. Chirality 28:514-520, 2016. © 2016 Wiley Periodicals, Inc.

Purification of single-chain antibody fragments exploiting pH-gradients in simulated moving bed chromatography.

This paper deals with the theoretical design and experimental validation of an affinity-based continuous multi-column chromatography process for the purification of single-chain Fragment variable (scFv) antibodies. An open-loop 3-zone pH-gradient simulated moving bed (SMB) process was investigated exploiting the highly specific affinity of metal ions toward histidine-tagged recombinant proteins. The separation problem was simplified by considering the cell culture supernatant as a pseudo-binary mixture. The influence of mobile phase pH on the adsorption isotherm parameters was estimated by the inverse method using recorded pH-gradient batch elution profiles. Suitable operating parameters for the SMB process were identified using an equilibrium stage model and subsequently validated in a lab-scale SMB unit. Finally, the performance of the pH-gradient SMB process was compared against a non-optimized batch process. Biologically active single-chain Fragment variable antibody formats were purified continuously with 9% more recovery, 11 times more productivity (576 mg of purified scFv per day and liter stationary phase in SMB) and enriched by a factor of 2.5 compared to those obtained in the non-optimized batch process.

Racemization of undesired enantiomers: Immobilization of mandelate racemase and application in a fixed bed reactor.

Production of optically pure products can be based on simple unselective synthesis of racemic mixtures combined with a subsequent separation of the enantiomers; however, this approach suffers from a 50% yield limitation which can be overcome by racemization of the undesired enantiomer and recycling. Application of biocatalyst for the racemization steps offers an attractive option for high-yield manufacturing of commercially valuable compounds. Our work focuses on exploiting the potential of racemization with immobilized mandelate racemase. Immobilization of crude mandelate racemase via covalent attachment was optimized for two supports: Eupergit(®) CM and CNBr-activated Sepharose 4 Fast Flow. To allow coupling of enzymatic reaction with enantioselective chromatography, a mobile phase composition compatible with both processes was used in enzymatic reactor. Kinetic parameters obtained analyzing experiments carried out in a batch reactor could be successfully used to predict fixed-bed reactor performance. The applicability of the immobilized enzyme and the determined kinetic parameters were validated in transient experiments recording responses to pulse injections of R-mandelic acid. The approach investigated can be used for futher design and optimization of high yield combined resolution processes. The characterized fixed-bed enzymatic reactor can be integrated e.g. with chromatographic single- or multicolumn steps in various configurations.

Advanced process for precipitation of lignin from ethanol organosolv spent liquors.

An advanced process for lignin precipitation from organosolv spent liquors based on ethanol evaporation was developed. The process avoids lignin incrustations in the reactor, enhances filterability of the precipitated lignin particles and significantly reduces the liquor mass in downstream processes. Initially, lignin solubility and softening properties were understood, quantified and exploited to design an improved precipitation process. Lignin incrustations were avoided by targeted precipitation of solid lignin at specific conditions (e.g. 100 mbar evaporation pressure, 43°C and 10%wt. of ethanol in lignin dispersion) in fed-batch operation at lab and pilot scale. As result of evaporation the mass of spent liquor was reduced by about 50%wt., thus avoiding large process streams. By controlled droplet coalescence the mean lignin particle size increased from below 10 µm to sizes larger than 10 µm improving the significantly filterability.

Are the Crystal Structures of Enantiopure and Racemic Mandelic Acids Determined by Kinetics or Thermodynamics?

Mandelic acids are prototypic chiral molecules where the sensitivity of crystallized forms (enantiopure/racemic compound/polymorphs) to both conditions and substituents provides a new insight into the factors that may allow chiral separation by crystallization. The determination of a significant number of single crystal structures allows the analysis of 13 enantiopure and 30 racemic crystal structures of 21 (F/Cl/Br/CH3/CH3O) substituted mandelic acid derivatives. There are some common phenyl packing motifs between some groups of racemic and enantiopure structures, although they show very different hydrogen-bonding motifs. The computed crystal energy landscape of 3-chloromandelic acid, which has at least two enantiopure and three racemic crystal polymorphs, reveals that there are many more possible structures, some of which are predicted to be thermodynamically more favorable as well as slightly denser than the known forms. Simulations of mandelic acid dimers in isolation, water, and toluene do not differentiate between racemic and enantiopure dimers and also suggest that the phenyl ring interactions play a major role in the crystallization mechanism. The observed crystallization behavior of mandelic acids does not correspond to any simple "crystal engineering rules" as there is a range of thermodynamically feasible structures with no distinction between the enantiopure and racemic forms. Nucleation and crystallization appear to be determined by the kinetics of crystal growth with a statistical bias, but the diversity of the mandelic acid crystallization behavior demonstrates that the factors that influence the kinetics of crystal nucleation and growth are not yet adequately understood.