Multicomponent Chiral Quantification with Ultraviolet Circular Dichroism Spectroscopy: Ternary and Quaternary Phase Diagrams of Levetiracetam.

Chiral molecules are challenging for the pharmaceutical industry because although physical properties of the enantiomers are the same in achiral systems, they exhibit different effects in chiral systems, such as the human body. The separation of enantiomers is desired but complex, as enantiomers crystallize most often as racemic compounds. A technique to enable the chiral separation of racemic compounds is to create an asymmetry in the thermodynamic system by generating chiral cocrystal(s) using a chiral coformer and using the solubility differences to enable separation through crystallization from solution. However, such quaternary systems are complex and require analytical methods to quantify different chiral molecules in solution. Here, we develop a new chiral quantification method using ultraviolet-circular dichroism spectroscopy and multivariate partial least squares calibration models, to build multicomponent chiral phase diagrams. Working on the quaternary system of (R)- and (S)-2-(2-oxopyrrolidin-1-yl)butanamide enantiomers with (S)-mandelic acid in acetonitrile, we measure accurately the full quaternary phase diagram for the first time. By understanding the phase stabilities of the racemic compound and the enantiospecific cocrystal, the chiral resolution of levetiracetam could be designed due to a large asymmetry in overall solubility between both sides of the racemic composition. This new method offers improvements for chiral molecule quantification in complex multicomponent chiral systems and can be applied to other chiral spectroscopy techniques.

The unexpected dominance of secondary over primary nucleation.

It is still a challenge to control the formation of particles in industrial crystallization processes. In such processes, new crystals can be generated either by primary or secondary nucleation. While in continuous stirred tank crystallization processes, secondary nucleation is thought to occur due to the shear or attrition of already present larger crystals; in antisolvent crystallization processes, where mixing at the inlets locally causes high supersaturations, primary nucleation is understood to be the main mechanism. We aim to show here that secondary nucleation is the dominant nucleation mechanism, even under conditions that are generally considered to be dominated by primary nucleation mechanisms. Measurements of primary and secondary nucleation rates under similar industrial crystallization conditions of sodium bromate in water, sodium chloride in water, glycine in water and isonicotinamide in ethanol show that the secondary nucleation rate is at least 6 orders of magnitude larger in all these systems. Furthermore, seeded fed-batch and continuous antisolvent crystallizations of sodium bromate under high local supersaturation, seeded with crystals of a specific handedness, result in a close to chirally pure crystalline product with the same handedness. This shows that indeed, enantioselective secondary nucleation is the dominant mechanism in these antisolvent crystallizations. It is even possible to use the enantioselective secondary nucleation mechanism to control the product chirality in such a process, making antisolvent crystallization a viable crystallization-enhanced deracemization technique, having a superior productivity compared to other crystallization-enhanced deracemization methods. Our finding of a dominant secondary nucleation mechanism, rather than primary nucleation, will have a strong impact on nucleation control strategies in industrial crystallization processes.

Simultaneous Chiral Resolution of Two Racemic Compounds by Preferential Cocrystallization*.

We tap into an unexplored area of preferential crystallization, being the first to develop simultaneous chiral resolution of two racemic compounds by preferential cocrystallization. We highlight how the two racemic compounds RS-mandelic acid (MAN) and RS-etiracetam (ETI) can be combined together as enantiospecific R-MAN⋅R-ETI and S-MAN⋅S-ETI cocrystals forming a stable conglomerate system and subsequently develop a cyclic preferential crystallization allowing to simultaneous resolve both compounds. The developed process leads to excellent enantiopurity both for etiracetam (ee>98 %) and mandelic acid (ee≈95 %) enantiomers.

Combining Incompatible Processes for Deracemization of a Praziquantel Derivative under Flow Conditions.

An efficient deracemization method for conversion of the racemate to the desirable (R)-enantiomer of Praziquantel has been developed by coupling incompatible racemization and crystallization processes. By a library approach, a derivative that crystallizes as a conglomerate has been identified. Racemization occurs via reversible hydrogenation over a palladium on carbon (Pd/C) packed column at 130 °C, whereas deracemization is achieved by alternating crystal growth/dissolution steps with temperature cycling between 5-15 °C. These incompatible processes are combined by means of a flow system resulting in complete deracemization of the solid phase to the desired (R)-enantiomer (98 % ee). Such an unprecedented deracemization by a decoupled crystallization/racemization approach can readily be turned into a practical process and opens new opportunities for the development of essential enantiomerically pure building blocks that require harsh methods for racemization.

Assessing Crystallisation Kinetics of Zr Metal-Organic Frameworks through Turbidity Measurements to Inform Rapid Microwave-Assisted Synthesis.

Controlling the crystallisation of metal-organic frameworks (MOFs), network solids of metal ions or clusters connected by organic ligands, is often hindered by the significant number of synthetic variables inherent to their synthesis. Coordination modulation, the addition of monotopic competing ligands to solvothermal syntheses, can allow tuning of physical properties (particle size, porosity, surface chemistry), enhance crystallinity, and select desired phases, by modifying the kinetics of self-assembly, but its mechanism(s) are poorly understood. Herein, turbidity measurements were used to assess the effects of modulation on the solvothermal synthesis of the prototypical Zr terephthalate MOF UiO-66 and the knowledge gained was applied to its rapid microwave synthesis. The studied experimental parameters-temperature, reagent concentration, reagent aging, metal precursor, water content, and modulator addition-all influence the time taken for onset of nucleation, and subsequently allow microwave synthesis of UiO-66 in as little as one minute. The simple, low cost turbidity measurements align closely with previously reported in situ synchrotron X-ray diffraction studies, proving their simplicity and utility for probing the nucleation of complex materials while offering significant insights to the synthetic chemist.

On the Effect of Secondary Nucleation on Deracemization through Temperature Cycles.

Herein, the pivotal role of secondary nucleation in a crystallization-enhanced deracemization process is reported. During this process, complete and rapid deracemization of chiral conglomerate crystals of an isoindolinone is attained through fast microwave-assisted temperature cycling. A parametric study of the main factors that affect the occurrence of secondary nucleation in this process, namely agitation rate, suspension density, and solute supersaturation, confirms that an enhanced stereoselective secondary nucleation rate maximizes the deracemization rate. Analysis of the system during a single temperature cycle showed that, although stereoselective particle production during the crystallization stage leads to enantiomeric enrichment, undesired kinetic dissolution of smaller particles of the preferred enantiomer occurs during the dissolution step. Therefore, secondary nucleation is crucial for the enhancement of deracemization through temperature cycles and as such should be considered in further design and optimization of this process, as well as in other temperature cycling processes commonly applied in particle engineering.

Enabling Direct Preferential Crystallization in a Stable Racemic Compound System.

The preparative resolution by preferential crystallization (PC) of proxyphylline has been achieved despite the existence of a stable racemic compound. This is enabled through the careful selection of a solvent in which both the racemic compound and the metastable conglomerate possess a low nucleation rate. Induction time measurements in isobutyl alcohol show that a highly supersaturated solution (ß = 2.3) remains clear for almost 1 h at 20 mL scale, revealing a slow nucleation rate. Seeding the supersaturated solution with the pure enantiomer triggered its crystallization both isothermal and polythermic modes of PC were successfully implemented. Alongside the reported case of diprophylline, this study opens opportunities to broaden the application of PC toward slowly crystallizing racemic compounds.

Prenucleation Self-Assembly and Chiral Discrimination Mechanisms during Solution Crystallisation of Racemic Diprophylline.

The crystallisation behaviour of (RS)-diprophylline (DPL) in two different solvents is investigated to assess the incidence of solvated pre-associations on nucleation, crystal growth and chiral discrimination. In the solvated state, Raman spectroscopy shows that dimeric associations similar to those depicted in the crystalline solid solution (ssRII) predominate in isopropanol (IPA), which may account for the systematic spontaneous nucleation of this crystal form from this solvent. By contrast, spontaneous nucleation in DMF yields the stable racemic compound RI, consistently with the distinct features of the Raman spectrum collected in this solvent. A crystal growth study of ssRII in IPA reveals that the crystal habitus is impacted by the solution enantiomeric excess; this is explained by increased competition between homo- and heterochiral pre-associations. This is supported by a molecular modelling study on the enantiomeric selectivity of the DPL crystal lattices. The combination of assessment methods on solution chemistry, nucleation and chiral discrimination provides methodological tools from which the occurrence of solid solutions can be rationalised.

Solid Separation from a Mixed Suspension through Electric-Field-Enhanced Crystallization.

When applied to a pure component suspension in an apolar solvent, a strong inhomogeneous electric field induces particle movement, and the particles are collected at the surface of one of the two electrodes. This new phenomenon was used to separately isolate two organic crystalline compounds, phenazine and caffeine, from their suspension in 1,4-dioxane. First, crystals of both compounds were collected at different electrodes under the influence of an electric field. Subsequent cooling crystallization enabled the immobilization and growth of the particles on the electrodes, which were separately collected after the experiment with purities greater than 91 %. This method can be further developed into a technique for crystal separation and recovery in complex multicomponent suspensions of industrial processes.

Measuring induction times and crystal nucleation rates.

A large variation is observed in induction times measured under equal conditions in 1 ml solutions. Ruling out experimental errors, this variation originates from the nucleation process. The induction time distribution is explained by the stochastic nature of nucleation if the number of nuclei formed is approaching 1 per vial. Accurate heterogeneous crystal nucleation rates were determined from the induction time distributions on a 1 ml scale for racemic diprophylline in two solvents. The difference in nucleation behaviour in the two solvents originates from the energy barrier for nucleation, which is much higher in the solvent in which induction times are much longer. In addition the pre-exponential factor for the crystal nucleation rate in both solvents is rather low compared to predictions using Classical Nucleation Theory. Unfortunately, concentration and surface characteristics of the effective heterogeneous particles are not known which clouds a further molecular interpretation.

Self-association during heterogeneous nucleation onto well-defined templates.

We investigated the interplay between self-associates in solution and surface templating by studying the crystallization behavior of isonicotinamide (INA) and 2,6-dihydroxybenzoic acid (DHB) in the presence of self-assembled monolayers (SAM). The end group of the SAM as well as the hydrogen-bonding capabilities of the solvent and self-association of INA and DHB were found to be important in polymorph crystallization on SAMs. In the case of INA in ethanol, both chain and dimer self-associates are present in the solution. In the absence of SAMs the polymorph form II (dimer structure) is the crystallization outcome. In ethanol the 4-mercaptopyridine and 4-mercaptobenzoic acid SAMs organize INA chain associates at the template surface and enable the crystallization of form I while the 16-mercaptohexadecanoic acid SAM results in the crystallization of form II. Raman spectroscopy suggests that molecular interactions between INA and the SAM are responsible for the formation of specific polymorphs. XRPD results in the identification of the orientation of the crystal on the surface that further verified the results obtained by Raman spectroscopy. In nitrobenzene and nitromethane INA associates in solution only as chains and crystallization results in the formation of form IV and form I, respectively (both chain forms). The crystals formed in the bulk solution and on SAMs were the same, which seems to indicate that the self-association in nitrobenzene and nitromethane is not influenced by the presence of templates. In the case of DHB in toluene and chloroform, all three SAMs nucleated only one type of polymorph (stable form 2). In the case of toluene the polymorphic outcome was stable form 2 instead of metastable form 1, which is favored in toluene in the absence of the SAMs. Again, Raman spectroscopy and XRPD suggest that DHB-SAM molecular interactions may be responsible for the formation of form 2.

Nucleation of organic crystals--a molecular perspective.

The outcome of synthetic procedures for crystalline organic materials strongly depends on the first steps along the molecular self-assembly pathway, a process we know as crystal nucleation. New experimental techniques and computational methodologies have spurred significant interest in understanding the detailed molecular mechanisms by which nuclei form and develop into macroscopic crystals. Although classical nucleation theory (CNT) has served well in describing the kinetics of the processes involved, new proposed nucleation mechanisms are additionally concerned with the evolution of structure and the competing nature of crystallization in polymorphic systems. In this Review, we explore the extent to which CNT and nucleation rate measurements can yield molecular-scale information on this process and summarize current knowledge relating to molecular self-assembly in nucleating systems.

Machine Learning-Derived Correlations for Scale-Up and Technology Transfer of Primary Nucleation Kinetics.

Scaling up and technology transfer of crystallization processes have been and continue to be a challenge. This is often due to the stochastic nature of primary nucleation, various scale dependencies of nucleation mechanisms, and the multitude of scale-up approaches. To better understand these dependencies, a series of isothermal induction time studies were performed across a range of vessel volumes, impeller types, and impeller speeds. From these measurements, the nucleation rate and growth time were estimated as parameters of an induction time distribution model. Then using machine learning techniques, correlations between the vessel hydrodynamic features, calculated from computational flow dynamic simulations, and nucleation kinetic parameters were analyzed. Of the 18 machine learning models trained, two models for the nucleation rate were found to have the best performance (in terms of % of predictions within experimental variance): a nonlinear random Forest model and a nonlinear gradient boosting model. For growth time, a nonlinear gradient boosting model was found to outperform the other models tested. These models were then ensembled to directly predict the probability of nucleation, at a given time, solely from hydrodynamic features with an overall root mean square error of 0.16. This work shows how machine learning approaches can be used to analyze limited datasets of induction times to provide insights into what hydrodynamic parameters should be considered in the scale-up of an unseeded crystallization process.

A workflow for the development of template-assisted membrane crystallization downstream processing for monoclonal antibody purification.

Monoclonal antibodies (mAbs) are commonly used biologic drugs for the treatment of diseases such as rheumatoid arthritis, multiple sclerosis, COVID-19 and various cancers. They are produced in Chinese hamster ovary cell lines and are purified via a number of complex and expensive chromatography-based steps, operated in batch mode, that rely heavily on protein A resin. The major drawback of conventional procedures is the high cost of the adsorption media and the extensive use of chemicals for the regeneration of the chromatographic columns, with an environmental cost. We have shown that conventional protein A chromatography can be replaced with a single crystallization step and gram-scale production can be achieved in continuous flow using the template-assisted membrane crystallization process. The templates are embedded in a membrane (e.g., porous polyvinylidene fluoride with a layer of polymerized polyvinyl alcohol) and serve as nucleants for crystallization. mAbs are flexible proteins that are difficult to crystallize, so it can be challenging to determine the optimal conditions for crystallization. The objective of this protocol is to establish a systematic and flexible approach for the design of a robust, economic and sustainable mAb purification platform to replace at least the protein A affinity stage in traditional chromatography-based purification platforms. The procedure provides details on how to establish the optimal parameters for separation (crystallization conditions, choice of templates, choice of membrane) and advice on analytical and characterization methods.

Manufacture of tablets with structurally-controlled drug release using rapid tooling injection moulding.

With advancements in the pharmaceutical industry pushing more towards tailored medicines, novel approaches to tablet manufacture are in high demand. One of the main drivers towards micro-scale batch production is the ability to fine-tune drug release. This study demonstrates the use of rapid tooling injection moulding (RTIM) for tablet manufacture. Tablets were manufactured with varying structural features to alter the surface area whilst maintaining the same volume, resulting in differing specific surface area (SSA). The precision of this technique is evaluated based on eleven polymer formulations, with the tablets displaying <2% variability in mass. Further tablets were produced containing paracetamol in three different polymer-based formulations to investigate the impact of SSA on the drug release. Significant differences were observed between the formulations based on the polymers polyvinyl alcohol (PVA) and Klucel ELF. The polymer base of the formulation was found to be critical to the sensitivity of the drug release profile to SSA modification. The drug release profile within each formulation was modified by the addition of structural features to increase the SSA.

Comparing and Quantifying the Efficiency of Cocrystal Screening Methods for Praziquantel.

Pharmaceutical cocrystals are highly interesting due to their effect on physicochemical properties and their role in separation technologies, particularly for chiral molecules. Detection of new cocrystals is a challenge, and robust screening methods are required. As numerous techniques exist that differ in their crystallization mechanisms, their efficiencies depend on the coformers investigated. The most important parameters characterizing the methods are the (a) screenable coformer fraction, (b) coformer success rate, (c) ability to give several cocrystals per successful coformer, (d) identification of new stable phases, and (e) experimental convenience. Based on these parameters, we compare and quantify the performance of three methods: liquid-assisted grinding, solvent evaporation, and saturation temperature measurements of mixtures. These methods were used to screen 30 molecules, predicted by a network-based link prediction algorithm (described in Cryst. Growth Des. 2021, 21(6), 3428-3437) as potential coformers for the target molecule praziquantel. The solvent evaporation method presented more drawbacks than advantages, liquid-assisted grinding emerged as the most successful and the quickest, while saturation temperature measurements provided equally good results in a slower route yielding additional solubility information relevant for future screenings, single-crystal growth, and cocrystal production processes. Seventeen cocrystals were found, with 14 showing stability and 12 structures resolved.

Template-Assisted Crystallization Behavior in Stirred Solutions of the Monoclonal Antibody Anti-CD20: Probability Distributions of Induction Times.

We present a method to determine the template crystallization behavior of proteins. This method is a statistical approach that accounts for the stochastic nature of nucleation. It makes use of batch-wise experiments under stirring conditions in volumes smaller than 0.3 mL to save material while mimicking larger-scale processes. To validate our method, it was applied to the crystallization of a monoclonal antibody of pharmaceutical interest, Anti-CD20. First, we determined the Anti-CD20 phase diagram in a PEG-400/Na2SO4/water system using the batch method, as, to date, no such data on Anti-CD20 solubility have been reported. Then, the probability distribution of induction times was determined experimentally, in the presence of various mesoporous silica template particles, and crystallization of Anti-CD20 in the absence of templates was compared to template-assisted crystallization. The probability distribution of induction times is shown to be a suitable method to determine the effect of template particles on protein crystallization. The induction time distribution allows for the determination of two key parameters of nucleation, the nucleation rate and the growth time. This study shows that the use of silica particles leads to faster crystallization and a higher nucleation rate. The template particle characteristics are shown to be critical parameters to efficiently promote protein crystallization.

Cocrystals of Praziquantel: Discovery by Network-Based Link Prediction.

Cocrystallization has been promoted as an attractive early development tool as it can change the physicochemical properties of a target compound and possibly enable the purification of single enantiomers from racemic compounds. In general, the identification of adequate cocrystallization candidates (or coformers) is troublesome and hampers the exploration of the solid-state landscape. For this reason, several computational tools have been introduced over the last two decades. In this study, cocrystals of Praziquantel (PZQ), an anthelmintic drug used to treat schistosomiasis, are predicted with network-based link prediction and experimentally explored. Single crystals of 12 experimental cocrystal indications were grown and subjected to a structural analysis with single-crystal X-ray diffraction. This case study illustrates the power of the link-prediction approach and its ability to suggest a diverse set of new coformer candidates for a target compound when starting from only a limited number of known cocrystals.

Effect of Chirality on the Compression of 2-(2-Oxo-1-pyrrolidinyl)butyramide: A Tale of Two Crystals.

Understanding polymorphism in chiral systems for drug manufacturing is essential to avoid undesired therapeutic effects. Generally, polymorphism is studied through changes in temperature and solution concentration. A less common approach is the application of pressure. The goal of this work is to investigate the effect of pressure on levetiracetam (pure enantiomer) and etiracetam (racemic compound). Anisotropic compressions of levetiracetam and etiracetam are observed to 5.26 and 6.29 GPa, respectively. The most compressible direction for both was identified to be perpendicular to the layers of the structure. Raman spectroscopy and an analysis of intermolecular interactions suggest subtle phase transitions in levetiracetam (∼2 GPa) and etiracetam (∼1.5 GPa). The stability of etiracetam increases with respect to levetiracetam on compression; hence, the chiral resolution of this system is unfavorable using pressure. This work contributes to the ongoing efforts in understanding the stability of chiral systems.