Co-Crystallization as a Separation Technology: Controlling Product

Feb 5, 2010 - Co-crystallization is known as a product formulation technology, but it can also be used as a tool to solve crystallization problems. Pr...
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DOI: 10.1021/cg9010778

Co-Crystallization as a Separation Technology: Controlling Product Concentrations by Co-Crystals

2010, Vol. 10 1171–1179

Johan Urbanus,*,† C. P. Mark Roelands,† Dirk Verdoes,† Peter J. Jansens,‡ and Joop H. ter Horst‡ †

Netherlands Organization for Applied Scientific Research (TNO), Department of Separation Technology, P.O. Box 6012, 2600 JA Delft, the Netherlands, and ‡Intensified Reaction & Separation Systems, Process & Energy Laboratory, Delft University of Technology, Leeghwaterstraat 44, 2628 CA Delft, the Netherlands Received September 4, 2009; Revised Manuscript Received December 18, 2009

ABSTRACT: Co-crystallization is known as a product formulation technology, but it can also be used as a tool to solve crystallization problems. Product removal by co-crystallization in fermentations is used as a showcase to demonstrate the potential of co-crystallization as a separation technique. In fermentations, the product solubility is often higher than the limiting concentration. Hence, the solubility should be decreased to below this limiting concentration to enable product removal by crystallization. Co-crystallization has the potential to accomplish this goal since co-crystals can have solubilities lower than pure component solubilities. Cinnamic acid (CA) forms co-crystals with 3-nitrobenzamide (NBA). The phase diagram of CA, NBA, and water shows that CA-NBA co-crystals can be formed at CA mole fractions seven times below the solubility of pure CA, facilitating the removal of CA from the solution at concentrations well below the pure CA product solubility. Co-crystal finetuning may lead to a substantially extended decrease of the CA co-crystal product solubility, so that product removal by cocrystallization can take place at even lower product concentrations. Co-crystallization experiments in a simulated fed-batch fermentation were performed by slowly adding a saturated solution of CA, simulating the production of CA in the fermentation process, to a suspension of pure co-former NBA. In time, the co-former crystals completely transform to CA-NBA co-crystals. During the transformation, a stationary state is achieved in which the CA and NBA mole fractions are constant because the added CA, together with the NBA from the dissolving co-former crystals, is captured in the co-crystals. In the stationary state, the process is operated near the 3-phase equilibrium point with equilibrium between co-crystals, co-former crystals, and solution. Co-crystals thus not only offer a route toward fine-tuned crystal properties of active pharmaceutical ingredients. This paper shows that, moreover, co-crystals offer solutions to crystallization problems.

Introduction Co-crystallization is an increasingly popular research topic for the pharmaceutical industry, since it offers the opportunity to improve and tailor physicochemical properties of pharmaceuticals.1 A co-crystal is defined as a crystal that is built up out of two or more organic compounds, that are, in their pure forms, solid under ambient conditions.2,3 Examples of improved properties as a result of co-crystallization are shelf life,4 dissolution rate,5 and bioavailability.6,7 Additionally, the solubility of co-crystals is generally distinct from that of the pure components.8-10 The modified co-crystal solubility can facilitate crystallization at decreased concentrations where pure components do not crystallize. Furthermore, co-crystal systems introduce an added degree of freedom, which is the concentration or mole fraction of the co-former: the product solubility can be tuned with the amount of co-former added to the systems. This feature enables crystallization at conditions below pure component solubilities, and therefore, co-crystallization can offer solutions to crystallization problems. An example of such a problem is encountered in (bio) chemistry. Chemical reactions can be limited by equilibrium,11 while biobased processes, where sustainable feedstock is transformed into products using enzymatic or whole-cell conversions, can be limited by enzyme inhibition and/or cell toxicity at high product concentrations.12,13 For the optimization of such reactions, it would be beneficial to isolate the *Corresponding author. E-mail: [email protected]. r 2010 American Chemical Society

product as soon as it is formed. This is of particular interest in biobased processes, where product removal alleviates inhibition and toxicity issues which results in increased volumetric productivities and yield. In principle, crystallization can be used to isolate the product.14-17 Often, however, the product solubility is higher than the limiting product concentration18 and crystallization does not occur. To facilitate product removal by crystallization, the product solubility has to be decreased below the limiting product concentration, for example, the product concentration at which enzymes are inhibited in fermentation processes. This can be achieved by isolating the product in the form of a salt or a co-crystal rather than the use of pure component crystals. By carefully selecting the counterion or co-former, the solubility of such a complex can be tuned to the desired value. Conventionally, calcium has predominantly been selected as counterion for product isolation (e.g., calcium citrate19). However, for other interesting chemical building blocks produced by fermentation, such as fumaric acid, the solubility of the salt exceeds the solubility of the neutral component (21.120 > 721 g 3 L-1). Consequently, in those cases, calcium cannot be used to achieve product removal. In principle, a suitable quaternary amine can be selected with which the product solubility can be decreased below the limiting product concentration, such that product removal by crystallization is established. Generally, however, regeneration of the salt to obtain the pure product implies recrystallization with an acid. This results in the formation of an undesired conjugated waste salt (the anion from the acid forms a complex with the cation Published on Web 02/05/2010

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Figure 1. Molecular structure of cinnamic acid (CA, left) and 3-nitrobenzamide (NBA, right). The carboxylic acid of CA and the amide of NBA can form the well-known amide-carboxylic acid synthon.

Figure 2. Schematic representation of a ternary phase diagram of fermentation product A and co-former B. A solution composition in region LþAB eventually results in equilibrium between the solution and AB co-crystals. In regions LþAþAB and LþBþAB, the solution comes into equilibrium with AB co-crystals and the solid phase of A or B, respectively. Solution compositions move to the 3phase equilibrium points indicated with blue dots. These points are intercepts of the co-crystal solubility line (xA 3 xB)* with lines of pure component solubilities (xA*, xB*).

selected for product removal). The use of co-crystallization also requires a regeneration of the co-crystal to obtain the pure product. Since neutral molecules rather than ions are used as the co-former,22 regeneration of the co-crystal provides both the product and the co-former, such that the production of a waste salt is prevented. Therefore, co-crystallization has the advantage over the use of salts for product isolation. We demonstrate the potential of co-crystallization for the model system cinnamic acid (CA - Figure 1). This compound can be produced by fermentation.23 It is furthermore known that CA and 3-nitrobenzamide (NBA - Figure 1) co-crystallize to form a 1:1 CA-NBA co-crystal.10 First, we determine the solubility decrease for the co-crystal compared to the pure component crystals of CA. Then we show in a simulated fermentation experiment that the solubility decrease can be maintained. Theory Figure 2 presents a schematic isothermal phase diagram for product A and co-former B in a solvent. The straight vertical and horizontal lines indicated with xA* and xB* represent solubility lines of A and B, respectively, while the curved line (xA 3 xB)* represents the solubility line of the AB co-crystal. The other solid lines indicate boundaries between compositional regions in which different equilibria are established. A solution composition in region LþA, LþB, or LþAB will eventually equilibrate to a position on the xA*, xB*, or (xA 3 xB)* solubility line. A solution composition in region

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LþAþAB or LþBþAB will equilibrate in one of the 3-phase equilibrium points (indicated by a blue dot). These are the solution compositions at which equilibrium can be established between the solution, the co-crystals, and one of the pure component crystalline phases. The apparent solubility xA# of product A is defined as the mole fraction at which the solution is in equilibrium with a solid phase containing A. This solid phase can either be pure crystals of product A or AB co-crystals including component A. At small fractions of B, up to the 3-phase equilibrium point at the right, the apparent solubility is equal to the pure component solubility (xA# = xA*). At higher fractions of B within zone LþAB, xA# decreases and is inversely proportional to the fraction of B via the solubility product of the AB co-crystal. This fraction of B has an upper limit due to the solubility of co-former B at the 3-phase equilibrium point at the left. Consequently, the corresponding apparent solubility of product A is at its minimum (xA#,min, see dashed arrow in Figure 2 pointing to blue square). The phase diagram shows that, as a result of co-crystallization, it should be possible to establish product concentrations below the pure component solubility in fermentation processes, when these are operated at the 3-phase equilibrium point. The diagonal dashed line in Figure 2 is the operating line of co-crystallization during a fermentation process; it describes a possible trajectory of overall mole fractions (including solids) of fermentation product A and co-former B. At the start of the fermentation, xA equal to zero, a certain amount of co-former is added to the fermentation broth, represented by the green dot at the co-former axis. xA increases as a result of the biological conversion of feedstock into product. Co-crystallization of the fermentation product starts as soon as xA exceeds xA#,min. When the overall composition enters region LþAB, xB becomes smaller than xB* which causes xA# to increase. Cocrystallization proceeds as the conversion of feedstock into product is continued. Eventually, the solution composition moves along the solubility lines toward the product axis (xB ∼ 0 and xA ∼ 1). In this article, a fed-batch fermentation with co-crystallization is experimentally simulated, to show that the solubility decrease as defined in the phase diagram can be maintained during such fermentation processes. For this purpose, a reactor containing a co-former suspension (composition represented by the green dot) resembles the fermenter where micro-organisms convert the feedstock into product. The actual production was simulated by the continuous addition of a solution saturated with the fermentation product (composition represented by the red dot). Consequently, the final solution composition will, after infinite dilution, become equal to the solubility of pure product A. In these experiments, the overall composition moves along the operating line (dash-dot). A simple equilibrium based model was developed to describe the course of these simulated fed-batch (SFB) experiments. The mole balance for fermentation product A, accounting for the continuous addition of A and the formation of AB co-crystals, is given by MA ðtÞ ¼ MA ðt -1Þ þ xA cT φfeed Δt -MAB ðtÞ

ð1Þ

where MA is the amount (mol) of product A present in the reactor, xA the mole fraction of product A and φfeed the flow (m3 3 s-1) of the saturated solution. cT is the total concentration (5.55  104 mol 3 m-3) in the system, consisting of the solvent (water), A, B, and AB. The small contributions of A, B, and AB to the total concentration are neglected. With a 1:1

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stoichiometric AB co-crystal, the amounts (mol) of product A and co-former B inside the co-crystal are equivalent and equal to the amount (mol) of co-crystalline material formed (MAB). The mole balance for co-former B is given by MB ðtÞ ¼ MB ðt -1Þ -MAB ðtÞ

ð2Þ

where MB is the amount (mol) of co-former B present in the reactor. MAB can be calculated from Δx of either one of the involved components due to the 1:1 stoichiometry of the AB co-crystal. Our approach, in accordance with the phase diagram, is that xB determines xA# via the solubility product K (K = xA* 3 xB*). Then xB# is equal to xB. Therefore, MAB can be calculated using ΔxA:   ð3Þ MAB ðtÞ ¼ VðtÞcT xA ðtÞ -x#A ðtÞ where V is the total volume (m3) of the solution inside the reactor, xA is the actual mole fraction (mol 3 mol-1) of fermentation product A and xA# is the apparent solubility. The total volume increases by the addition of feed: ð4Þ VðtÞ ¼ Vðt -1Þ þ φfeed Δt The actual mole fraction of A is a function of MA, V, and cT: MA ðtÞ ð5Þ xA ðtÞ ¼ VðtÞcT The apparent solubility of fermentation product A is determined by xB as stated above: K ð6Þ x#A ðtÞ ¼ xB ðtÞ where K is the solubility product (mol2 3 mol-2) of the cocrystal, while xB is calculated like eq 5: xB ðtÞ ¼

MB ðtÞ VðtÞcT

ð7Þ

The two 3-phase equilibrium points define boundary conditions of mole fractions of A and B. When the outcome of eq 7 exceeds the co-former pure component solubility (xB > xB*), at low fractions of fermentation product, xB = xB*. Accordingly, the upper limit of xA# is the solubility of component A. This equilibrium based model was used to explain our experimental results and proved to be useful in understanding effects of co-former mole fraction and simulated product formation rates. Experimental Section Cinnamic acid (99% purum) and 3-nitrobenzamide (98%) were purchased from Sigma-Aldrich and used as received. Demineralized water and 96% ethanol were used as solvents. X-ray Powder Diffraction (XRPD). XRPD patterns were recorded on a Bruker GADDS diffractometer equipped with a Hi-Star area detector. The pattern was calibrated using silver behenate for the long d-spacing and corundum for the short d-spacing. Data collection was carried out at room temperature using monochromatic Cu KR radiation (λ = 1.5418 A˚) in the 2θ region between 1.5° and 41.5°. Phase Diagram. CA and NBA are slightly soluble in water. Pure component solubilities in water at 30 °C were determined by equilibrating an excess amount of pure component with water. Equilibrium was assumed to be established overnight. 0.45 μm filters (Whatson) were used to obtain solid-free samples. These samples were instantaneously diluted with demineralized water to prevent cooling crystallization of the pure components. Concentrations of CA and NBA were measured with HPLC at 278 nm using a C18 column. Retention times (flow 1.5 mL 3 min-1) were approximately 2.5 and 4 min for NBA and CA, respectively. This method was also used to determine the phase diagram of the co-crystal

Figure 3. XRPD patterns of CA-NBA co-crystals in ethanol10 (top) and water (bottom). system CA-NBA in water of 30 °C. For this purpose, different ratios of CA over NBA, ranging from low to high CA mole fractions with corresponding high to low NBA mole fractions, were used to obtain a good scatter of experimental data. Solvent-Mediated Transformation. Images of co-crystals in ethanol and water were recorded following procedures of solventmediated transformation experiments.24,25 For co-crystallization in ethanol 28 mmol 3 mol-1 CA (approximately 72 mg 3 mL-1) and 4.4 mmol 3 mol-1 NBA (approximately 13 mg 3 mL-1) was used. Crystals were directly observed. In water 0.07 mmol 3 mol-1 CA (approximately 0.57 mg 3 mL-1) and 0.15 mmol 3 mol-1 NBA (approximately 1.5 mg 3 mL-1) was used to establish the solventmediated transformation. To obtain conditions where co-crystallization could well be observed, the suspension in water was heated to dissolve all the NBA crystals and subsequently cooled to obtain the co-crystals. Simulated Fed-Batch (SFB) Experiment. A CA fermentation process was experimentally simulated by the dropwise addition (3.2 mL 3 min-1) of a saturated CA solution (0.069 mmol 3 mol-1, 0.57 g 3 L-1) to a suspension of 1, 1.25, or 1.5 g of NBA in 100 mL of water of 30 °C. Solution samples were taken in time using 0.45 μm filters, and the CA and NBA concentrations were determined using HPLC. Additionally, using Pasteur pipettes, suspension samples were obtained in time, which where directly placed under a light microscope to collect pictures of the solid phase during these SFB experiments. Similar SFB experiments using a Raman probe (Hololab 5000 series of Kaiser optics) were performed to follow the transformation from a co-former to a co-crystal suspension. According to Ono et al,26 the in situ Raman technique is well suited to observe solvent-mediated transformations. Peaks at 1575 and 1633 cm-1 were representative for solid NBA and solid CA-NBA, respectively. The solid fraction, as plotted in Figure 6b, was obtained by dividing the area of either NBA or CA-NBA by the total area of those two peaks. These in situ Raman measurements were validated by offline XRPD measurements, to confirm the solid-state transition. For this purpose, SFB experiments were halted at approximately 35 and 100 min. Solids were collected by filtration of the complete suspension using 0.45 μm filters and XRPD patterns of collected solids were recorded.

Results The first section investigates the existence of CA-NBA cocrystals in aqueous solutions. In the second section, the phase

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Figure 4. Optical microscopy images of CA-NBA co-crystals from ethanol (left) and water (right). The CA-NBA co-crystals prepared in ethanol are more platelike while those prepared from water are more needlelike.

diagram of the CA-NBA co-crystal system in water is described. Proof-of-principle of product removal by co-crystallization is the subject of the third section, which demonstrates that product removal by co-crystallization is feasible at product concentrations well below the pure CA solubility. CA-NBA Co-Crystals. Recently, it was shown that CA and NBA form co-crystals in ethanol.10 It is not evident that these co-crystals are formed or are stable in water, since solvate crystals can be formed that might be more stable than the co-crystal. To establish the existence of CA-NBA cocrystals in water, the crystalline product from solutions of CA and NBA in water was investigated. Figure 3 compares the XRPD patterns of CA-NBA co-crystals formed in ethanol10 and water (the overall solution composition in the phase diagram is indicated by the diamond in Figure 5). The patterns of the co-crystals from both solvents are identical, which demonstrates that co-crystals formed in water have the same crystal structure as those formed in ethanol. Figure 4 shows optical microscopy images of co-crystals from both solvents. The co-crystal morphology from ethanol is platelike while that from water is more needlelike. This might be caused by different growth rates of crystal faces that determine the morphology of the co-crystal. Generally, growth rates of crystal faces are affected by solvent choice (ethanol versus water) and supersaturation (S), which was slightly higher in the case of the solvent water (S = 1.8 > S = 1.3, calculated with the solubility products 0.00346 and 69.9 mmol2 3 mol-2 for water and ethanol,10 respectively). These initial supersaturations were calculated from the ratio of the product of mole fractions and the solubility product. The Phase Diagram of CA-NBA Co-Crystals in Water. To determine the decrease in apparent solubility of CA when

using co-crystals, the phase diagram of CA, NBA, and water was determined. Figure 5 shows the ternary phase diagram of CA and NBA in water measured at 30 °C. The lines indicated with xCA* and xNBA* represent the solubility lines of pure CA and NBA, respectively. At 30 °C CA has an experimentally determined solubility of 0.069 mmol 3 mol-1 and NBA has a solubility of 0.357 mmol 3 mol-1 in water. The solubility line of the co-crystal, indicated by (xCA 3 xNBA)*, was drawn with the experimentally determined solubility product K = (xCA 3 xNBA)* = 0.00346 ( 0.00053 mmol2 3 mol-2 (experimental points represented by black squares). The low pure component solubility in water is responsible for the difference between the general phase diagram depicted in Figure 2 and the specific phase diagram of CA-NBA co-crystals presented in Figure 5. The acronyms S, A, and B in Figure 5 refer to the vertices of the ternary phase diagram that correspond to pure solvent, product, and co-former. In region L, the solution is the single stable phase. In regions LþCA, LþNBA, and LþCA-NBA, an equilibrium between the solution and respectively CA, NBA, or CANBA crystals will be established. A solution composition will eventually equilibrate to a position on the CA (xCA*), NBA (xNBA*), or CA-NBA (xCA 3 xNBA)* line. In regions LþCAþCA-NBA and LþNBAþCA-NBA, the solution will equilibrate to the 3-phase equilibrium point, indicated by the blue dots in Figure 5. In these points, where co-crystal and pure component solubility lines intercept, the clear solution L, the co-crystal CA-NBA, and one of the pure component crystalline phases (CA or NBA) are in equilibrium. This is illustrated by the black dot, representing a composition in region LþNBAþCA-NBA, that equilibrates to the 3-phase equilibrium point with the solution, cocrystals CA-NBA and pure component NBA. The dashed

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Figure 5. The ternary phase diagram at 30 °C of cinnamic acid (CA) and 3-nitrobenzamide (NBA) in water. Solid lines indicated with xCA* and xNBA* are solubility lines corresponding to experimentally determined pure component solubilities. The solid line indicated with (xCA 3 xNBA)* is the co-crystal solubility line drawn with the experimentally determined solubility product K. Other solid lines define boundaries between compositional regions in which different equilibria are established: region L: the solution is the singe stable phase; regions L+CA, L+NBA, and L+CA-NBA: equilibrium between solution and crystals of respectively CA, NBA, or CA-NBA co-crystals; regions L+CA+CA-NBA and L+NBA+ CA-NBA: equilibrium between solution, co-crystals and crystals of one of the pure component crystalline phases (CA or NBA). The blue dots indicate the solution compositions of the 3-phase equilibrium points. The blue square represents the minimum value of the apparent solubility xCA#,min. The black dot refers to a composition that equilibrates with its corresponding 3-phase equilibrium point. Dashed arrows indicate that crystallization of NBA and the co-crystal occur simultaneously. The open diamond in region LþCA-NBA indicates the solution composition from which co-crystals were grown for the XRPD analysis depicted in Figure 3. The dashed line represents the operating line of the SFB experiment. The green dot at xNBA = 1 mmol 3 mol-1 indicates the overall mole fraction of NBA at the start of the experiment. The red dot at xCA = 0.069 mmol 3 mol-1 is the CA mole fraction in the solution continuously fed to the suspension. Four stages are identified as the operating line crosses four different regions of the phase diagram. A description of these stages is given in the section Product Removal by Co-Crystallization.

arrow that connects the black dot with the corresponding 3-phase equilibrium point is split into two parts to indicate that co-crystals are formed according to the 1:1 stoichiometry while simultaneously NBA crystals are formed (vertical dashed arrow). The phase diagram, with its compositional regions, defines the operating window for co-crystallization of CA and NBA at 30 °C. It shows whether and which crystalline material is created from each region, together with the corresponding solution composition. According to the theoretical section, the apparent CA solubility xCA# = K/xNBA (eq 6) can be tuned with the coformer mole fraction xNBA to have values between those in the two 3-phase equilibrium points. At small fractions of NBA, up to a value of 0.0498 mmol 3 mol-1 at the 3-phase equilibrium point, the apparent solubility xCA# equals the pure component solubility of CA (xCA# = xCA* = 0.069 mmol 3 mol-1). At

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higher NBA fractions, limited by the pure component solubility of NBA, xCA# decreases. The vertical dashed arrow in Figure 5 connects the 3-phase equilibrium point, the upper limit of xNBA, with the blue square, indicating the minimal apparent CA solubility xCA#,min=K/xNBA*=0.0097 mmol 3 mol-1. The minimal apparent solubility xCA#,min of CA is therefore approximately seven times lower than the pure component solubility xCA* of cinnamic acid. Product Removal by Co-Crystallization. The goal of this research is to establish product removal by co-crystallization at concentrations well below the pure component solubility xCA*. This could be achieved if co-crystallization can be performed at CA mole fractions around the minimum apparent solubility xCA#,min. To achieve these CA mole fractions, a NBA mole fraction of around the solubility value xNBA* should be present. This is the case around the 3-phase equilibrium point where NBA crystals, co-crystals, and solution are in equilibrium. When co-crystallizing near the 3-phase equilibrium point both CA and NBA are removed from the solution and the solution mole fractions decrease. In order to maintain the mole fractions at levels near the 3-phase equilibrium point both CA and NBA have to be provided to the solution. In fermentations, CA would be produced by the biocatalyst, while NBA would have to be added. By using a suspension of NBA crystals, exactly this would be achieved. The suspended NBA crystals act as a NBA buffer. The biocatalyst produces CA which co-crystallizes with NBA in the solution. The decreased levels of NBA are increased again by dissolution of NBA crystals from the NBA buffer. Both CA and NBA mole fractions are then maintained at a constant level near the 3-phase equilibrium point. This was tested in an experimentally simulated fermentation process, intended to simulate a fed-batch fermentation. The reactor containing the NBA suspension represents the STR in which the fed-batch fermentation is performed. The SFB experiments started with an NBA suspension having an overall NBA fraction xNBA = 1 mmol 3 mol-1. This value is indicated by the green dot in the phase diagram in Figure 5. A saturated CA solution, xCA*= 0.069 mmol 3 mol-1, was added dropwise to the NBA suspension, representing the CA production by the biocatalyst as well as the addition of feed. The red dot in Figure 5 indicates the mole fraction of CA in the feed. The dashed line in Figure 5 is the operating line which describes the overall composition in the STR during the SFB experiment. At the start with a suspension of NBA crystals, the overall composition is located at the upper left part of the dashed line. During the process, the overall CA mole fraction increases due to addition of the saturated CA solution, while the overall NBA mole fraction decreases due to dilution of the NBA suspension with the saturated CA solution. From the position of the operating line in the phase diagram, four stages can be identified in the SFB experiment. Starting at the NBA suspension without the presence of CA to a saturated CA solution, the operating line passes through the regions LþNBA (stage 1), LþNBAþCA-NBA (stage 2), LþCA-NBA (stage 3), and L (stage 4), respectively . In stage 1, NBA is the most stable crystalline phase. Stage 1 starts with a pure NBA suspension in water. If we assume equilibrium between the suspended NBA crystals and the solution, then the mole fraction of NBA in solution xNBA = xNBA*. Upon the addition of saturated CA solution, the CA

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mole fraction xCA increases, while NBA crystals dissolve to maintain a saturated NBA solution with a mole fraction xNBA = xNBA*. The solution composition therefore would follow the solubility line xNBA* toward larger CA mole fractions until reaching the 3-phase equilibrium point where stage 2 starts. Stage 2 takes place in the region of the phase diagram where three phases can co-exist: solution, NBA crystals, and CA-NBA co-crystals. The addition of CA causes the CA mole fraction to rise which in turn is consumed by the formation of co-crystals. The NBA mole fraction is decreasing not only because of the dilution due to the addition of saturated CA solution but also due to the removal of NBA by co-crystallization. The decrease of xNBA is counteracted by the dissolution of NBA crystals acting as a NBA buffer, such that during stage 2 a stationary state is achieved where mole fractions of CA and NBA are maintained at the 3-phase equilibrium point (xCA = xCA#,min and xNBA = xNBA*). Stage 3 starts when the NBA buffer is completely depleted. In stage 3, the co-crystal is the stable crystalline phase. As the addition of saturated CA solution continues, the consumption of NBA, as a result of dilution and co-crystallization, is no longer counteracted since the NBA buffer is exhausted. Consequently, the mole fraction of NBA decreases. This affects the apparent CA solubility via the solubility product (xCA# = K/xNBA): as xNBA decreases, xCA# increases. The solution composition will follow the solubility line up to the point where the operating line intersects with the solubility line. This intersection marks the start of stage 4. In stage 4, the clear solution is the most stable phase. The original suspension from previous stages is diluted with saturated CA solution to such extent that all mole fractions are below their apparent solubilities. Eventually, with continued CA addition, when the suspension present at the start of the SFB experiment is infinitely diluted, the solution composition becomes equal to the red dot in Figure 5, which is the composition of the saturated CA solution itself. Figures 6-8 show that these stages, as deducted from the phase diagram and the operating line of the SFB experiment, can be recognized in the experimental results. Figure 6a presents the solution composition during the SFB experiment in time. Open and closed triangles are experimental data for the CA and NBA mole fractions xCA and xNBA, respectively, while the solid lines are calculated mole fractions using the model described in the theoretical section. The conversion in time of solid NBA into solid CA-NBA is monitored in situ by Raman measurements and given in Figure 6b. The solid transformation is furthermore confirmed by off-line XRPD measurements of solid samples collected during SFB experiments (Figure 7). Furthermore, microscope images of crystals observed in the different stages are given in Figure 8. In stage 1, see red triangles in Figure 6a, the mole fraction of NBA xNBA remained constant, while the mole fraction of CA xCA increased. The solution composition thus moved along the solubility line xNBA* toward the 3-phase equilibrium point. Raman (Figure 6b) and XRPD (Figure 7) analysis confirmed that NBA was the only crystalline phase in stage 1. Figure 8a shows NBA crystals collected during stage 1. Initially, in stage 2 (blue triangles) co-crystals were not present (see Figure 6b) and the CA mole fraction xCA increased to above the minimum value of the apparent CA

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Figure 6. (a) Solution composition as a function of time of the SFB experiment. Open and closed triangles represent experimental mole fractions of CA and NBA, respectively, while solid lines are calculated using a mass balance and the co-crystal solubility. The horizontal parts in these solid lines refer to the minimal apparent solubility xCA#, min of CA and the pure component solubility xNBA* of NBA. Numbers 1-3 refer to the stages in the SFB experiments. See text for a detailed description. (b) Solid composition in the suspension as a function of time of the SFB experiment. In stage 1 a suspension of pure NBA crystals is present, in stage 2 these NBA crystals are transformed into co-crystals, and in stage 3 a suspension of pure co-crystals is present. The solid composition was determined using in situ Raman spectroscopy.

solubility xCA#,min. This behavior is expected since a certain minimum supersaturation is required to induce nucleation and growth of the co-crystal. The equilibrium based model, with which the lines in Figure 6a are constructed, predicts the conversion of NBA into CA-NBA taking place as soon as xCA increases above xCA#,min. In the SFB experiment however, the first co-crystals were only observed after 15 min when the CA mole fraction reached a value of xCA = 0.024 mmol 3 mol-1. Once co-crystals were formed (as observed with Raman and microscopy and indicated in Figure 6a with an arrow), the CA concentration decreased to slightly above the minimal apparent CA solubility xCA#, min. Raman analysis shows that during stage 2 NBA crystals dissolved, while the fraction of co-crystals increased. Additionally, the XRPD pattern of a sample from stage 2 proves that a mixture of NBA crystals and CA-NBA co-crystals was present. As a result of both co-crystallization and dissolution of the NBA buffer, the solution composition remained in the stationary state: constant mole fractions of CA and NBA located near the 3-phase equilibrium point (see horizontal lines in Figure 6a). Figure 8b shows a mixture of NBA crystals (rectangular shaped) and co-crystals (needle-like morphology). It appears

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Figure 7. XRPD patterns of identified stages in the SFB experiment. The pattern from stage 1 is identical to that of pure NBA crystals. The pattern from stage 2 is a mixture of NBA crystals and co-crystals. The pattern of stage 3 is identical to that of co-crystals as given in Figure 3. The patterns of both polymorphs of CA are provided in order to validate that the samples do not contain pure cinnamic acid crystals.

that these co-crystals might form at the surface of NBA crystals. This would be due to the high NBA concentration at the surface of the dissolving NBA crystals. The new co-crystal nuclei consume all the CA present in the vicinity of the NBA crystal preventing the formation of new nuclei on the surface of that NBA crystal. This might allow NBA to dissolve, such that the simultaneous dissolution of NBA crystals and the formation of CA-NBA co-crystals are guaranteed. The start of stage 3 (green triangles) is marked by the complete dissolution of NBA crystals (indicated by Raman analysis). Because of the continued addition of the saturated CA solution, solid NBA dissolves to compensate for the dilution and co-crystallization. Finally, when all NBA crystals are consumed, the NBA mole fraction xNBA starts to decrease while that of CA starts to increase as can be observed in Figure 6a. The solution composition moves along the solubility line (xCA 3 xNBA)* toward the 3-phase equilibrium point due to further addition of CA and consequent dilution of NBA. An XRPD pattern of a crystalline sample from stage 3 is identical to that of pure co-crystals. The XRPD measurement, which analyzes the complete crystal and not only its surface, therefore indicates that no NBA is present at the end of stage 3, proving that in this stage only co-crystals were present. A microscope image of pure cocrystals from stage 3 is depicted in Figure 8c. The experiment was stopped before the less interesting stage 4 was reached. The results prove that crystallization of CA into CA-NBA co-crystals can take place at CA concentrations seven times lower than the pure component CA solubility. Furthermore, the results showed that the solution composition remained in the stationary state near the 3-phase equilibrium point as long as NBA crystals were present in the suspension. Figure 9 shows three SFB experiments with different initial amounts of solid NBA (numbers account for both dissolved and solid

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NBA). Open and solid symbols represent mole fractions of CA and NBA, respectively. Triangles correspond to the SFB experiment displayed in Figure 6a, with an initial amount of 1 g of NBA. Diamonds and circles represent SFB experiments with initial amounts of 1.25 and 1.5 g of NBA, respectively. Solid lines refer to the equilibrium based model described by eqs 1-7, with different initial amounts of NBA. The dashed line refers to model calculations without NBA added to the system. Obviously, without NBA, co-crystallization would not take place and the CA mole fraction xCA increases and eventually becomes equal to the CA pure component solubility. These results show that higher amounts of initial NBA (green compared to black) resulted in prolonged periods of constant solution compositions at or close to the 3-phase equilibrium point with a minimum apparent CA concentration xCA#,min. It can therefore be concluded that low concentrations of CA can be established using co-crystallization, while a stationary state can be maintained when enough solid co-former is provided. NBA crystals thus act as a storage buffer, which indicates that the duration of the stationary state can be tuned with the initial amount of solid NBA. Discussion. Because of the application of co-crystallization with NBA as co-former, CA can be crystallized at concentrations seven times below its solubility. The minimum concentration for co-crystallization of CA is determined by the position of the 3-phase equilibrium point, which depends on the pure co-former solubility. Since there are many possible co-formers for the particular case of CA, fine-tuning of the crystalline phase may lead to substantially extended decreases of the concentration at which crystallization of CA occurs. The pure co-former solubility, therefore, is the first selection criterion for co-former screening. The SFB experiments were intended to simulate fed-batch fermentations. It is recognized that in actual fermentations, production and dilution are not coupled as in the SFB experiment. Obviously, the product is produced within the STR, while a limited amount of feed is used to provide extra substrate for the conversion of feedstock into product. For co-crystallization inside a fermenter, this would practically mean that less solid NBA is consumed to compensate for the dilution by the feed. Consequently, fewer NBA crystals are required to maintain low product concentrations within the fermenter. When co-crystallization is applied in continuous fermentations, NBA should be provided in the feed. This is because the initial excess of NBA will quickly be consumed due to continuous dilution by the feed and co-crystallization with the product. A second general selection criterion for co-formers in fermentations is related to the toxicity of the co-former toward the micro-organism producing the fermentation product. As co-crystallization is applied to minimize effects of product inhibition, the co-former that is used should not be toxic at all, since an excess amount of co-former is present to guarantee low product concentrations. The log P value of the co-former is a useful tool in evaluating the toxicity effect of the component on micro-organism.27,28 With product removal by co-crystallization, the desired product ends up in the co-crystal, which is suspended in the reaction mixture. For fermentations, this is a complex mixture of water, carbon-source, minerals, micro-organisms, crystalline co-former, and co-crystals. In order to obtain the desired product, the co-crystals should first be separated from this mixture. This can in principle be achieved with

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Figure 8. Optical microscopy images of crystalline samples from stage 1 (a), 2 (b), and 3 (c) in an SFB experiment. In stage 1, platelike NBA crystals are present, in stage 2 a mixture of NBA crystals and CA-NBA co-crystals (needles) coexist, and in stage 3 only CA-NBA co-crystals are present. The observed crystalline phases are confirmed by Raman and XRPD as shown in Figures 6b and 7.

of salts is generally conducted by recrystallization29 or electrodialysis.30 In principle, the regeneration of the product from the co-crystalline phase can be achieved by affinity separations or selective recrystallization. The separation of product and co-former is the subject of future research. Conclusion

Figure 9. Mole fractions of CA (open symbols) and NBA (solid symbols) in time during several SFB experiments with different initial amounts of NBA. Numbers given are total amounts: both dissolved (∼0.33 g) and solid NBA (∼0.67, 0.92, and 1.17 g). Solid lines represent the modeled solution compositions, according to eqs 1-7. The dashed line refers to a system without NBA, which would result in an increasing CA mole fraction xCA up to its pure component solubility.

rather straightforward solid-liquid separations that do account for (size-based) separation of co-crystals and microorganism. The morphology of co-crystals therefore is important, which is influenced by the co-former. Consequently, the co-crystal morphology is the third selection criterion for co-former screening. The required solid-solid separation of co-former crystals and co-crystals, following the solid-liquid separation, can be prevented with the use of some sort of filter bag that contains the excess of solid NBA. Additionally, this separation can be avoided with the proper selection of the initial amount of NBA, which should be such that the solution composition at the moment of product separation lies within stage 3, where all solid NBA is consumed. Furthermore, a separation of product and co-former is required. Nowadays, a lot of research is focused on acquiring new co-crystals, while little is known about the separation of co-crystals into their pure components. A fourth selection criterion for fine-tuning of the co-crystalline phase for application in processes therefore is the ease of separation of the co-crystal into its pure components. This separation has similarities with the breaking of salts, required to obtain the isolated product. The breaking

The 1:1 CA-NBA co-crystal discovered in ethanol was tested in the solvent water and proved to be the most stable crystalline phase considering equimolar amounts of pure components. The structure of co-crystals from water determined with XRPD was identical to the structure belonging to co-crystals crystallized in ethanol. The ternary phase diagram of CA, NBA, and water was determined. The phase diagram of CA-NBA co-crystals showed a 7-fold decrease of the apparent solubility of CA at solution compositions near the 3-phase equilibrium point. The apparent solubility of components can in general be decreased when a co-former suspension is present. The SFB experiment proved that low product concentrations were established by the application of co-crystallization at conditions near the 3-phase equilibrium point. Furthermore, low product concentrations could be maintained when enough solid co-former was present. Modeling and experimental results demonstrated that co-crystallization successfully prevented high product concentrations. In fermentations, this would prevent inhibition of the biocatalyst, thus improving the bioprocess. Selection criteria for co-former screening, to enable and improve product removal by co-crystallization, are the coformer pure component solubility, the toxicity of the coformer expressed as log P value, the morphology of the co-crystal, and the ease of separation of the co-crystal into pure product and pure co-former. These selection criteria, developed in the context of biobased processes, can easily be translated to the broader context of chemical reactions. Here, the application of co-crystallization can prevent equilibriumrelated limitations, thus improving the production process. Acknowledgment. This project is financially supported by the Netherlands Ministry of Economic Affairs and the B-Basic partner organizations (www.b-basic.nl) through B-Basic, a public-private NWO-ACTS programme (ACTS = Advanced Chemical Technologies for Sustainability). This research was made possible by a Casimir grant obtained by J.t.H. from NWO (Netherlands Organisation for Scientific Research). J.t.H. gratefully acknowledges the hospitality that he enjoyed at Avantium Technologies during the Casimir

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project. The authors thank Sukanya Srisanga, Sulivan Djamad, Jarek Mazurek, and Avantium Technologies for sample preparation and recording the XRPD patterns.

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