Comparative Study on Seeded and Unseeded Bulk Evaporative Batch

May 8, 2017 - ... of Lysozyme. Huaiyu Yang , Peter Peczulis , Pavan Inguva , Xiaoyu Li , Jerry Y.Y. Heng. Chemical Engineering Research and Design 201...
1 downloads 0 Views 4MB Size
Download PDF
Article pubs.acs.org/crystal

Comparative Study on Seeded and Unseeded Bulk Evaporative Batch Crystallization of Tetragonal Lysozyme Michael Barros Groß and Matthias Kind* Thermal Process Engineering, Karlsruhe Institute of Technology (KIT), Kaiserstraße 12, 76131 Karlsruhe, Germany S Supporting Information *

ABSTRACT: Seeding is highlighted as a promising tool to improve controllability, reproducibility, and quality of the process and of the product in bulk protein crystallization. In this experimental study, hen egg white lysozyme is crystallized from its buffered aqueous solution in a stirred tank by evaporative batch crystallization at low pressure. Experiments with and without the use of lysozyme seed crystals are evaluated and compared in terms of nucleation behavior, supersaturation evolution, and product quality. The supersaturations needed for spontaneous nucleation are shown to vary strongly in between the unseeded experiments, resulting in a large variation of the supersaturation evolutions during the experiments and the final crystal size distributions. In contrast, seeding is proved to lead to controlled and reproducible secondary nucleation. The reproducibility of the product crystal size distribution is demonstrated to be dramatically increased by seeding compared to the unseeded experiments. Moreover it is shown that the supersaturation evolution and the crystal size distribution of the seeded experiments can be governed by the seed quantity and the evaporation rate. The findings are expected to pave the way for the application of seeding in future protein crystallization processes.

■

INTRODUCTION The demand for both therapeutic proteins and industrial enzymes for use in the pharmaceutical, biotechnological, and food industry is increasing rapidly.1 Crystalline proteins often offer advantages compared to the amorphous or dissolved form in terms of shelf life, product handling, and drug release properties.2,3 Besides the formulation aspect, crystallization can serve as a cost-effective and highly selective purification step and possibly replace one or more chromatographic steps in the downstream processing of proteins.4 Therefore, lots of effort was taken recently to develop appropriate techniques for bulk crystallization of these macromolecules.5−11 Knowledge of nucleation and growth behavior is of fundamental importance to design and control such a crystallization process and, thus, to determine the quality and quantity of the produced crystals.12−14 However, thorough investigations on the metastable zone width for primary and secondary nucleation in bulk protein crystallization are still hardly available, and seeding strategies to control protein crystal nucleation and the final crystal size distribution are, so far, not published in the literature. A solution containing more solute than it would contain in the thermodynamic solid−liquid equilibrium is supersaturated with respect to the solid and in a metastable state. The probability of primary, spontaneous nucleation in such a particle-free solution is strongly dependent on the supersaturation and increases with increasing supersaturation. To design a crystallization process, it is common and useful to use a phase diagram and divide this supersaturated region into a © XXXX American Chemical Society

region of high nucleation probability and a region of low nucleation probability. This dividing line is called the metastable zone limit (MZL). Between the solubility and the MZL, spontaneous nucleation is highly improbable, whereas spontaneous nucleation is likely to occur in the labile zone above the MZL. Unlike the thermodynamic solubility, the MZL is kinetic in nature, time dependent, and hence affected, among others, by the rate of supersaturation generation.13,15 In general, the MZL is determined in crystallization experiments by the supersaturation at which the crystals are first detected visually or nephelometrically.12,16 Knowledge of the MZL for the used process conditions can be used to guide the crystallization in industrial production and to avoid massive nucleation yielding small product crystals. The required supersaturations for nucleation are expected to be considerably higher for proteins than for small molecules due to their complicated surface structure being accompanied by a purely statistical mechanical difficulty to crystallize them.17,18 So far, there are only a few studies to be found in the literature determining MZLs for proteins. Specifying the MZL as a supersaturation ratio, which is the ratio of the solute concentration to the solute solubility concentration, MZLs of 4.4−4.9,19 2.5−4.0 (extracted from ref 20), and 3.0−5.0 (extracted from ref 7) have been reported for the protein hen egg-white lysozyme. All these MZLs were determined in small volume experiments of between 200 and Received: March 29, 2017 Revised: May 5, 2017 Published: May 8, 2017 A

DOI: 10.1021/acs.cgd.7b00456 Cryst. Growth Des. XXXX, XXX, XXX−XXX

Crystal Growth & Design

Article

1600 μL by salting-out the protein with sodium chloride. A broad metastable zone up to a MZL of 50 has been found for ovalbumin when salting-out the protein with ammonium sulfate in 100 mL vessels.5 However, reliable MZLs determined in bulk protein crystallization with operating volumes of some liters to hectoliters have not been published yet. In small molecule crystallization, seed crystals of the solute to be crystallized are often used for controlled nucleation at reduced supersaturation instead of spontaneous nucleation. Thereby, the start of crystallization is very well-defined, and the reproducibility of the process and its outcome can be enhanced, enabling the process engineer to gain control over important product properties as the particle size and shape. As a rule of thumb, 0.1−1% of the final crystal mass should be added as seed crystals to a supersaturated solution considerably below the metastable limit for primary nucleation.21 In a seeded crystallization process, as well as in the course of an unseeded crystallization process, secondary nucleation by attrition, contact, or shear nucleation is the predominant mechanism of the generation of new crystals.22 Thus, knowledge of the supersaturation range in which controlled secondary nucleation occurs is essential for an optimum performance of a crystallizer. Comparable to primary nucleation, secondary nucleation will only be induced when the solution supersaturation exceeds a certain minimum below the MZL, the so-called secondary nucleation threshold (SNT).23 Just like the MZL, the SNT is a dynamic boundary and can depend on experimental conditions like the rate of supersaturation generation and the size, morphology, purity, and quantity of the used seed crystals.21−23 Therefore, a suitable seeding strategy has to be found for each substance to be crystallized and for given process conditions. Whereas seeding is an established tool to increase the crystal size in single crystal protein crystallization for X-ray diffraction studies,24−27 it is hardly used in bulk crystallization of the macromolecules. More fungal lipase crystals have been reported to grow in a shorter time when 0.1% seeds were added prior to crystallization in a 500 mL vessel.6 A SNT of 20 has been determined in ovalbumin crystallization studies with the use of 0.1% seeds in a 100 mL vessel.5 The seed crystal size (105 or 210 μm) and quantity (0.2 or 0.3%) have been reported to not influence the secondary nucleation rate of lysozyme in a stirred tank of 1 L volume.28 However, thorough studies on the influence of seeding on nucleation, the supersaturation evolution during crystallization, and the process outcome, i.e., the crystal size distribution and morphology, are still missing. In order to overcome the lack of relevant experimental data on both spontaneous and controlled nucleation behavior and its relevance to final product properties in bulk protein crystallization, the bulk evaporative crystallization of tetragonal lysozyme, recently introduced by the authors,11 is characterized thoroughly, and unseeded and seeded experiments are compared in detail. It is hypothesized that the MZL depends on the rate of supersaturation generation, i.e., the rate of solvent evaporation, and, thus, that the evaporation rate influences both the supersaturation evolution of the process and the crystal size distribution of the product. Further it is presumed that nucleation can be controlled by the use of seed crystals and, thereby, that the reproducibility of the crystal size distribution can be dramatically increased. It is further expected that for this controlled, secondary nucleation a minimum supersaturation, i.e., the SNT, is necessary, which is a function of the evaporation rate and the seed quantity. Finally, it is hypothesized that the extent of forming secondary nuclei can

be regulated by varying the seed mass and the supersaturation, at which the seed particles are added to the protein solution, and, thus, that the crystal size distribution can be governed. The aim of this work is to corroborate or to disprove the presented hypotheses and to point out seeding as a promising option to improve controllability, reproducibility, and quality of the process and of the product in bulk protein crystallization. Hen egg white lysozyme was chosen as the model protein for the experimental studies. It was crystallized by evaporative crystallization in a stirred and baffled tank with a total volume of 3 L at a boiling temperature of 24 °C to not denature the enzyme (as shown in Groß and Kind11). Experiments with and without the use of lysozyme seed crystals were conducted. The influence of the evaporation rate, the seed mass, and the seeding supersaturation on the MZL, the SNT, the supersaturation evolution, and the product crystal size distribution and morphology is reported and discussed.

■

EXPERIMENTAL SECTION

Experimental Setup. The setup of the evaporative crystallization experiments is depicted in Figure 1.

Figure 1. Experimental setup. A double-walled and baffled glass vessel with an inner diameter of 0.1 m and a total volume of 3 L was used as crystallizer. In order to mix the crystallizing suspension, a three-blade propeller stirrer (diameter 0.064 m, blade angle 40°) was mounted. A demister was inserted to prevent foam and droplet entrainment. The crystallizer jacket was connected to a thermostat to control the heat flow into the crystallizing solution and, thereby, to adjust the evaporation rate. The system pressure and, thus, the boiling temperature of the solution were adjusted by the use of a membrane vacuum pump. A turbidity sensor (FSC402, Mettler Toledo, USA) was immersed into the crystallizing solution to determine the nucleation onset. The sensor was calibrated by a two-point-calibration with diluted lysozyme crystal suspensions to a crystal content of between 0.1% and 0.2% (corresponding to 0% and 100% turbidity). A stainless steel cannula (inner diameter 750 μm) was mounted to the crystallizer and immersed into the solution for both sampling and seeding via a connected valve and a syringe. An iced water-cooled condenser was connected to the vapor pipe to condense the vapor. The condensate was collected in a glass bottle, which was placed on a balance to determine the evaporation rate inline. Experimental Procedure. Spray-dried and agglomerated hen egg white lysozyme powder was acquired from Ovobest Eiprodukte GmbH & Co. KG (Germany). High lysozyme purity (>98.9%) with respect to other proteins was determined by high-throughput capillary gel electrophoresis as described in ref 11, and the lysozyme powder was used without further purification. Citric acid anhydrate (>99.5%) and sodium hydroxide (>99%) were purchased from Carl Roth GmbH & Co. KG (Germany). In order to prepare the feed solution for the experiments, an aqueous lysozyme solution was filtrated through a 1.6 B

DOI: 10.1021/acs.cgd.7b00456 Cryst. Growth Des. XXXX, XXX, XXX−XXX

Crystal Growth & Design

Article

μm glass microfiber filter (VWR, USA) to remove any insoluble impurities and gently mixed with a citric acid/sodium hydroxide buffer stock solution to give the required concentrations. This feed solution was already slightly supersaturated at 24 °C (supersaturation ratio S = 1.5). At the beginning of each experiment, 1.8 kg of the feed solution containing 14.4 wt % lysozyme (LSZ) and 0.9 wt % buffer (B) at pH 4.5 were filled into the crystallizer, and the stirrer was adjusted to a stirring rate of 170 rpm. Then, the crystallizer was slowly evacuated to an absolute pressure of 2.5 kPa to start evaporation. Foam formation was observed at the start-up phase, and foam migration into the vapor pipe was prevented by decreasing the pressure step by step over a time period of approximately 30 min as described in Groß and Kind.11 The boiling temperature of the solution was adjusted to 24 °C with a maximum temperature fluctuation from average of ±1 °C. Constant evaporation rates of between 2 and 6 g min−1 were realized. When 0.9 kg condensate was collected in the glass bottle, the vacuum pump was disabled, and the vacuum was released to prevent further evaporation. After the break of the vacuum, the stirred suspensions were kept at ambient pressure and 24 °C for the supersaturation to decay. Seeding Procedure. In the case of seeded crystallization experiments, the experimental procedure was expanded by a seeding step. The seeding procedure was as follows: at a certain evaporation progress a syringe filled with seed suspension was attached to the seeding device, the valve was opened and the seed suspension was soaked into the crystallizer instantly. Afterward, the cannula was rinsed with a small amount of deionized water to avoid encrustation. The seed suspension mass Mseed varied between 0.3 and 9.0 g, corresponding to 0.03 and 1.0% of the final crystal suspension mass. The mass fraction based seeding supersaturation ratios Sseed varied between 1.5 and 3.0. A summary of the experimental parameters is given in Table 1.

Scientific, USA) at 280 nm using the Lambert−Beer law with a decadic absorption coefficient of 265 L g−1 m−1.29,30 Concentrations were converted into mass fractions and supersaturation ratios S were calculated by dividing the so-determined lysozyme mass fraction in the liquid phase “L” by the lysozyme solubility mass fraction at 24 °C: S=

variation of

seed type

seed mass Mseed/g

* = 0.01 + 0.37e−157xB x LSZ

2, 4, 6

B

0.9

2.1

4

A

Mseed

4

B

1.5 2.1 2.1

Sseed Sseed

4 4

A B

4.5, 9.0 0.9, 4.5 0.3, 0.7, 0.9, 2.3, 4.5, 9.0 4.5 0.9

(2)

Additionally, theoretical supersaturation ratios Sth were calculated at certain time steps by dividing the total lysozyme mass fraction of the suspension, which is the ratio of the total lysozyme mass in the suspension to the suspension mass, by the lysozyme solubility mass fraction:

Sth =

total x LSZ * x LSZ

(3)

Product Characterization. In order to evaluate the morphology of the produced crystals, suspension samples were analyzed via a light microscope. Volume-based crystal size distributions of diluted suspension samples were measured 3-fold via dynamic image analysis (Camsizer XT, Retsch Technologies, Germany) 2 h after the break of the vacuum, when the protein concentration in the solution was depleted mostly to supersaturations S below 2.5. A saturated lysozyme solution was used to dilute the samples during the measurement preventing growth and dissolution of the crystals. The diameter d used to evaluate the crystal sizes is the diameter of the area equivalent circle to the particle projection.31

■

RESULTS AND DISCUSSION Spontaneous Nucleation in Unseeded Experiments. To gain insight into the metastable zone width for primary nucleation, a first series of unseeded experiments with identical conditions (evaporation rate 4 g min−1) was performed. Because of water evaporation, the lysozyme concentration and the buffer concentration increase with increasing evaporation progress η, defined as the ratio of the condensate mass at the moment to the total condensate mass at the end of evaporation. Thus, the theoretical supersaturation ratio Sth evolves from 1.5 at the beginning of the experiments (η = 0) to 9.4 at the end of the experiments (η = 1). At a certain evaporation progress η, the supersaturation is high enough to cause spontaneous nucleation and subsequent crystal growth, which finally results in a turbidity increase. The onset of turbidity increase was defined as the start of primary nucleation, since no seed particles were added to the system, and the corresponding supersaturation ratio Snucl was determined as shown in Figure 2. The onset of each turbidity curve can be found clearly, and the signals increase continuously up to the upper calibration limit of 100%. Spontaneous nucleation took place at an evaporation progress η of between 0.53 and 0.82, corresponding to supersaturation ratios Snucl of between 3.1 and 6.0. The variance of primary nucleation supersaturations is conspicuous, since the experimental conditions were identical within this series of experiments, but it was, somehow, expected due to the stochastic nature of nucleation. Nucleation supersaturation ratios of all unseeded experiments as a function of the evaporation rate are given in Figure 3. From Figure 3 there is no evidence for an influence of the evaporation rate, i.e., the velocity of supersaturation generation,

seeding supersaturation ratio Sseed/−

evaporation rate evaporation rate Mseed

(1)

Lysozyme solubility mass fractions at 24 °C were determined from eq 2,11 considering the prevailing buffer mass fraction:

Table 1. Experimental Conditions of the Evaporative Crystallization Experiments evaporation rate/g min−1

L x LSZ * x LSZ

2, 3, 4, 5, 6

1.5, 2.1, 2.4, 2.6 1.6, 2.1, 2.5, 3.0

Two seed suspensions A and B were used, each prepared by evaporative crystallization as described above, filled into syringes and stored in a refrigerator for up to 2 months until use. Samples of each seed suspension were taken at appropriate time intervals to determine the crystal size distribution of the isometric-shaped, tetragonal lysozyme crystals, which were found to be almost unchanged within the time frame of each series of experiments with a medium size d50 of 39.9 ± 0.9 μm for seed A and 45.5 ± 1.4 μm for seed B, respectively (see Figure S1 for microscopic pictures and crystal size distributions of the seed suspensions). The seed suspensions could not be used for longer time periods than 2 months when they began moldering. Supersaturation Evolution. To monitor the supersaturation evolution of the solution during the crystallization experiments, small suspension samples were taken from the crystallizer through the sampling device at certain time steps and subsequently filtrated through a 0.2 μm cellulose acetate filter (VWR, USA) to separate the solution from the crystals. Lysozyme concentrations in the filtrate were determined as triplicates by measuring the absorption of diluted solution samples in a spectrophotometer (Genesys 10S, Thermo C

DOI: 10.1021/acs.cgd.7b00456 Cryst. Growth Des. XXXX, XXX, XXX−XXX

Crystal Growth & Design

Article

start of evaporation (η = 0.04, Sseed = 1.5) are depicted in Figure 4a−c.

Figure 2. Determination of the nucleation supersaturation ratios Snucl (triangles) from turbidity signals of unseeded experiments with an evaporation rate of 4 g min−1. The theoretical supersaturation evolution is depicted by a dashed line in gray. Only three turbidity signals are shown for reasons of clarity.

Figure 3. Nucleation supersaturation ratios Snucl (triangles) and metastable zone (box) of the unseeded crystallization experiments.

Figure 4. Turbidity signals of seeded crystallization experiments (Mseed = 4.5 g, seed A, evaporation rate 4 g min−1), (a−c) determination of SNT, Sseed = 1.5, (d) seeding between SNT and MZL, Sseed = 2.1 (left green line), 2.4 (intermediate red line), and 2.6 (right blue line).

on the metastable zone width. The metastable zone, where spontaneous nucleation is highly improbable, reaches from the solubility (S = 1) up to the MZL of ca. 3.1. This means that under the investigated experimental conditions, the lysozyme solutions must contain at least three times as much of the solute as in equilibrium before spontaneous, primary nucleation will occur. It is remarkable that this MZL has the same magnitude as published before for small scale lysozyme crystallization trials,7,19,20 although the crystallization volume is by orders of magnitude higher and the crystallization pathway is completely different. Controlled Nucleation in Seeded Experiments. To obtain a better controllability of nucleation, seeded experiments were performed. The turbidity signals of crystallization experiments seeded with 4.5 g of seed A shortly after the

It can be seen that adding the seed crystals to the solution leads to a distinct peak of the turbidity curve. This is because the crystals are not suspended ideally at once. At a certain evaporation progress η of between 0.07 and 0.29, the turbidity signal starts to increase due to secondary nucleation followed by crystal growth. Since the product crystals of the seeded experiments are smaller than the seed crystals (see Figures 12−15), the number of product crystals has to be much greater than the number of seed crystals. Thus, plenty of new crystals are generated in the process by secondary nucleation by attrition, contact, or shear nucleation. The onset of the curves was evaluated, and corresponding nucleation supersaturation ratios Snucl were found to range between 1.6 and 2.1. For these experimental conditions, the maximum nucleation supersaturation Snucl of 2.1 (η = 0.29) was defined as the SNT, D

DOI: 10.1021/acs.cgd.7b00456 Cryst. Growth Des. XXXX, XXX, XXX−XXX

Crystal Growth & Design

Article

to secondary nucleation at a higher supersaturation and, thus, to a higher SNT. Supersaturation Evolution in Unseeded Experiments. Typically, low supersaturations are desired in a crystallization process to suppress massive nucleation and to obtain large product crystals. The solution supersaturation evolution during selected unseeded crystallization experiments with different evaporation rates of 2, 3, 4, and 6 g min−1 is displayed in Figure 6 together with the theoretical supersaturation evolution of

above which secondary nucleation is likely to occur in the presence of seed crystals. After Threlfall and Coles,23 the region between the SNT and the MZL gives the process engineer the opportunity for controlled nucleation by seeding. This was validated in dedicated experiments, where the same seed mass (4.5 g) was injected into the crystallizer at an evaporation progress η of 0.29, 0.35, and 0.43, corresponding to supersaturation ratios Sseed of 2.1, 2.4, and 2.6. The corresponding turbidity signals are depicted in Figure 4d. There it can be seen that directly after the introduction of seed crystals to the solution (seeding peak) the turbidity signal of each of the experiments starts to increase. Hence, it is shown that via seeding in between the SNT and the MZL nucleation can be controlled by inducing secondary nucleation and spontaneous uncontrolled nucleation can be avoided. The kinetic nature of the SNT was investigated both by varying the seed mass and the rate of supersaturation generation, i.e., the evaporation rate. The dependencies are illustrated in Figure 5.

Figure 6. Supersaturation evolution of unseeded crystallization experiments with different evaporation rates: 2 g min−1 (blue squares), 3 g min−1 (magenta down-pointing squares), 4 g min−1 (green circles), and 6 g min−1 (red triangles). The connecting solid lines serve as guides to the eye. The nucleation supersaturation Snucl of each experiment is depicted by a star of the same color, and the theoretical supersaturation evolution of each experiment is depicted by a dashed line of the same color. The diagram is divided in two parts, the evaporation phase (t < 0, shaded in white) and the phase after evaporation (t > 0, shaded in gray).

each experiment. The time t = 0 was defined as the time when an evaporation progress η of 1 was reached, i.e., at break of the vacuum. Thus, the diagram is divided in two parts, the evaporation phase (t < 0, shaded in white) and the phase after evaporation (t > 0, shaded in gray). For all experiments it is evident that after nucleation took place the solution supersaturation increases further due to water evaporation but gradually detaches from the theoretical supersaturation curve because of crystal mass deposition. Once a maximum solution supersaturation Smax is reached, the supersaturation starts to deplete continuously aiming equilibrium (S = 1). At the end of the evaporation phase (t = 0), each suspension is still supersaturated at St=0 of between 2.5 (blue curve) and 6.8 (green curve). When comparing the supersaturation curves thoroughly, an influence of both the evaporation rate and the nucleation supersaturation can be detected. First, the lowest evaporation rate (2 g min−1, blue squares) leads to the lowest Smax and St=0, even though nucleation occurred at nearly the same supersaturation in the experiment with the evaporation rate of 3 g min−1 (magenta down-pointing squares). Second, Smax and St=0 coincide nearly for the experiments at 4 g min−1 (green circles) and 6 g min−1 (red triangles), which might be explained by different nucleation supersaturations. Hence, it is obvious that the crystallization period τcryst, which is actually the time period between nucleation and the end of the evaporation phase, plays

Figure 5. Nucleation supersaturation ratios Snucl (triangles) and SNT (dashed lines), (a) variation of the seed mass (evaporation rate 4 g min−1, Sseed = 2.1, seed B), (b) variation of the evaporation rate (Mseed = 0.9 g, Sseed = 2.1, seed B).

As expected, the SNT increases distinctly with decreasing seed mass because less secondary nuclei are formed resulting in a longer time necessary to approach the detection limit of the turbidity sensor. Interestingly, the SNT is even higher than the MZL at a low seed mass of 0.9 g and below. When planning to seed with that low seed masses, seeding above the SNT is not advisable. In this case seeding has to take place below the SNT and the MZL to not risk spontaneous, uncontrolled nucleation. Then, secondary nucleation will occur, but not instantaneously. Seeding above the SNT with instantaneous secondary nucleation is only an option for higher seed masses for which the SNT lies within the metastable zone. Besides the seed mass, the SNT is a function of the evaporation rate as well, as shown in Figure 5b. As expected, an increasing evaporation rate leads E

DOI: 10.1021/acs.cgd.7b00456 Cryst. Growth Des. XXXX, XXX, XXX−XXX

Crystal Growth & Design

Article

the key role in determining the residual supersaturation ratio St=0, since it strongly depends on both the evaporation rate and the nucleation supersaturation. This is illustrated in Figure 7.

Figure 7. Residual supersaturation ratio St=0 of unseeded crystallization experiments with different nucleation supersaturations: Snucl < 3.5 (black down-pointing triangles), 3.5 < Snucl < 4.0 (magenta downpointing squares), 4.0 < Snucl < 4.5 (blue right-pointing triangles), 4.5 < Snucl < 5.0 (red up-pointing triangles), 5.0 < Snucl < 6.0 (olive circles), 6.0 < Snucl (orange squares).

The residual supersaturations St=0 vary between 2.5 and 9.5, with the highest values at low crystallization periods below 60 min. Higher crystallization periods and, thus, lower residual supersaturations only arise at nucleation supersaturations lower than 4.5. However, it is clear that the nucleation supersaturation cannot be controlled without seeding, and, consequently, the residual supersaturation St=0 cannot be controlled either. This implies a batch-to-batch variation of the additional time needed to equilibrate the suspension after evaporation, since the residual supersaturation St=0 is mainly responsible therefore. For economic reasons, low residual supersaturations and low equilibration times would certainly be desired to increase process efficiency. Supersaturation Evolution in Seeded Experiments. In order to reduce the supersaturation during the crystallization process and especially the residual supersaturation at the end of the evaporation phase, the use of seed crystals was investigated. The solution supersaturation evolution during seeded crystallization experiments with different seed masses Mseed of between 0.3 and 9.0 g is depicted in Figure 8a together with the theoretical supersaturation evolution of the experiments. For a better comparability, the supersaturation evolution of an unseeded experiment with the same evaporation rate (4 g min−1) is shown as well. Analogous to Figure 6, the diagram is divided in two parts, the evaporation phase (t < 0, shaded in white) and the phase after evaporation (t > 0, shaded in gray). It is apparent that all the supersaturation curves of the seeded experiments lie distinctly below the one of the unseeded experiment (olive pentagons) and that an increasing seed mass leads to lower supersaturations. Further it is remarkable that the supersaturation evolutions of all three experiments with the same seed mass (0.9 g) are virtually identical. For a more precise evaluation, characteristic supersaturation ratios Snucl, Smax, and St=0 are plotted as a function of the seed mass in Figure 8b. Herein it is clearly shown that a higher seed mass

Figure 8. (a) Supersaturation evolution of seeded crystallization experiments (evaporation rate 4 g min−1, Sseed = 2.1, seed B) with different seed masses Mseed: 0 g (olive pentagons), 0.3 g (magenta down-pointing squares), 0.9 g (red, brown and green triangles), 2.3 g (blue squares), 4.5 g (cyan stars), and 9.0 g (black circles). The connecting solid lines serve as guides to the eye. The theoretical supersaturation evolution is depicted by a dashed line. (b) Characteristic supersaturations Snucl (black triangles), Smax (olive squares), and St=0 (blue circles) of seeded crystallization experiments (evaporation rate 4 g min−1, Sseed = 2.1, seed B).

leads to a lower nucleation supersaturation Snucl. Besides, a higher seed mass leads to a larger amount of secondary nuclei, which then grow to crystals under reduction of the solute concentration, resulting in a lower maximum supersaturation Smax and a lower residual supersaturation St=0. The influence of the evaporation rate and the seeding supersaturation ratio on the characteristic supersaturation ratios Snucl, Smax, and St=0 is shown in Figure 9. It can be seen in Figure 9a that a higher evaporation rate not only leads to a higher nucleation supersaturation Snucl, but also to a higher Smax and St=0. This is because the available time to reduce the solute concentration, and thereby the supersaturation, is lowered at higher evaporation rates. Whereas the influence of the evaporation rate on the characteristic supersaturation ratios is apparent, there is no clear influence to be found of the seeding supersaturation ratio Sseed, when seeding occurs below the SNT (SNT = 3.8, Figure 9b). However, when seeding is conducted above the SNT, an increasing seeding supersaturation Sseed enhances the nucleation F

DOI: 10.1021/acs.cgd.7b00456 Cryst. Growth Des. XXXX, XXX, XXX−XXX

Crystal Growth & Design

Article

Figure 10. Microscopic pictures of the diluted product suspensions of crystallization experiments with an evaporation rate of 4 g min−1, (a and b) unseeded, (c) Mseed = 0.9 g, Sseed = 2.1, seed B, (d) Mseed = 4.5 g, Sseed = 1.5, seed A.

The crystals obtained from the unseeded experiments are well-defined and isometric shaped (Figure 10a,b). Whereas the crystals from the experiment seeded with 0.9 g of seed B have the same optical appearance (Figure 10c), the crystals from the experiment seeded with 4.5 g of seed A are obviously smaller and of lower optical quality (Figure 10d). The seed mass seems to have a great impact on the final crystal sizes, which will be discussed in further detail later (see Figure 15). In Figure 11, crystal size density distributions q3(d) of the product suspensions of unseeded and seeded crystallization experiments with an identical evaporation rate of 4 g min−1 are displayed. As can be seen in Figure 11a, the crystal size distributions of the unseeded experiments are generally monomodal with sizes up to 90 μm. However, great size variances in between the experiments are apparent, despite identical experimental conditions. This might be explained by the differences in nucleation supersaturation (see Figure 3). Seeding not only leads to a controlled nucleation and defined start of crystallization, but also to highly reproducible crystal size distributions of the product, as can be seen in Figure 11b,c for seeding with seed A or B, respectively. In order to compare the absolute crystal sizes, characteristic crystal sizes d10, d50, and d90 and distribution widths (d90 − d10)/d50 of the size distributions obtained from all of the unseeded and seeded experiments are depicted in Figure 12. In Figure 12a it can be seen that the absolute sizes of the product crystals being produced in the seeded experiments are generally smaller compared to the unseeded experiments. For example, whereas the medium diameter d50 reaches values of above 50 μm in some of the unseeded experiments, a maximum d50 of only 35 μm is obtained in the seeded experiments. However, the ranges of the characteristic sizes of the seeded and unseeded experiments overlap largely, so that in some of the seeded experiments the product crystals are bigger than in some of the seeded experiments. Seeding further leads to size distributions with a narrow width of between 0.7 and 1.2, whereas the size distribution widths of the unseeded experiments vary between 0.7 and 3.0 as it is shown in Figure 12b. In order to find an appropriate way to govern the crystal size distributions in such an evaporative crystallization experiment, each of the parameters evaporation rate, seeding supersaturation, and seed mass was varied; meanwhile the remaining parameters were kept constant. Crystal size distributions were

Figure 9. Characteristic supersaturations Snucl (black triangles), Smax (olive squares), and St=0 (blue circles) of seeded crystallization experiments, (a) variation of the evaporation rate (Sseed = 2.1, Mseed = 0.9 g, seed B), (b) seeding below the SNT (SNT = 3.8, Mseed = 0.9 g, seed B, evaporation rate 4 g min−1), (c) seeding above the SNT (SNT = 2.1, Mseed = 4.5 g, seed A, evaporation rate 4 g min−1).

supersaturation Snucl resulting in higher Smax and St=0 (SNT = 2.1, Figure 9c). In conclusion, the seed mass, the seeding supersaturation, and the evaporation rate can be eligible process parameters to guide the supersaturation evolution in seeded evaporative crystallization and can be used to increase the process efficiency. Crystal Size Distribution and Product Morphology. In industrial crystallization, the crystal morphology and size distribution are the main quality criteria of the product, since they affect the filtration and rheological properties of the suspension and the flow and dissolution properties of the crystals.14 In order to discuss the influence of seeding on the crystal morphology and size distribution of the product, seeded and unseeded experiments were carried out, and microscopic pictures of the product were taken and crystal size distributions were determined. The results are shown in the following. Figure 10 shows microscopic images of the diluted product suspensions of unseeded and seeded crystallization experiments with an identical evaporation rate of 4 g min−1. G

DOI: 10.1021/acs.cgd.7b00456 Cryst. Growth Des. XXXX, XXX, XXX−XXX

Crystal Growth & Design

Article

determined, and characteristic values d10, d50, and d90 are plotted in Figures 13−15.

Figure 13. Characteristic crystal sizes d10 (blue down-pointing triangles), d50 (black squares), and d90 (red up-pointing triangles) as a function of the evaporation rate, (a) unseeded experiments, (b) seeded experiments (Mseed = 0.9 g, Sseed = 2.1, seed B).

As expected, no clear influence of the evaporation rate can be detected for the unseeded experiments, since the characteristic sizes vary strongly even between experiments of the same evaporation rate (Figure 13a). Thus, the evaporation rate is not an appropriate parameter to control the crystal size distribution in unseeded crystallization. In contrast, the evaporation rate seems to manipulate the crystal size distribution of the product in seeded experiments, where an increasing evaporation rate leads to smaller characteristic sizes and a smaller width of the size distribution (Figure 13b). This might be explained by the higher supersaturations arising in the experiments of higher evaporation rates (see Figure 9a), which possibly leads to enhanced crystal nucleation yielding smaller product crystals. However, for both seeds A and B there is no clear influence to be found of the seeding supersaturation on the size distribution of the product (Figure 14a,b). In Figure 15 it is shown that when decreasing the seed mass from 9 to 0.9 g, the characteristic crystal sizes increase 1.5−3fold for both seeds A and B. This was expected, since more seed crystals should produce more secondary nuclei, e.g., by attrition, resulting in smaller crystals. However, a lower seed mass has also been shown to increase the supersaturations in the crystallization experiments (see Figure 8b), which would be indicative for enhanced nucleation yielding smaller crystals. Obviously, the effect of supersaturation on the formation of secondary nuclei can be neglected compared to the opposite, strong effect of the seed mass. Thus, the seed quantity turns out to be the main influencing parameter on the product size distribution in seeded crystallization. For visualization, a series of microscopic pictures shows the morphology and size evolution of two seeded experiments with different seed masses of 0.9 g (Figure 16, on the left) and 4.5 g (Figure 16, on the right).

Figure 11. Crystal size distributions obtained from crystallization experiments with an evaporation rate of 4 g min−1, (a) eight unseeded experiments, (b) three seeded experiments (Mseed = 4.5 g, Sseed = 1.5, seed A), (c) five seeded experiments (Mseed = 0.9 g, Sseed = 2.1, seed B).

Figure 12. (a) Characteristic crystal sizes d10 (blue down-pointing triangles), d50 (black squares), and d90 (red up-pointing triangles) obtained from 18 unseeded and 23 seeded experiments, (b) size distribution widths (d90 − d10)/d50 obtained from 18 unseeded and 23 seeded experiments. H

DOI: 10.1021/acs.cgd.7b00456 Cryst. Growth Des. XXXX, XXX, XXX−XXX

Crystal Growth & Design

Article

Figure 14. Characteristic crystal sizes d10 (blue down-pointing triangles), d50 (black squares), and d90 (red up-pointing triangles) as a function of the seeding supersaturation ratio obtained from seeded crystallization experiments with an evaporation rate of 4 g min−1, (a) Mseed = 4.5 g, seed A, (b) Mseed = 0.9 g, seed B.

Figure 16. Microscopic pictures taken during seeded crystallization experiments (evaporation rate 4 g min−1, Sseed = 2.1, seed A) with different seed masses, left: 0.9 g, right: 4.5 g.

■

CONCLUSIONS In this study, the unseeded and seeded bulk evaporative crystallization processes of the protein lysozyme were investigated and compared in terms of nucleation behavior, supersaturation evolution, and product quality. This is the first dedicated study to use seeding in bulk protein crystallization to control nucleation and govern the product crystal size distribution. It was shown that the metastable zone limit for primary nucleation (MZL) is comparable to the MZLs published for small volume crystallization experiments. However, the nucleation supersaturation was found to vary strongly in between the unseeded experiments despite identical experimental conditions. As a consequence, the supersaturation evolution during the unseeded experiments and the residual supersaturation at the end of the evaporation phase were shown to vary from experiment to experiment, resulting in differences in the additional time needed to equilibrate the suspension and in strong variations of the product crystal size distributions. In contrast, seeding was proven to lead to controlled and reproducible secondary nucleation above the SNT, which depends on the seed quantity and the evaporation rate. Further, the seed quantity, the seeding supersaturation, and the evaporation rate were demonstrated to be eligible process parameters to guide the supersaturation evolution in seeded evaporative crystallization. Moreover it was validated that the reproducibility of the crystal size distribution of the product suspension can be dramatically increased by seeding compared to the unseeded experiments, while the well-defined outer shape of the crystals is of similar optical quality. Finally, the crystal size distributions of the seeded experiments can be governed mainly by variation in seed mass and evaporation rate,

Figure 15. Characteristic crystal sizes d10 (blue down-pointing triangles), d50 (black squares), and d90 (red up-pointing triangles) as a function of the seed mass obtained from seeded crystallization experiments with an evaporation rate of 4 g min−1, (a) Sseed = 2.1 (filled symbols) or Sseed = 1.5 (open symbols), seed A, (b) Sseed = 2.1, seed B.

As can be seen, around 25 min after nucleation took place, small crystals become visible in both experiments. With increasing experiment time, the number and size of the crystals increases. It is obvious that more crystals are produced in the experiment with the higher seed mass of 4.5 g resulting in smaller product crystals. I

DOI: 10.1021/acs.cgd.7b00456 Cryst. Growth Des. XXXX, XXX, XXX−XXX

Crystal Growth & Design

Article

MZL metastable zone limit SNT secondary nucleation threshold

whereas an influence of the seeding supersaturation could not be detected. In combination, the results highlight seeding as a promising tool for bulk protein crystallization to improve controllability, reproducibility, and quality of the process and of the product. Thus, this work paves the way for the application of seeding in future protein crystallization processes.

■

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.cgd.7b00456. Microscopic pictures and crystal size distributions of the seed suspensions A and B (PDF)

■

AUTHOR INFORMATION

Corresponding Author

*Phone: +49 721 608 42390. Fax: +49 721 608 43490. E-mail: [email protected]. Web: www.tvt.kit.edu. ORCID

Matthias Kind: 0000-0002-7203-1776 Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors thank the German Research Foundation (DFG) for the financial support of this work within the SPP 1934 DiSPBiotech. We also wish to thank Marius Böser, Elena Cava, Sarah Klein, and Felix Pitzal for their valuable experimental contributions.

■

NOTATION

Symbols

d d10 d50 d90 Mseed q3 S Smax Snucl Sseed St=0 Sth t xB xtotal LSZ xLLSZ x*LSZ η τcryst

REFERENCES

(1) Walsh, G. Proteins Biochemistry and Biotechnology; John Wiley & Sons: Chichester, U.K., 2002. (2) Jen, A.; Merkle, H. Diamonds in the Rough: Protein Crystals from a Formulation Perspective. Pharm. Res. 2001, 18, 1483−1488. (3) Elkordy, A. A.; Forbes, R. T.; Barry, B. W. Stability of Crystallised and Spray-Dried Lysozyme. Int. J. Pharm. 2004, 278, 209−219. (4) Hekmat, D. Large-Scale Crystallization of Proteins for Purification and Formulation. Bioprocess Biosyst. Eng. 2015, 38, 1209−1231. (5) Judge, R. A.; Johns, M. R.; White, E. T. Protein Purification by Bulk Crystallization: the Recovery of Ovalbumin. Biotechnol. Bioeng. 1995, 48, 316−323. (6) Lee, T. S.; Vaghjiani, J. D.; Lye, G. J.; Turner, M. K. A Systematic Approach to the Large-Scale Production of Protein Crystals. Enzyme Microb. Technol. 2000, 26, 582−592. (7) Hekmat, D.; Hebel, D.; Schmid, H.; Weuster-Botz, D. Crystallization of Lysozyme: From Vapor Diffusion Experiments to Batch Crystallization in Agitated ml-Scale Vessels. Process Biochem. 2007, 42, 1649−1654. (8) Hebel, D.; Ü rdingen, M.; Hekmat, D.; Weuster-Botz, D. Development and Scale up of High-Yield Crystallization Processes of Lysozyme and Lipase Using Additives. Cryst. Growth Des. 2013, 13, 2499−2506. (9) Yu, X.; Wang, J.; Ulrich, J. Purification of Lysozyme from Protein Mixtures by Solvent-Freeze-Out Technology. Chem. Eng. Technol. 2014, 37, 1353−1357. (10) Neugebauer, P.; Khinast, J. G. Continuous Crystallization of Proteins in a Tubular Plug-Flow Crystallizer. Cryst. Growth Des. 2015, 15, 1089−1095. (11) Groß, M.; Kind, M. Bulk Crystallization of Proteins by LowPressure Water Evaporation. Chem. Eng. Technol. 2016, 39, 1483− 1489. (12) Ulrich, J.; Strege, C. Some Aspects of the Importance of Metastable Zone Width and Nucleation in Industrial Crystallizers. J. Cryst. Growth 2002, 237−239, 2130−2135. (13) Mullin, J. W. Crystallization, 4th ed.; Butterworth-Heinemann: Oxford, MA, 2001. (14) Myerson, A. S. Handbook of Industrial Crystallization, 2nd ed.; Butterworth-Heinemann: Boston, 2002. (15) García-Ruiz, J. M. Nucleation of Protein Crystals. J. Struct. Biol. 2003, 142, 22−31. (16) Nývlt, J.; Rychlý, R.; Gottfried, J.; Wurzelová, J. Metastable Zone-Width of Some Aqueous Solutions. J. Cryst. Growth 1970, 6, 151−162. (17) Nanev, C. N. On the Slow Kinetics of Protein Crystallization. Cryst. Growth Des. 2007, 7, 1533−1540. (18) Durbin, S. D.; Feher, G. Protein Crystallization. Annu. Rev. Phys. Chem. 1996, 47, 171−204. (19) Lu, J.; Wang, X.-J.; Ching, C.-B. Batch Crystallization of Soluble Proteins: Effect of Precipitant, Temperature and Additive. Prog. Cryst. Growth Charact. Mater. 2002, 45, 201−217. (20) Ildefonso, M.; Revalor, E.; Punniam, P.; Salmon, J. B.; Candoni, N.; Veesler, S. Nucleation and Polymorphism Explored via an Easy-toUse Microfluidic Tool. J. Cryst. Growth 2012, 342, 9−12. (21) Beckmann, W. Crystallization; Wiley-VCH: Weinheim, Germany, 2013. (22) Agrawal, S. G.; Paterson, A. H. J. Secondary Nucleation: Mechanisms and Models. Chem. Eng. Commun. 2015, 202, 698−706. (23) Threlfall, T. L.; Coles, S. J. A Perspective on the Growth-Only Zone, the Secondary Nucleation Threshold and Crystal Size Distribution in Solution Crystallization. CrystEngComm 2016, 18, 369−378. (24) Bergfors, T. Seeds to Crystals. J. Struct. Biol. 2003, 142, 66−76. (25) Bergfors, T. Protein Crystallization, 2nd ed.; International University Line: La Jolla, CA, 2009.

[μm] crystal size (diameter of the area equivalent circle to the particle projection) [μm] characteristic crystal size, where 10% of the volume distribution lies below [μm] characteristic crystal size, where 50% of the volume distribution lies below [μm] characteristic crystal size, where 90% of the volume distribution lies below [g] seed mass [μm−1] volume distribution density [−] supersaturation ratio [−] maximum supersaturation ratio [−] nucleation supersaturation ratio [−] seeding supersaturation ratio [−] residual supersaturation ratio at the end of the evaporation phase (t = 0) [−] theoretical supersaturation ratio [min] time [−] buffer mass fraction [−] total lysozyme mass fraction of the suspension [−] lysozyme mass fraction in the liquid phase [−] lysozyme solubility mass fraction [−] evaporation progress [min] crystallization period between nucleation and end of evaporation

Abbreviations

B buffer LSZ lysozyme J

DOI: 10.1021/acs.cgd.7b00456 Cryst. Growth Des. XXXX, XXX, XXX−XXX

Crystal Growth & Design

Article

(26) Durbin, S. D.; Feher, G. Crystal Growth Studies of Lysozyme as a Model for Protein Crystallization. J. Cryst. Growth 1986, 76, 583− 592. (27) Forsythe, E.; Ewing, F.; Pusey, M. Studies on Tetragonal Lysozyme Crystal Growth Rates. Acta Crystallogr., Sect. D: Biol. Crystallogr. 1994, 50, 614−619. (28) Tait, S.; White, E. T.; Litster, J. D. A Study on Nucleation for Protein Crystallization in Mixed Vessels. Cryst. Growth Des. 2009, 9, 2198−2206. (29) Sophianopoulos, A. J.; Rhodes, C. K.; Holcomb, D. N.; Van Holde, K. E. Physical Studies of Lysozyme. J. Biol. Chem. 1962, 237, 1107−1112. (30) Gill, S. C.; von Hippel, P. H. Calculation of Protein Extinction Coefficients from Amino Acid Sequence Data. Anal. Biochem. 1989, 182, 319−326. (31) ISO 13322−2 Particle size analysis: Image analysis methods Part 2: Dynamic image analysis methods, 2006.

K

DOI: 10.1021/acs.cgd.7b00456 Cryst. Growth Des. XXXX, XXX, XXX−XXX