Electric-Field-Assisted Protein Crystallization in Continuous Flow

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Electric-field-assisted protein crystallization in continuous flow Fei Li, and Richard Lakerveld Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.8b00095 • Publication Date (Web): 09 Apr 2018 Downloaded from http://pubs.acs.org on April 9, 2018

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Crystal Growth & Design

Electric-field-assisted protein crystallization in continuous flow Fei Li and Richard Lakerveld∗ Department of Chemical and Biological Engineering, The Hong Kong University of Science and Technology, Hong Kong, China E-mail: [email protected] Abstract Crystallization is an attractive method for separation and purification of large biomolecules such as proteins due to inherent advantages related to product composition, stability, and process throughput. However, the lack of control over the nucleation and growth of protein crystals complicates design and operation. Electric fields have been used to improve control over the location and induction time of protein nucleation in small batches. To adopt electric-field-assisted protein crystallization as a separation and purification method, a novel device based on continuous flow is presented. The application of continuous flow allows for a large surface-to-volume ratio to be exploited for the electrode design and aligns with the current trend in (bio)pharmaceutical industry toward continuous manufacturing. The device features millimeter-sized channels with coplaner electrodes to study the influence of non-uniform electric fields on protein crystallization in continuous flow. The results demonstrate that the induction time for protein nucleation can be reduced in continuous flow compared to batch crystallization irrespective of the application of an electric field. A higher yield can be attained for electric-field-assisted protein crystallization in continuous flow compared to control flow experiments without an electric field only when a longer residence time is applied,

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which is attributed to increased secondary nucleation at lower supersaturation. The device for electric-field-assisted protein crystallization in continuous flow can produce a large number of crystals of a small size, which is attractive for applications that require a large surface-to-volume ratio or for the use as seeds in downstream crystallizers optimized for crystal growth.

Introduction The global biopharmaceutical market is increasing rapidly. The competitive environment calls for products with consistent quality, short development times and high flexibility in manufacturing. A key method to potentially achieve step-wise improvements in the manufacture of biopharmaceuticals is to switch from traditional batch-wise to continuous manufacturing. 1 Continuous processing offers several potential advantages such as a reduction in the required processing time, simplified scale-up, and the ability to implement more effective process control strategies. 2 Crystallization is an attractive separation and purification technique for biopharmaceuticals. 3 Compared to conventional packed-bed chromatography, crystallization can potentially process a higher protein titer and larger volume from upstream processes, provide flexible scale-up, and reduce the overall cost. 4–7 However, the design and control of crystallization processes involving large biomolecules such as proteins remains notoriously difficult. Protein crystallization can be performed in continuous flow mode in a well-mixed vessel or in a tubular crystallizer. 8–14 The shear force from stirring or convective flow can influence both nucleation and crystal growth. For example, the nucleation of ferritin, apoferritin, and lysozyme can be manipulated by varying the solution flow rate. 15 Byington et al. 16 showed that a flow with a shear rate larger than 10 s−1 can increase the size of lysozyme clusters, which may act as nucleation precursors, but suppresses the population volume of the lysozyme clusters, which suggests that nucleation would be inhibited. The nucleation rate and crystal yield of insulin crystallization can be increased by applying an intermittent 2


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oscillatory flow compared to the stationary mode. 12 For example, with an oscillatory flow velocity from 6 to 16 mm/min, the nucleation rate grows with the flow velocity. 12 The response of crystal growth to shear depends on various factors. Durbin and Feher 17 found that the growth rate of lysozyme crystals is not altered by applying a flow of 40-600 µm/s while Pusey et al. 18 reported that a flow velocity from 18 to 40 µm/s leads to a rapid decrease in the growth rate of crystals smaller than 20 µm. Growth cessation of lysozyme crystals was observed by Nyce and Rosenberger 19 in a closed loop thermosyphon with a velocity of 5-15 mm/s compared to that in a stagnant batch. Vekilov and Rosenberger 20 investigated lysozyme crystal growth at a velocity from 0 to 2000 µm/s and noticed that the growth rate not only depends on the flow velocity, but also on the level of impurities and supersaturation. For example, in their experiments, a critical flow velocity existed such that for high-purity solutions the average growth rate increased when the velocity was smaller than the critical velocity, but decreased when the velocity was larger than the critical velocity. Crystal growth decelerated at any velocity for solutions containing ∼ 1% impurities. Finally, improvement in crystal quality by a forced flow has also been reported by several groups, which is probably due to the resulting higher kinetic stability for crystals grown by a spiral growth mechanism or the reduction of step growth rate for crystals grown by a 2D-nucleation growth mechanism. 20–24 In addition to adjusting global variables like pH, 25 temperature, 26 or solvent composition, 27 other strategies to promote protein crystallization, such as templates, 28 freezing out solvents, 29 switching a laser on and off, 30 using microwave, 31 ultrasound, 32 electric, 33 and magnetic fields, 34 have been explored. The utilization of electric fields is of particular interest due to some demonstrated control over the nucleation rate and location and the high flexibility in shaping the electric field spatially and dynamically. 35–39 Design variables such as electrode type, material and spatial arrangement as well as operational variables such as electric field strength and frequency can be adjusted flexibly. 40–45 Electric-field-assisted protein crystallization has been studied extensively in batch mode, often with the objective

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of producing high-quality material for crystallography or biomedical analysis. 46,47 However, electric-field-assisted crystallization is also attractive as a potential separation and purification technique for proteins in view of the challenges that currently exist related to the lack of control over protein nucleation in conventional crystallizers. Furthermore, the combination of electric-field-assisted protein crystallization and continuous flow is attractive, as in contrast to a batch process, in a continuous process not all material is processed at the same time. Therefore, scale-up limitations related to the need of applying an electric field to a large volume in a vessel can be mitigated in a flow process by exploiting the large surfaceto-volume ratio of a flow channel with patterned electrodes. However, to the best of our knowledge, such electric-field-assisted crystallization approach for the separation and purification of proteins from solution in continuous flow has not been reported. Consequently, insights into the feasibility and guidelines for design are lacking. The objective of this work is to characterize the feasibility and performance of electricfield-assisted protein crystallization in continuous flow. A transparent microfluidic device (MFD) made of polydimethylsiloxane (PDMS) with patterned electrodes is used to crystallize the model protein lysozyme in continuous flow. The dimensions of the flow channel are in the millimeter range instead of the conventional micrometer range 48 to avoid clogging. An additional benefit of those dimensions is that molds made from machining or 3D printing can be used with sufficient precision, avoiding more complicated procedures based on soft lithography. Our earlier work 45 on protein crystallization in non-uniform alternating current (AC) electric fields in batch mode provides the basis for design of the MFD with continuous flow in the present work. In particular, it was discovered for the MFDs operated in batch mode that indium tin oxide (ITO) can act as a template for nucleation. Furthermore, dielectrophoresis (DEP), which is a frequency-dependent phenomenon, can affect lysozyme nucleation. Negative DEP (i.e., when molecules tend to accumulate at minima of the electric field, which occurred at the glass surface) appeared to be influential for some cases at a frequency of 1 MHz. 45 To avoid fouling in the channels induced by the templating effect of

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ITO, thin layers of Ti/Au instead of ITO are used as electrodes. From a practical point of view, Ti/Au also allows for easier alignment of the electrodes with the flow channel, as ITO electrodes would be transparent. Furthermore, a coplanar configuration instead of parallelplate configuration is used as the basis for design of the electrodes. A coplanar configuration allows for better control over the gap between both electrodes, and thus over the strength of the electric field, without having to compromise on the flow conditions due to restrictions on the height of the channel. Consequently, a much smaller gap between the electrodes (200 µm) can be created with a coplanar design compared to when using a parallel-plate configuration (0.8 mm) so that a stronger electric field can be induced. To take advantage of the possible negative DEP response to concentrate proteins, an electric field with a frequency of 1 MHz is applied and the gap between the electrodes (glass surface) is aligned in the center of the flow channel where the liquid velocity is highest such that crystals can be dragged by the fluid in the device efficiently. Finally, since the objective of this work is to investigate the feasibility and performance of electric-field-assisted protein crystallization in continuous flow, the protein solution is recirculated as a practical way to vary the residence time in the MFD while maintaining flow conditions.

Experimental section Materials and solution preparation Lysozyme from chicken egg white (≥ 98%, L4919) and acetate buffer solution (pH 4.65, ∼0.22 M, Fluka 31103) were purchased from Sigma Aldrich. Sodium chloride was purchased from VWR. PDMS kit (Sylgard 184) was purchased from Dow Corning. All the chemicals were used without further purification. Solutions of lysozyme (60 mg/mL and 80 mg/mL) and NaCl (6% w/v, 8% w/v, 10% w/v and 12 % w/v) were prepared in acetate buffer and filtered (0.22 µm filters) before use.

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Solubility and metastable zone width measurement The solubility of lysozyme in the acetate buffer was obtained by provoking crystallization in a mixture containing 40 mg/mL lysozyme and 3% NaCl in an instrument for automated solubility measurements (Crystal16r , Technobis Crystallization Systems). The mixture was stirred at 25◦ C for 6 days after nucleation to ensure that saturation was reached. The slurry was then centrifuged (Genespeed X1, Gene) at 15000 g for 5 minutes and the protein concentration in the supernatant was determined by measuring the absorbance at 280 nm using NanoDrop 2000C spectrophotometer (ThermoFisher Scientific) and comparison with protein standards. The measurements were repeated four times and the average solubility of lysozyme in the acetate buffer (0.22 M, pH 4.65) was determined to be 6.3±0.2 mg/mL, which is about half the value as reported by Cacioppo and Pusey for lysozyme (12.4 mg/mL) in a 0.1 M sodium acetate buffer (pH 4.6) with 3% NaCl at 25◦ C. 49 This difference is expected as solubility decreases with the ionic strength of the solution. 49,50 The metastable zone width was characterized as a function of salt concentration to assure that the chosen solvent composition for the crystallization experiments provided a sufficiently large metastable zone width to observe any effect of electric fields and flow conditions. To determine the metastable zone width, equal volumes of lysozyme solution (60 mg/mL) and NaCl solution of various concentrations (6%, 8%, 10% and 12%) were mixed and kept at 25◦ C for three days within the Crystal16r parallel crystallizer under stirred conditions (900 rpm). The turbidity of the mixture was monitored continuously to determine the induction time for protein crystallization. Four identical experiments were performed for each salt concentration. For the mixtures with a salt concentration of 3% and 4%, no increase in turbidity was detected during three days, which indicates that the induction time under the tested conditions is at least three days. In contrast, for mixtures with a salt concentration of 5% and 6%, a sharp increase in turbidity was measured within one day due to the formation of protein crystals. Our results are consistent with literature, as a concentration of 29.5 mg/ml has been reported by others as the boundary of the metastable zone for lysozyme 6


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crystallization in micro-batch with a similar solvent environment (3% NaCl in 0.2 M acetate buffer with pH 4.7) but lower temperature (20◦ C). 51 In general, the metastable zone width depends on operating conditions and equipment type. Therefore, it is expected that the metastable zone width will change when we conduct lysozyme crystallization in a continuousflow device. A lysozyme solution with a concentration of 30 mg/mL and 3% NaCl has been selected for flow experiments. In batch such conditions do not lead to nucleation within at least three days, which provides a good reference if any reduction in induction time can be achieved in an electric-field-assisted flow device.

Fabrication of the MFD The workflow for fabrication of the MFD is illustrated in Figure 1(a-c). The MFD consisted of two coplanar Ti/Au electrodes on a glass wafer and an S-shaped microchannel in PDMS. The coplanar electrodes were fabricated using standard micro-fabrication techniques. A glass wafer of 4 inches in diameter was cleaned in acetone, isopropyl alcohol and water by sonicating for 15 minutes each and dehydrated in an oven at 120◦ C for 10 minutes. Then, the wafer was primed with HMDS, spin-coated with positive photoresist (HPR504, thickness of ∼1.5 µm), and exposed to UV light under a mask (designed in LayoutEditor, Juspertor GmbH, Germany). The exposed photoresist was dissolved in FHD-5 developer and the wafer was hard-baked at 120◦ C for 30 minutes before a thin layer of Ti/Au (10/100 nm) was sputtered on the surface. Finally, the wafer was sonicated in acetone to remove the unwanted Ti/Au and residual photoresist. To fabricate the microchannel, PDMS base agent and curing agent were mixed at a mass ratio of 10:1 and poured onto an aluminum mold made using a computer numerical control machining centre (Mikron VCP600). The mixture was degassed in a vacuum desiccator before being cured in the oven at 70◦ C for 1 hour. The cured PDMS was peeled from the mold and holes were punched to create an inlet and outlet. Uncured liquid PDMS with a mixing ratio of 10:3 was used as the adhesive to bond the cured PDMS piece with the electrode glass wafer. 52 The cured PDMS was placed on a silicon wafer 7



spin-coated with a thin layer of uncured PDMS (∼3 µm by spinning at 4000 rpm for 200 s) and quickly removed for attachment with the electrode glass wafer. The whole device was left to cure at room temperature for 48 hours before use. An example of the fabricated MFD can be seen in Figure 1(d). The PDMS channel with a square cross-section (width of 1.6 mm) was aligned with the coplanar electrodes so that the gap between the electrodes (200 µm) was kept nearly at the center of the channel (Figure 1(e)).

Crystallization experiments For crystallization, 0.8 mL of 60 mg/mL lysozyme solution was mixed with 0.8 mL of 6% NaCl solution in a 2 mL Eppendorf tube. The solution was pumped into the MFD (see Figure 2) via a two-channel peristaltic pump (Masterflex L/S, Cole Parmer) with a tubing c (L/S 13, Tygon Chemical) with an inner diameter of 0.8 mm and recirculated in the system

at a constant flow rate of 0.015 mL/min creating laminar-flow conditions (average velocity of ∼98 µm/s inside the MFD). PTFE tubing with an inner diameter of 0.56 mm and an outer diameter of 1.06 mm was used to connect the MFD with the pump tubing and the solution reservoir. The pump tubing was placed at the bottom of the Eppendorf tube and the PTFE tubing was positioned slightly above the top liquid surface to ensure the recirculation of the bulk solution. Subsequently, each of the two coplanar electrodes was connected to a function generator (33500B, Keysight Technologies) via copper tape with conductive adhesive (Agar Scientific) to apply an electric field (sine wave, 10V peak-to-peak, 1 MHz) between both electrodes. Two series of experiments were conducted, each with a different residence time, (1) 24 hours; (2) 48 hours. The volume of the flow channel was around 1 mL, so during the experiments 63% of the solution was subject to the electric field. For all tested conditions, control experiments were conducted in the absence of an electric field in, 1) the MFD, 2) in a batch system (Eppendorf tube) without agitation and, 3) in a batch system (total solution volume of 1 mL, in Crystal16r ) with agitation (900 rpm). All of the experiments were carried out at room temperature (around 25◦ C) in triplicate. Acetate buffer was used to clean the 8


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(a)

(b)

coplanar electrodes

PDMS channel

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MFD

UV glass wafer photoresist mask Ti/Au Al mold uncured PDMS cured PDMS (d)

(e) PDMS channel

gap between coplanar electrodes

coplanar electrodes

inlet

200 μm

outlet 1.6 mm

coplanar electrodes on glass wafer

PDMS channel

Figure 1: Fabrication workflow of (a) the coplanar electrodes on a glass wafer, (b) the Sshaped microchannel in PDMS, and (c) the MFD. (d) An image of the fabricated MFD and (e) a zoom-in image of the dashed area in (d) to demonstrate the alignment between the electrodes and the channel.

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system before each run. During the experiments, the MFDs were occasionally observed using a magnifying glass (60X) to identify the presence of the crystals. Although the MFDs were transparent, real-time observation with an optical microscope and digital camera to record the formation of crystals frequently in a specific part of the channel was practically limited for most of the experiments. In particular, the produced crystals were small and the thickness of the MFD prevented the use of a high-magnification objective due to the limited working distance. Furthermore, flow experiments in the presence of electric fields and control experiments were conducted with the same solution at the same time. Therefore, devices would have to be moved regularly when using an optical microscope, which might have caused disturbances. Nevertheless, we conducted one more set of experiments (identical to experiments no. 1 and 2, see Table 1) using two optical microscopes at the same time to record a series of in-situ images. In particular, one optical microscope (Nikon, Ni-U, 20X objective) with camera (Nikon, Digital Sight Qi2 cooled camera system) was used for the experiment with an electric field and another optical microscope (Nikon, Eclipse E100, 10X objective) with camera (Nikon, Digital Sight Fi1) was used for the control experiment. At the end of each experiment, samples were pumped out of the MFDs and collected in tubes for analysis. The sample slurry was placed on microscope slides and observed under an optical microscope (Nikon, Ni-U, 40X and 60X objectives) with digital camera (Nikon, Digital Sight Qi2 cooled camera system) to determine the crystal size distribution based on analysis of more than 800 crystals from the images (using ImageJ software). The protein concentration of the mother liquor was measured using a spectrophotometer (NanoDrop 2000C, ThermoFisher Scientific) as described earlier. The crystal yield was estimated based on the difference in protein concentration between the initial feed solution and the final mother liquor. The influence of the applied electric field on the lysozyme crystallization behavior in the MFD was characterized in terms of induction time, crystal yield and crystal size distribution.

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MFD

pump

solution

F Figure 2: Process flow diagram of the electric-field-assisted protein crystallization in continuous flow.

Results and discussion Almost no crystals were formed in any of the batch control experiments (static and agitated cases) with hardly any change in the protein concentration between the start and end of the experiments. When the solution was recirculated inside the system, crystals were formed in the runs for all experiments regardless of any application of an electric field (please see Table 1 for experimental conditions). Figure 3 shows in-situ images of crystals formed in continuous flow with and without an electric field. Small crystals can be observed in the MFD after 24 hours both for the experiment with electric-field-assisted crystallization and for the control experiment without an electric field. The reduction of the induction time in flow mode compared to the batch quiescent mode of operation is consistent with earlier reports on flow-enhanced crystallization for lysozyme and insulin. 12,15 If the shear rate is too high, such as in the batch experiment with high-speed agitation, nucleation may be inhibited by the shear flow. 16 For all the experiments, the initial protein concentration (c0 ) is 30 mg/mL, which corresponds to a relative supersaturation of 4.8. According to the

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metastability measurement, this supersaturation created at the beginning of each experiment is expected to fall in the metastable zone of the stirred batch system, where no spontaneous nucleation is expected. Thus, the formation of crystals in the MFD indicates that the metastable concentration is lower in the MFD than in batch. control

200 µm with EF

Figure 3: Microscope images of the channel of the MFD for experiments conducted under optical microscopes after 24 hours with (bottom) and without (top) an electric field. The areas with crystals visible is the gap between the coplanar electrodes.

Table 1 lists the average final protein concentration cf and supersaturation Sf for all the experiments conducted in the MFD. The crystallization yield y is defined as

y=

c0 − cf , c0 − c∗ 12


(1)

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Table 1: Average Final Protein Concentration (mg/mL) and supersaturation for experiments in the MFD. no. residence electric field time (h) 1 24 no 2 24 yes 3 48 no 4 48 yes

c0 cf Sf mg/mL mg/mL 30 25.5±3.6 4.1±0.6 30 25.3±2.0 4.0±0.3 30 26.9±2.8 4.3±0.4 30 21.8±0.2 3.5±0.03

where c∗ is the saturated concentration. The calculated yield is plotted in Figure 4 as a function of the residence time in the system. The solid vertical lines correspond to the 90% confidence intervals of the experiments conducted in triplicate. For a residence time of 24 hours, although a narrower confidence interval exists when the electric field is applied, no statistically meaningful difference in the yield obtained from electric-field-assisted protein crystallization in flow and the control experiments without electric field can be seen as the error bars overlap. The large variability is caused by a significant outlier for both cases, which can be expected in a system that is dominated by primary nucleation. When the residence time is increased from 24 to 48 hours, a significant increase in the yield obtained by the electric-field-assisted protein crystallization in flow can be seen. Furthermore, the variability of the yield obtain by the runs with the electric field is much lower. In contrast, extending the residence time from 24 to 48 hours does not make a significant difference in the average yield and confidence interval for the control experiment without the electric field. The reduction of the protein concentration may be attributed to the growth of existing crystals or to the nucleation of new crystals during the experiments. The lack of increase in yield for the control case suggests that in absence of the electric field no significant crystal growth or nucleation occurs in the second half of each run, whereas in the presence of electric fields apparently additional growth or nucleation occurs in the second half of each run. Figure 5 shows representative microscope images of crystals formed in the experiments conducted in the MFD (see Table 1 for descriptions). Although the edges of the crystals are not very clear (due to the small size and the limitation of the optical microscope), it

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50

control with EF

40

Yield (%)

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30

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10

0

0

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Residence time (h)

50

60

Figure 4: Average crystal yield as a function of the residence time for experiments in the MFD with an electric field of 10 Vpp and 1 MHz (circle) and without an electric field (diamond). can be seen that the crystals have a tetragonal or hexagonal shape. The average crystal size distribution is shown in Figure 6 with the 90% confidence intervals, which were calculated from the triplicate experiments. Despite the differences in experimental conditions, the crystal size distributions exhibit the same features for all experiments with the majority of crystals in the range of 3-7 µm. It is likely that differences in crystal distribution would be observed if the increase in yield was caused by significant additional crystal growth. Therefore, any significant changes in yield are most likely due to the formation of new crystals. No statistically meaningful differences between the size distributions of the crystals obtained from experiment 1 and 2 can be seen (top panel of Figure 6). Since no significant difference could be seen for the yields as well, it is concluded that applying the electric field does not influence the average crystal size and number for experiments in the MFD when a residence time of 24 hours is applied. When extending the residence time to 48 hours (bottom panel of Figure 6), no statistically meaningful difference in the crystal size distribution between the experiment with and without an electric field can be observed. Therefore, it is most likely that the increase in the crystal yield that only occurs when extending the residence time of the electric-field-assisted protein crystallization in flow should

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be attributed to the formation of new crystals rather than to the growth of existing crystals. Our results imply that the secondary nucleation rate in continuous flow is higher under the application of the electric field compared to the same conditions without an electric field. Therefore, although crystals will be formed initially for both the cases with and without the electric field, in the second half of a long experiment, when supersaturation is lower, protein nucleation will only be significant when the electric field is applied. Furthermore, our results imply that the crystal growth of proteins is restricted under the current flow conditions, as the difference in the average crystal size distribution observed for all the experiments in flow is negligible. In principle, the used peristaltic pumps to circulate the suspensions could be a source of attrition, which may also limit the growth of the crystals. However, the formed crystals are very small and, therefore, less prone to attrition, as similar experiments in literature have demonstrated that the mechanical stress caused by such pumps is typically not sufficient to limit the crystal size even of much larger lysozyme crystals. 53 Impurities may also restrict the growth of protein crystals. The purity of lysozyme used in this work is more than 98%, which is comparable to the purity commonly used for lysozyme crystallization in the literature. 15,38,54 In our previous work, we used the same lysozyme powder for electricfield-assisted crystallization in micro-batches without agitation and obtained high-quality crystals with sizes of up to several hundreds of microns after 48 hours. 45 Convective transport of the solute and of impurities can cause a reduction of lysozyme crystal growth under forced solution flow, 20 which may explain our observation of restricted crystal growth under flow conditions in the MFD compared to the crystal growth under stagnant conditions in batch from earlier work.

Conclusions Electric-field-assisted protein crystallization in continuous flow has been realized in a MFD with patterned microelectrodes in a coplanar configuration. The flow conditions enable a

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24 h, control

24 h, with EF

48 h, control

48 h, with EF

Figure 5: Microscope images for crystals formed in experiments conducted in the MFD as described in Table 1.

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0.4

24 h, control 24 h, with EF

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Frequency

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0.15 0.1

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Crystal Size (µm)

48 h, control 48 h, with EF

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0.25 0.2

0.15 0.1 0.05 0

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Crystal Size (µm)

Figure 6: Average crystal size distribution for experiments conducted in the MFD as described in Table 1.

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shorter induction time compared to batch experiments. Furthermore, at least under the tested conditions, operation in continuous flow restricts the maximum attainable crystal size. Applying an electric field allows for a higher secondary nucleation rate throughout an experiment and, consequently, for a larger total number of crystals to be formed. Therefore, electric-field-assisted protein crystallization in continuous flow can increase the crystallization yield compared to control cases without electric fields and batch experiments. For future work, this novel approach for protein crystallization in continuous flow has still much room for optimization, as various parameters such as flow rate (shear), flow direction, distribution of the electric-field potential and frequency can be adjusted. Furthermore, although the volume of solution that can be handled by the MFD is larger than what is typically reported for other electric-field-assisted crystallization setups in literature, understanding the scale-up behavior is an important question for future research, which can benefit from inherent scale-up advantages of continuous flow such as easy parallelization. In addition, during the experiments, 63% of the solution was subject to the electric field and the rest was in the tubing or the solution reservoir. Therefore, the nett time that the solution was subject to the electric field was either 15 hours or 30 hours. Thus, besides increasing the total residence time to extend the exposure of the solution to the electric field, practical strategies such as using shorter tubing for connections or integrating multiple MFDs in a single line may be adopted. The MFD may be integrated with other crystallization equipment downstream, using the large number of small crystals formed as seeds with large surface-tovolume ratio for further growth in a dedicated growth compartment. Such integration allows for the decoupling of crystal formation and growth, which is of fundamental importance for the optimal design of future protein crystallization processes. Finally, electric-field-assisted protein crystallization in continuous flow could potentially be of interest for biopharmaceutical applications for which a high process yield and a large surface-to-volume ratio of the produced crystals is desirable to facilitate a high bioavailability.

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Acknowledgement The authors thank Prof. Fei Sun from the Hong Kong University of Science and Technology for support with the NanoDrop 2000C spectrophotometer. The work described in this paper was supported by a grant from the Research Grants Council of the Hong Kong Special Administrative Region, China (Project No. 16242916).

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For Table of Contents Use Only

Electric-field-assisted protein crystallization in continuous flow Fei Li and Richard Lakerveld∗ Department of Chemical and Biological Engineering, The Hong Kong University of Science and Technology, Hong Kong, China E-mail: [email protected]

protein solution 48 h test

control, yield=13.3%±11.3%

EF, yield=34.4%±0.7%

Synopsis A microfluidic device with coplanar electrodes has been developed for electric-fieldassisted protein crystallization in continuous flow. Protein crystallization is enhanced with reduced induction time in flow compared to batch. The application of an electric field increases crystallization yield in continuous flow by promoting secondary nucleation.

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Electric-Field-Assisted Protein Crystallization in Continuous Flow

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