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Enhancement of lysozyme crystallization under ultrasound field
Journal Pre-proofs Enhancement of Lysozyme Crystallization under Ultrasound Field Yafei Mao, Fei Li, Ting Wang, Xiaowei Cheng, Guiping Li, Danning Li, Xiunan Zhang, Hongxun Hao PII: DOI: Reference:
S1350-4177(19)31779-1 https://doi.org/10.1016/j.ultsonch.2020.104975 ULTSON 104975
To appear in:
Ultrasonics Sonochemistry
Received Date: Revised Date: Accepted Date:
7 November 2019 14 January 2020 14 January 2020
Please cite this article as: Y. Mao, F. Li, T. Wang, X. Cheng, G. Li, D. Li, X. Zhang, H. Hao, Enhancement of Lysozyme Crystallization under Ultrasound Field, Ultrasonics Sonochemistry (2020), doi: https://doi.org/10.1016/ j.ultsonch.2020.104975
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© 2020 Published by Elsevier B.V.
Enhancement of Lysozyme Crystallization under Ultrasound Field
Yafei Maoa, Fei Lia, Ting Wanga, Xiaowei Chenga, Guiping Lia, Danning Lia, Xiunan Zhanga, Hongxun Hao a,b*
a
National Engineering Research Center of Industrial Crystallization Technology,
School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China b
Collaborative Innovation Center of Chemical Science and Engineering (Tianjin),
Tianjin 300072, China
*Corresponding
author.
E-mail address: [email protected] (H. X. Hao)
(Submitted to: Ultrasonics Sonochemistry)
ABSTRACT: With the increasing demand for biopharmaceuticals, a method to crystallize biomolecule products with high quality, high yield and uniform size distribution as well as regular crystal habit is needed. In this work, ultrasound was used as a nucleation accelerator to decrease the energy barrier for lysozyme crystal formation. Crystallization experiments on egg-white lysozyme were carried out with and without ultrasound. The effect of ultrasound on induction time, metastable zone width, crystal size and morphology and process yield was investigated in detail. The nucleation-promoting effect produced by ultrasound is illustrated by the reduction of metastable zone width and induction time. By inducing faster nucleation, ultrasound leads to protein crystals grow at lower supersaturation levels with shorter induction time. It was found that ultrasound could result in uniform size distribution of the product due to the preventing of aggregation. However, long time continuous application of ultrasound could result in smaller particle size. Hence, ultrasonic-stop method was found to be a more appropriate strategy to enhance the crystallization process of proteins such as lysozyme.
Keywords: Ultrasound; protein crystallization; lysozyme; nucleation
1. Introduction With the fast developing of the global biopharmaceutical market, products with high quality, short development times and ideal morphology in manufacturing are urged for the competitive environment. As an effective separation and purification unit operation, crystallization has already been successfully used in biopharmaceutical industry [1-3]. Compared to conventional packed-bed chromatography, crystallization has several advantages, such as higher protein titer and larger volume from upstream processes, thus leading to a more flexible scale-up [4-6]. Among many crystallization approaches [7-9], the most common one in protein crystallization is salting-out precipitation [10]. However, this process usually results in a broad crystal size distribution (CSD) and low crystal quality [11, 12]. It often causes difficulties for downstream process such as tableting, filtration and washing, leading to reduced recovery rate and poor final product quality [13]. Hence, in order to get products of better stability, fluidity and bioavailability, the control and enhancement of this crystallization process, usually nucleation [14-16] and growth process [17], is extremely important. Recently, different methods are explored to strengthen the process of crystallization, In addition to regulating common variables such as pH [18], temperature [19], and solvent composition [20], other strategies, such as templates [21], freezing out solvents [22], adding laser [23], and using microwave [24], ultrasound [25], electric [26], magnetic fields [27], and membrane crystallization [28], have been discussed. Among these approaches, sonocrystallization, which applies ultrasound in crystallization process, has been proven to be an effective way to enhance nucleation process. In recent years, ultrasound has received widespread attentions because of its unique physical attribution and huge energy in transient. Since the promoting effect of sonocrystallization was first observed in 1927 [29], many benefits of sonocrystallization have been illustrated: decreased metastable zone width (MSZW), shorter induction time, narrowed crystal size distribution, higher crystal purity, improved product homogeneity, faster crystallization time, preferential
polymorph formation and better process repeatability [30-33]. However, the promoting effect of ultrasound is mainly investigated for inorganic and small organic molecules [34, 35]. Application of ultrasound in biomacromolecule especially for protein crystallization was seldom investigated. Crespo et al. [25] has demonstrated the potential use of ultrasound by using plate transducer, and described the change of MSZW and the PXRD of lysozyme crystals. However, the mechanism of nucleation and growth in protein sonocrystallization process is not illustrated. Furthermore, despite the effect of process parameters such as temperature and solute concentration on protein crystallization has been discussed, the effect of sonication parameters like power and sonication time on protein crystallization has not been well studied. Besides, the effect of ultrasonic irradiation on lysozyme yield has not been investigated yet. Hence, in this work, the effect of ultrasound on the crystallization of was investigated from both thermodynamic and kinetic perspectives. The influences of different sonication parameters (power and sonication time) were also explored. Specifically, the effect of ultrasound on MSZW, induction time, crystal size distribution, crystal morphology and the yield of protein crystallization process were investigated by using lysozyme as model compound. More importantly, the mechanism of how ultrasound affects nucleation and growth process was discussed in detail.
2 Experimental 2.1. Materials and solution Lysozyme from chicken egg white ( ≥ 98%, L4919) was purchased from Sigma-Aldrich. Other chemicals used were of analytical reagent grade. Acetate and sodium acetate anhydrous were purchased from Aladdin Industrial Co., China. Sodium chloride was purchased from Tianjin Yuxiang Chemical Reagent Co., Ltd., China. Water used for all experiments was supplied by the Millipore water system
(Milli-Q) with a resistivity of 18.2 MΩ cm at 298 K. All of the chemicals were used without further purification. Acetate buffer solution, which was fixed at pH=4.60 (Mettler Toledo-FE28) and 0.1 M, is consisted of acetate and sodium acetate anhydrous. All solutions of lysozyme and NaCl were prepared in acetate buffer and filtered (0.22 μm filters) before use. 2.2. Metastable zone width and induction time measurement To determine the MSZW, NaCl solutions with various concentrations (5%-15%) were quickly added into various volumes of lysozyme solution (60 mg/mL). Namely, through controlling the mixture ratio of the two solutions, different lysozyme and NaCl concentration could be achieved. In this way, the metastable zone curves could be obtained from the minimum NaCl concentration needed for the appearance of crystals at different lysozyme concentrations. Meantime, the induction time was also recorded. The experiments were carried out by using batch crystallization at 293 K under magnetic stirred conditions in order to guarantee constant supersaturation until the moment of crystal formation. Each salt concentration was measured for three times. Since the induction time was going to be longer when the concentration of lysozyme decreased, 24 h was chosen as the maximum time to reach phase equilibrium in this study. The experiments apparatus were shown in Fig. 1. Ultrasonic irradiation frequency is fixed at 20 kHz using an ultrasonic homogenizer workstation (Nanjing, China ATPIO-SM200). The horn depth (2 cm) and the tip diameters (10 mm) were fixed in this work. The power was set to 20 W and 80 W. The temperature was kept constant (293 ± 0.5 K) by using thermostat with a thermoelectric controller (Nanjing, China ATPIO-2006N). Table 1 shows the experiment parameters of the metastable zone and induction time measurements.
Fig. 1. Schematic diagram for the metastable zone width and induction time experiments: (1) noise reduction box; (2) the solution of sodium chloride; (3) the solution of lysozyme; (4) magnetic stirrer; (5) ultrasonic horn; (6) thermometer; (7) 50 mL water-jacketed vessel; (8) cooling water.
Table 1 The Experimental Parameters used in Metastable Zone and Induction Time Measuring Experiments Exp. No.
frequency
1 2
power
temperature
0 20 KHz
3
20
293 K
80
2.3 Crystallization experiments After measuring the MSZW and induction time, 15 mL of 8% (w/v) NaCl solution was added to 15 mL of 60 and 70 mg/mL lysozyme solutions respectively in a 50 mL jacked crystallizer for subsequent crystallization experiments. The vessel was sealed during the whole experiment to prevent evaporation. The salt solution was pumped into the lysozyme solution via a peristaltic pump (Longer, BT-100-1F) with the rate of 0.3 ml/min in order to prevent the unexpected suddenly happening of nucleation.
When the mixing was finished, the ultrasonic was applied instantly. The electrical power to produce the ultrasound is fixed at 80 W. The period of the ultrasound burst is set to 2 s with a pause of 2 s. During the process, the temperature was kept at 293 ± 0.5 K by using two thermostats in parallel because the ultrasound would release so much energy that the temperature of solution would be increased. And the agitation rate was maintained at 300 rpm by using a magnetic stirring. To identify the moment of nucleation, the laser was used to measure the turbidity. At the time of nucleation, which could have enough turbidity to be measured by the laser, the solution began to be sampled at intervals of 20 min. After one hour, the sample is taken once an hour, and the whole sample time is controlled within five or fourteen hours. The sample was firstly observed under an optical microscope (Nikon, Eclipse E200MV, 10× and 40× objective) with a digital camera (Nikon, Digital Sight Fi1) to study the morphology. Then, crystal size distribution was determined on the basis of analysis of more than 800 crystals from the images (using ImageJ software). Immediately, the slurry was centrifuged (Xiangyi, H1650-W) at 15000 g for 3 min. The protein concentration in the supernatant was determined by measuring the absorbance at 280 nm using an ultraviolet spectrophotometer (Hitachi, U-3010). The crystal yield was calculated on the basis of the difference in protein concentrations between the initial feed solution and the final supernatant mother liquor. Every measurement was repeated three times. In the presence of ultrasound, the experiments were divided into two groups: the continuous ultrasonic group, in which the ultrasound was applied during entire experiment, and ultrasonic-stop group, in which the ultrasound was stopped when nuclei was formed. For all tested conditions, control experiments were performed in the absence of an ultrasonic field in the same batch system with the same experimental conditions. The experiments apparatus were shown in Fig. 2. All conditions and parameters in this part are shown in Table 2.
Fig. 2. Schematic diagram for crystallization experiments: (1) noise reduction box; (2) peristaltic pump; (3) the solution of lysozyme; (4) magnetic stirrer; (5) ultrasonic horn; (6) thermometer; (7) 50 mL water-jacketed vessel; (8) cooling water; (9) the solution of sodium chloride; (10) constant temperature water; (11) thermoelectric controller; (12) ultrasonic generation control system.
Table 2 The Experimental Parameters used in Crystallization Experiments
No.
1 2 3 4 5 6
group
lysozyme
NaCl
concentration
concentration
(mg/ml)
(w/v)
continuous ultrasonic
30
ultrasonic -stop
30
control
power
35
35
(W)
time
(KHz)
80
2s ultrasound with 2s pause
20
0
no
no
4%
30 35
sonication frequency
3 Results and Discussion 3.1 Effect of ultrasound on the width of metastable zone The influence of ultrasound on metastable zone is shown in Fig. 3. The metastable zone was characterized as a function of salt concentration since salting out crystallization technology was applied in this work. The solubility curve was obtained from Forsythe et al [36]. Crystals were obtained at lower protein concentrations when
using ultrasound, as MSZW is narrower. As shown in Fig. 3, although the power of 20 W had already narrowed the MSZW to some extents, 80 W power narrowed the width more obviously. This means that the nucleation energy barrier was decreased in the presence of ultrasound and hence the nucleation of lysozyme became easier. For example, at Clys = 30 mg/ml the concentration of NaCl needed to produce crystals in the presence of ultrasound (20 W) is about 60% of the corresponding concentration in the absence of irradiation. According to two-step nucleation theory [37, 38], the variation of supersaturation solution could lead to the formation of disordered protein-rich dense clusters firstly. Then, when the size of cluster of solute molecules reaches a sufficient size, the cluster will reorganize into an ordered structure, and finally the ordered crystalline nuclei will be formed. The dense liquid is usually unstable and has a higher free energy than the dilute solution and hence nucleation would likely occur from the protein-rich dense metastable molecular clusters [39, 40]. In the whole nucleation process, the first maximum free energy (ΔG1*) is needed to form the dense liquid. Then, the crystals were formed by crossing the second maximum free energy (ΔG2*). Generally, the primary nucleation of protein will only occur when the two critical energy of nucleation are overcome under certain temperature and protein concentration. When ultrasound is applied, the energy needed for nucleation could be overcome more quickly. The acoustic cavitation effect [30, 41-44] of ultrasound may contribute to this promotion. When ultrasonic wave spreads into solution, the pressure variation creates the cavitation phenomena, which is the formation, growth and collapse of bubbles [45]. When bubble collapses, it will cause rapid adiabatic compression of gases and vapors inside the bubble. At the meantime, very high pressure (up to tens of GPa) and temperature (up to tens of thousands K), as well as shock waves, microjets and micro-turbulences [46] will also be generated. Therefore, the energy released by the shock waves during the collapse of cavitation bubbles could overcome the critical energetic barrier required for spontaneous nucleation [47], and solid-liquid mass transfer would be enhanced so that nucleation is promoted. Besides, pressure gradients generate by oscillating bubbles could allow small solute molecules to
aggregate to an oscillating bubble wall. These solutes can then cluster and reach the sufficient cluster size necessary for crystal nucleation. When the solute clusters grow large enough, they are then pushed away from the oscillating bubble and into the bulk phase [48]. Hence, in the whole process, the supersolubility curve will drop down, and the MSZW would decrease. Solubility Supersolubility Without Ultrasound Supersolubility With Ultrasound-20 W Supersolubility With Ultrasound-80 W
50
Clys (mg/mL)
40
30
20
10
0 1
2
3
4
5
6
7
8
CNaCl (% w/v) Fig. 3. The metastable zone of lysozyme with various ultrasound power at 293 K and pH 4.6. 3.2 Effect of ultrasound on induction time The effect of ultrasound on induction time is shown in Fig. 4. In the absence of ultrasound, the induction time increases exponentially with the decreasing of the concentration of lysozyme. More importantly, when the ultrasonic was applied, the induction time was evidently decreased and the induction time of the sonicated experiments was significantly shorter than the control experiments, especially obvious at lower supersaturations. What’s more, it can also be found that the induction time after application of ultrasound was less affected by the concentration of the solutions. Generally, small solute molecules around the oscillating bubble could easily grow up
to the critical nucleation cluster size. When the bubbles collapse, the temperature would rapidly decrease, which will also enhance the nucleation. Therefore, it can be suggested that irradiating the solution with ultrasound favors the generation of acoustic cavitation, which could promote the formation of clusters and hence shorten induction time. Such approach would help to reduce the total time of the crystallization and hence increase the efficiency of the crystallization of protein.
600
Ultrasound—80W Ultrasound—20W Without ultrasound
500
tind/min
400 300 200 100 0 25
30
35
Clys/mg·ml-1 Fig. 4. The histogram of induction time of the salt concentration of 4% and the lysozyme concentration of 25, 30 and 35 mg/ml at 293 K and pH 4.6 with the presence and absence of ultrasonic. 3.3 Effect of ultrasound on morphology and size distribution Lysozyme crystals morphology and size distribution obtained by batch crystallization under different conditions are shown in Fig. 5 and Fig. 6 respectively. It can be seen that most of lysozyme crystals obtained from control experiments (without ultrasound) show non-uniform size distribution and aggregation (Fig. 5c, f). The crystal size in this group was counted by using the mean size of the aggregation and calculated in the triplicate experiments. The mean size of the aggregation is 43.7
μm in the 35 mg/ml lysozyme concentration, and the size distribution is broad as shown in Fig. 6. This might be attributed to unexpected secondary nucleation and aggregation. Interestingly, compared to the broad size distribution of aggregated crystals in the control group, the crystals of lysozyme obtained from ultrasonic group and ultrasonic-stop group showed sharper and narrower distributions as shown in Fig.6. The results show that the ultrasound can significantly improve the size uniformity. Meanwhile, from Fig. 5, it can be seen that, although the crystals obtained from control group are larger than crystal obtained from the continuous ultrasound and ultrasound-stop group, most of them are aggregated crystals, especially under the condition of C0=30 mg/ml. The mode of ultrasonic can also affect the morphology and size distribution of lysozyme crystals. From Fig. 5 and Fig. 6, it can be seen that, when continuous ultrasound is applied, the size of the final products are quite small (only several micrometers) although the size distributions are quite narrow. Especially in the condition of 30 mg/ml lysozyme concentration, the morphology of the crystals was even too small to be clearly observed in the view of regular microscope (Fig. 5d). On one hand, ultrasound could enhance both primary nucleation and secondary nucleation, which will result in more nuclei. On the other hand, ultrasound could also restrain the growth of lysozyme crystals and result in breakage of crystals [49]. These two reasons can explain the appearance of very tiny crystals (Fig. 5d) when continuous ultrasound was applied. The possible explanation for the improved size distribution is the ameliorating of aggregating by ultrasound. The shock wave [50], which is caused by cavitation, can shorten contact between crystals to an extent, precluding their bonding together and thus reducing the chance of aggregating. These small and well dispersed crystals generated by continuous ultrasonic could act as crystal seeds, which will be a good strategy for the industrial crystallization of some proteins. To overcome the shortcoming of continuous ultrasonic, ultrasonic-stop mode was applied. From the data presented in Fig. 5 and Fig. 6, it can be seen that the morphology of the obtained crystals (Fig. 5b, e) are clearly improved, with more
regular shape and much bigger size, without sacrificing the uniform size distribution. The possible explanation for the better shape and bigger size of the products from the ultrasonic-stop mode experiments is that the cease of ultrasound after nucleation can lower down the rate of secondary nucleation (or breakage) of the obtained crystals. Meanwhile, from comparison of Fig. 5(b) and Fig. 5(e), it can be seen that products obtained from higher concentration have bigger size. This possible reason might be that higher lysozyme concentration can provide more solute for the nucleated crystals to grow into larger size. In a word, ultrasound could effectively enhance nucleation and prevent aggregating. But long time ultrasound (too much energy injected) could also result in smaller particle size. Therefore, ultrasonic-stop mode will be a more effective approach for crystallizing protein with uniform and bigger size.
a)
b)
c)
d)
e)
f)
Fig. 5. Microscope images for lysozyme crystals formed at 293 K and pH 4.6. (a) continuous ultrasonic at C0=35 mg/ml. (b) ultrasonic-stop at C0=35 mg/ml. (c) in the absence of ultrasonic irradiation at C0=35 mg/ml. (d) Continuing ultrasonic at C0=30 mg/ml. (e) ultrasonic-stop at C0=30 mg/ml and (f) in the absence of ultrasonic irradiation at C0=30 mg/ml.
0.30
ultrasonic ultrasonic-stop control 35 mg/ml lys+ 4% NaCl
0.25
Frequence
0.20
0.15
0.10
0.05
0.00 0
10
20
30
40
50
60
70
80
90
100
110
120
130
Crystal size (μm) Fig. 6. The crystal size distribution of lysozyme under different conditons at 35 mg/ml lysozyme with 4% Nacl 3.4 Effect of ultrasound on yield Table 3 lists the average final protein concentration Cf and final supersaturation Sf for all the experiments. The crystallization yield y and S are defined as
𝑦=
𝐶0 ― 𝐶𝑓 𝐶0 𝐶
𝑆 = 𝐶∗
(1) (2)
where C0 is the initial protein concentration, C is actual concentration and C* is the equilibrium concentration of lysozyme. By using the data listed in Table 3, the yields of different crystallization experiments were calculated. The results are shown in Fig. 7. For the ultrasonic-stop condition, the yield of the process quickly reaches the maximum level after nucleation and the whole crystallization process could be finished in around 300 minutes. But for the continuous ultrasonic condition, the final yield of the process is lower than the yields of other two conditions although the nucleation could happen much earlier than no ultrasound condition. As for the control group, although its final yield was
approximately equal to the ultrasonic-stop condition, much longer time is needed to reach the same yield due to the long induction time. Table 3 Average Final Protein Concentration and Supersaturation expt no.
time (h)
ultrasonic field
C0 (mg/mL)
S0
Cf (mg/mL)
Sf
1
5
yes
35
11.4
13.1 ± 0.2
4.3 ± 0.06
2
5
only during nucleation
35
11.4
9.0 ± 0.3
3.0 ± 0.1
3
5
no
35
11.4
11.2 ± 0.9
3.7 ± 0.3
4
14
yes
30
9.8
13.4 ± 0.2
4.4 ± 0.06
5
14
only during nucleation
30
9.8
8.5 ± 0.3
2.8 ± 0.1
6
14
no
30
9.8
9.3 ± 1.1
3.0 ± 0.4
The faster process in the ultrasound-stop group could be due to the ultrasound cavitation effect which is able to enhance heat and mass transfer by triggering the micro-turbulence [46]. The cavitation effect can be responsible for increasing of micromixing and nucleation rate and for the generation of plenty of supersaturation locations. The higher yield of this condition could be attributed to the bigger size of the obtained crystals, which could reduce the loss of produce from following centrifuging and washing step. This explanation can be supported by the observation of the crystal morphology and size in the above discussion. For the phenomenon of lower yield in continuous ultrasonic group, the first possible reason is protein denaturation. Long time ultrasound would bring considerable energy to this system. Some studies have demonstrated that long-term ultrasound would damage the protein crystals [51-53] since protein is incredibly sensitive to high temperature which would be triggered by continuous energy input. Furthermore, the second possible explanation is higher solubility and more difficult solid-liquid separation due to smaller crystals. From Fig. 5 and Fig. 6, continuous ultrasound will result in much smaller crystal size. On one hand, particle size could
affect the solubility of the solute. When the particle size is too small (smaller than several microns), it would result in higher solubility and hence lower yield. On the other hand, small crystals can make the solid-liquid separation (centrifuge in this case) difficult. Because some tiny lysozyme crystals will remain in the supernatant and redissolve, the final yield of the crystallization process will be reduced. In addition, prolonging ultrasonic time will consume more electricity energy with no further obvious promotion, which has been proved by literatures [32, 49, 54]. Therefore, compared with protein denaturation and smaller crystal size caused by continuous ultrasonic and the lower efficiency in the control group, the ultrasonic-stop method can produce better lysozyme product with higher yield in a shorter time. Hence, ultrasonic-stop method will be a good strategy for crystallizing proteins such as lysozyme with lower cost and higher efficiency.
80
a
70 60
Yield (%)
50 40 30
ultrasonic ultrasonic-stop control 30 mg/ml lys + 4% NaCl
20 10 0 0
100
200
300
400
500
600
Sample time (min)
700
800
900
80
b
Yield (%)
60
40
ultrasonic ultrasonic-stop control 35 mg/ml lys + 4% NaCl
20
0 0
50
100
150
200
250
300
350
Sample time (min) Fig. 7. The yields at different time in control group, ultrasonic-stop group and continuous ultrasonic group at (a) 30 mg/ml lysozyme with 4% NaCl (T0=19 min) and (b) 35 mg/ml lysozyme with 4% NaCl (T0=15 min)
4. Conclusions This work demonstrated the positive nucleation enhancement effect of ultrasonic in lysozyme crystallization process. When ultrasound was applied, the metastable zone width was evidently narrowed and the induction time was significantly reduced. However, long time continuous ultrasonic would input too much energy which may cause lysozyme denaturation and ultrasound wave could also restrain the growth of crystals. Therefore, the ultrasonic-stop method, in which ultrasound is only applied during the nucleation process, was suggested. It was found that the ultrasonic-stop method can produce better lysozyme crystals with uniform size distribution and intact morphology. The crystallization time was also shortened and hence the efficiency of the whole crystallization process was improved.
Acknowledgements This work was financially supported by the Tianjin Natural Science Foundation (grant number 18JCYBJC40800).
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Highlights
Ultrasound was successfully applied to enhance lysozyme crystallization process.
The effect of ultrasound on induction time, metastable zone and yield was investigated.
The effect of ultrasound on crystal size and morphology was investigated.
The affecting mechanism of ultrasound on lysozyme crystallization was elucidated.
Ultrasonic-stop method was found to be more appropriate strategy.
S1350-4177(19)31779-1 https://doi.org/10.1016/j.ultsonch.2020.104975 ULTSON 104975
To appear in:
Ultrasonics Sonochemistry
Received Date: Revised Date: Accepted Date:
7 November 2019 14 January 2020 14 January 2020
Please cite this article as: Y. Mao, F. Li, T. Wang, X. Cheng, G. Li, D. Li, X. Zhang, H. Hao, Enhancement of Lysozyme Crystallization under Ultrasound Field, Ultrasonics Sonochemistry (2020), doi: https://doi.org/10.1016/ j.ultsonch.2020.104975
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© 2020 Published by Elsevier B.V.
Enhancement of Lysozyme Crystallization under Ultrasound Field
Yafei Maoa, Fei Lia, Ting Wanga, Xiaowei Chenga, Guiping Lia, Danning Lia, Xiunan Zhanga, Hongxun Hao a,b*
a
National Engineering Research Center of Industrial Crystallization Technology,
School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China b
Collaborative Innovation Center of Chemical Science and Engineering (Tianjin),
Tianjin 300072, China
*Corresponding
author.
E-mail address: [email protected] (H. X. Hao)
(Submitted to: Ultrasonics Sonochemistry)
ABSTRACT: With the increasing demand for biopharmaceuticals, a method to crystallize biomolecule products with high quality, high yield and uniform size distribution as well as regular crystal habit is needed. In this work, ultrasound was used as a nucleation accelerator to decrease the energy barrier for lysozyme crystal formation. Crystallization experiments on egg-white lysozyme were carried out with and without ultrasound. The effect of ultrasound on induction time, metastable zone width, crystal size and morphology and process yield was investigated in detail. The nucleation-promoting effect produced by ultrasound is illustrated by the reduction of metastable zone width and induction time. By inducing faster nucleation, ultrasound leads to protein crystals grow at lower supersaturation levels with shorter induction time. It was found that ultrasound could result in uniform size distribution of the product due to the preventing of aggregation. However, long time continuous application of ultrasound could result in smaller particle size. Hence, ultrasonic-stop method was found to be a more appropriate strategy to enhance the crystallization process of proteins such as lysozyme.
Keywords: Ultrasound; protein crystallization; lysozyme; nucleation
1. Introduction With the fast developing of the global biopharmaceutical market, products with high quality, short development times and ideal morphology in manufacturing are urged for the competitive environment. As an effective separation and purification unit operation, crystallization has already been successfully used in biopharmaceutical industry [1-3]. Compared to conventional packed-bed chromatography, crystallization has several advantages, such as higher protein titer and larger volume from upstream processes, thus leading to a more flexible scale-up [4-6]. Among many crystallization approaches [7-9], the most common one in protein crystallization is salting-out precipitation [10]. However, this process usually results in a broad crystal size distribution (CSD) and low crystal quality [11, 12]. It often causes difficulties for downstream process such as tableting, filtration and washing, leading to reduced recovery rate and poor final product quality [13]. Hence, in order to get products of better stability, fluidity and bioavailability, the control and enhancement of this crystallization process, usually nucleation [14-16] and growth process [17], is extremely important. Recently, different methods are explored to strengthen the process of crystallization, In addition to regulating common variables such as pH [18], temperature [19], and solvent composition [20], other strategies, such as templates [21], freezing out solvents [22], adding laser [23], and using microwave [24], ultrasound [25], electric [26], magnetic fields [27], and membrane crystallization [28], have been discussed. Among these approaches, sonocrystallization, which applies ultrasound in crystallization process, has been proven to be an effective way to enhance nucleation process. In recent years, ultrasound has received widespread attentions because of its unique physical attribution and huge energy in transient. Since the promoting effect of sonocrystallization was first observed in 1927 [29], many benefits of sonocrystallization have been illustrated: decreased metastable zone width (MSZW), shorter induction time, narrowed crystal size distribution, higher crystal purity, improved product homogeneity, faster crystallization time, preferential
polymorph formation and better process repeatability [30-33]. However, the promoting effect of ultrasound is mainly investigated for inorganic and small organic molecules [34, 35]. Application of ultrasound in biomacromolecule especially for protein crystallization was seldom investigated. Crespo et al. [25] has demonstrated the potential use of ultrasound by using plate transducer, and described the change of MSZW and the PXRD of lysozyme crystals. However, the mechanism of nucleation and growth in protein sonocrystallization process is not illustrated. Furthermore, despite the effect of process parameters such as temperature and solute concentration on protein crystallization has been discussed, the effect of sonication parameters like power and sonication time on protein crystallization has not been well studied. Besides, the effect of ultrasonic irradiation on lysozyme yield has not been investigated yet. Hence, in this work, the effect of ultrasound on the crystallization of was investigated from both thermodynamic and kinetic perspectives. The influences of different sonication parameters (power and sonication time) were also explored. Specifically, the effect of ultrasound on MSZW, induction time, crystal size distribution, crystal morphology and the yield of protein crystallization process were investigated by using lysozyme as model compound. More importantly, the mechanism of how ultrasound affects nucleation and growth process was discussed in detail.
2 Experimental 2.1. Materials and solution Lysozyme from chicken egg white ( ≥ 98%, L4919) was purchased from Sigma-Aldrich. Other chemicals used were of analytical reagent grade. Acetate and sodium acetate anhydrous were purchased from Aladdin Industrial Co., China. Sodium chloride was purchased from Tianjin Yuxiang Chemical Reagent Co., Ltd., China. Water used for all experiments was supplied by the Millipore water system
(Milli-Q) with a resistivity of 18.2 MΩ cm at 298 K. All of the chemicals were used without further purification. Acetate buffer solution, which was fixed at pH=4.60 (Mettler Toledo-FE28) and 0.1 M, is consisted of acetate and sodium acetate anhydrous. All solutions of lysozyme and NaCl were prepared in acetate buffer and filtered (0.22 μm filters) before use. 2.2. Metastable zone width and induction time measurement To determine the MSZW, NaCl solutions with various concentrations (5%-15%) were quickly added into various volumes of lysozyme solution (60 mg/mL). Namely, through controlling the mixture ratio of the two solutions, different lysozyme and NaCl concentration could be achieved. In this way, the metastable zone curves could be obtained from the minimum NaCl concentration needed for the appearance of crystals at different lysozyme concentrations. Meantime, the induction time was also recorded. The experiments were carried out by using batch crystallization at 293 K under magnetic stirred conditions in order to guarantee constant supersaturation until the moment of crystal formation. Each salt concentration was measured for three times. Since the induction time was going to be longer when the concentration of lysozyme decreased, 24 h was chosen as the maximum time to reach phase equilibrium in this study. The experiments apparatus were shown in Fig. 1. Ultrasonic irradiation frequency is fixed at 20 kHz using an ultrasonic homogenizer workstation (Nanjing, China ATPIO-SM200). The horn depth (2 cm) and the tip diameters (10 mm) were fixed in this work. The power was set to 20 W and 80 W. The temperature was kept constant (293 ± 0.5 K) by using thermostat with a thermoelectric controller (Nanjing, China ATPIO-2006N). Table 1 shows the experiment parameters of the metastable zone and induction time measurements.
Fig. 1. Schematic diagram for the metastable zone width and induction time experiments: (1) noise reduction box; (2) the solution of sodium chloride; (3) the solution of lysozyme; (4) magnetic stirrer; (5) ultrasonic horn; (6) thermometer; (7) 50 mL water-jacketed vessel; (8) cooling water.
Table 1 The Experimental Parameters used in Metastable Zone and Induction Time Measuring Experiments Exp. No.
frequency
1 2
power
temperature
0 20 KHz
3
20
293 K
80
2.3 Crystallization experiments After measuring the MSZW and induction time, 15 mL of 8% (w/v) NaCl solution was added to 15 mL of 60 and 70 mg/mL lysozyme solutions respectively in a 50 mL jacked crystallizer for subsequent crystallization experiments. The vessel was sealed during the whole experiment to prevent evaporation. The salt solution was pumped into the lysozyme solution via a peristaltic pump (Longer, BT-100-1F) with the rate of 0.3 ml/min in order to prevent the unexpected suddenly happening of nucleation.
When the mixing was finished, the ultrasonic was applied instantly. The electrical power to produce the ultrasound is fixed at 80 W. The period of the ultrasound burst is set to 2 s with a pause of 2 s. During the process, the temperature was kept at 293 ± 0.5 K by using two thermostats in parallel because the ultrasound would release so much energy that the temperature of solution would be increased. And the agitation rate was maintained at 300 rpm by using a magnetic stirring. To identify the moment of nucleation, the laser was used to measure the turbidity. At the time of nucleation, which could have enough turbidity to be measured by the laser, the solution began to be sampled at intervals of 20 min. After one hour, the sample is taken once an hour, and the whole sample time is controlled within five or fourteen hours. The sample was firstly observed under an optical microscope (Nikon, Eclipse E200MV, 10× and 40× objective) with a digital camera (Nikon, Digital Sight Fi1) to study the morphology. Then, crystal size distribution was determined on the basis of analysis of more than 800 crystals from the images (using ImageJ software). Immediately, the slurry was centrifuged (Xiangyi, H1650-W) at 15000 g for 3 min. The protein concentration in the supernatant was determined by measuring the absorbance at 280 nm using an ultraviolet spectrophotometer (Hitachi, U-3010). The crystal yield was calculated on the basis of the difference in protein concentrations between the initial feed solution and the final supernatant mother liquor. Every measurement was repeated three times. In the presence of ultrasound, the experiments were divided into two groups: the continuous ultrasonic group, in which the ultrasound was applied during entire experiment, and ultrasonic-stop group, in which the ultrasound was stopped when nuclei was formed. For all tested conditions, control experiments were performed in the absence of an ultrasonic field in the same batch system with the same experimental conditions. The experiments apparatus were shown in Fig. 2. All conditions and parameters in this part are shown in Table 2.
Fig. 2. Schematic diagram for crystallization experiments: (1) noise reduction box; (2) peristaltic pump; (3) the solution of lysozyme; (4) magnetic stirrer; (5) ultrasonic horn; (6) thermometer; (7) 50 mL water-jacketed vessel; (8) cooling water; (9) the solution of sodium chloride; (10) constant temperature water; (11) thermoelectric controller; (12) ultrasonic generation control system.
Table 2 The Experimental Parameters used in Crystallization Experiments
No.
1 2 3 4 5 6
group
lysozyme
NaCl
concentration
concentration
(mg/ml)
(w/v)
continuous ultrasonic
30
ultrasonic -stop
30
control
power
35
35
(W)
time
(KHz)
80
2s ultrasound with 2s pause
20
0
no
no
4%
30 35
sonication frequency
3 Results and Discussion 3.1 Effect of ultrasound on the width of metastable zone The influence of ultrasound on metastable zone is shown in Fig. 3. The metastable zone was characterized as a function of salt concentration since salting out crystallization technology was applied in this work. The solubility curve was obtained from Forsythe et al [36]. Crystals were obtained at lower protein concentrations when
using ultrasound, as MSZW is narrower. As shown in Fig. 3, although the power of 20 W had already narrowed the MSZW to some extents, 80 W power narrowed the width more obviously. This means that the nucleation energy barrier was decreased in the presence of ultrasound and hence the nucleation of lysozyme became easier. For example, at Clys = 30 mg/ml the concentration of NaCl needed to produce crystals in the presence of ultrasound (20 W) is about 60% of the corresponding concentration in the absence of irradiation. According to two-step nucleation theory [37, 38], the variation of supersaturation solution could lead to the formation of disordered protein-rich dense clusters firstly. Then, when the size of cluster of solute molecules reaches a sufficient size, the cluster will reorganize into an ordered structure, and finally the ordered crystalline nuclei will be formed. The dense liquid is usually unstable and has a higher free energy than the dilute solution and hence nucleation would likely occur from the protein-rich dense metastable molecular clusters [39, 40]. In the whole nucleation process, the first maximum free energy (ΔG1*) is needed to form the dense liquid. Then, the crystals were formed by crossing the second maximum free energy (ΔG2*). Generally, the primary nucleation of protein will only occur when the two critical energy of nucleation are overcome under certain temperature and protein concentration. When ultrasound is applied, the energy needed for nucleation could be overcome more quickly. The acoustic cavitation effect [30, 41-44] of ultrasound may contribute to this promotion. When ultrasonic wave spreads into solution, the pressure variation creates the cavitation phenomena, which is the formation, growth and collapse of bubbles [45]. When bubble collapses, it will cause rapid adiabatic compression of gases and vapors inside the bubble. At the meantime, very high pressure (up to tens of GPa) and temperature (up to tens of thousands K), as well as shock waves, microjets and micro-turbulences [46] will also be generated. Therefore, the energy released by the shock waves during the collapse of cavitation bubbles could overcome the critical energetic barrier required for spontaneous nucleation [47], and solid-liquid mass transfer would be enhanced so that nucleation is promoted. Besides, pressure gradients generate by oscillating bubbles could allow small solute molecules to
aggregate to an oscillating bubble wall. These solutes can then cluster and reach the sufficient cluster size necessary for crystal nucleation. When the solute clusters grow large enough, they are then pushed away from the oscillating bubble and into the bulk phase [48]. Hence, in the whole process, the supersolubility curve will drop down, and the MSZW would decrease. Solubility Supersolubility Without Ultrasound Supersolubility With Ultrasound-20 W Supersolubility With Ultrasound-80 W
50
Clys (mg/mL)
40
30
20
10
0 1
2
3
4
5
6
7
8
CNaCl (% w/v) Fig. 3. The metastable zone of lysozyme with various ultrasound power at 293 K and pH 4.6. 3.2 Effect of ultrasound on induction time The effect of ultrasound on induction time is shown in Fig. 4. In the absence of ultrasound, the induction time increases exponentially with the decreasing of the concentration of lysozyme. More importantly, when the ultrasonic was applied, the induction time was evidently decreased and the induction time of the sonicated experiments was significantly shorter than the control experiments, especially obvious at lower supersaturations. What’s more, it can also be found that the induction time after application of ultrasound was less affected by the concentration of the solutions. Generally, small solute molecules around the oscillating bubble could easily grow up
to the critical nucleation cluster size. When the bubbles collapse, the temperature would rapidly decrease, which will also enhance the nucleation. Therefore, it can be suggested that irradiating the solution with ultrasound favors the generation of acoustic cavitation, which could promote the formation of clusters and hence shorten induction time. Such approach would help to reduce the total time of the crystallization and hence increase the efficiency of the crystallization of protein.
600
Ultrasound—80W Ultrasound—20W Without ultrasound
500
tind/min
400 300 200 100 0 25
30
35
Clys/mg·ml-1 Fig. 4. The histogram of induction time of the salt concentration of 4% and the lysozyme concentration of 25, 30 and 35 mg/ml at 293 K and pH 4.6 with the presence and absence of ultrasonic. 3.3 Effect of ultrasound on morphology and size distribution Lysozyme crystals morphology and size distribution obtained by batch crystallization under different conditions are shown in Fig. 5 and Fig. 6 respectively. It can be seen that most of lysozyme crystals obtained from control experiments (without ultrasound) show non-uniform size distribution and aggregation (Fig. 5c, f). The crystal size in this group was counted by using the mean size of the aggregation and calculated in the triplicate experiments. The mean size of the aggregation is 43.7
μm in the 35 mg/ml lysozyme concentration, and the size distribution is broad as shown in Fig. 6. This might be attributed to unexpected secondary nucleation and aggregation. Interestingly, compared to the broad size distribution of aggregated crystals in the control group, the crystals of lysozyme obtained from ultrasonic group and ultrasonic-stop group showed sharper and narrower distributions as shown in Fig.6. The results show that the ultrasound can significantly improve the size uniformity. Meanwhile, from Fig. 5, it can be seen that, although the crystals obtained from control group are larger than crystal obtained from the continuous ultrasound and ultrasound-stop group, most of them are aggregated crystals, especially under the condition of C0=30 mg/ml. The mode of ultrasonic can also affect the morphology and size distribution of lysozyme crystals. From Fig. 5 and Fig. 6, it can be seen that, when continuous ultrasound is applied, the size of the final products are quite small (only several micrometers) although the size distributions are quite narrow. Especially in the condition of 30 mg/ml lysozyme concentration, the morphology of the crystals was even too small to be clearly observed in the view of regular microscope (Fig. 5d). On one hand, ultrasound could enhance both primary nucleation and secondary nucleation, which will result in more nuclei. On the other hand, ultrasound could also restrain the growth of lysozyme crystals and result in breakage of crystals [49]. These two reasons can explain the appearance of very tiny crystals (Fig. 5d) when continuous ultrasound was applied. The possible explanation for the improved size distribution is the ameliorating of aggregating by ultrasound. The shock wave [50], which is caused by cavitation, can shorten contact between crystals to an extent, precluding their bonding together and thus reducing the chance of aggregating. These small and well dispersed crystals generated by continuous ultrasonic could act as crystal seeds, which will be a good strategy for the industrial crystallization of some proteins. To overcome the shortcoming of continuous ultrasonic, ultrasonic-stop mode was applied. From the data presented in Fig. 5 and Fig. 6, it can be seen that the morphology of the obtained crystals (Fig. 5b, e) are clearly improved, with more
regular shape and much bigger size, without sacrificing the uniform size distribution. The possible explanation for the better shape and bigger size of the products from the ultrasonic-stop mode experiments is that the cease of ultrasound after nucleation can lower down the rate of secondary nucleation (or breakage) of the obtained crystals. Meanwhile, from comparison of Fig. 5(b) and Fig. 5(e), it can be seen that products obtained from higher concentration have bigger size. This possible reason might be that higher lysozyme concentration can provide more solute for the nucleated crystals to grow into larger size. In a word, ultrasound could effectively enhance nucleation and prevent aggregating. But long time ultrasound (too much energy injected) could also result in smaller particle size. Therefore, ultrasonic-stop mode will be a more effective approach for crystallizing protein with uniform and bigger size.
a)
b)
c)
d)
e)
f)
Fig. 5. Microscope images for lysozyme crystals formed at 293 K and pH 4.6. (a) continuous ultrasonic at C0=35 mg/ml. (b) ultrasonic-stop at C0=35 mg/ml. (c) in the absence of ultrasonic irradiation at C0=35 mg/ml. (d) Continuing ultrasonic at C0=30 mg/ml. (e) ultrasonic-stop at C0=30 mg/ml and (f) in the absence of ultrasonic irradiation at C0=30 mg/ml.
0.30
ultrasonic ultrasonic-stop control 35 mg/ml lys+ 4% NaCl
0.25
Frequence
0.20
0.15
0.10
0.05
0.00 0
10
20
30
40
50
60
70
80
90
100
110
120
130
Crystal size (μm) Fig. 6. The crystal size distribution of lysozyme under different conditons at 35 mg/ml lysozyme with 4% Nacl 3.4 Effect of ultrasound on yield Table 3 lists the average final protein concentration Cf and final supersaturation Sf for all the experiments. The crystallization yield y and S are defined as
𝑦=
𝐶0 ― 𝐶𝑓 𝐶0 𝐶
𝑆 = 𝐶∗
(1) (2)
where C0 is the initial protein concentration, C is actual concentration and C* is the equilibrium concentration of lysozyme. By using the data listed in Table 3, the yields of different crystallization experiments were calculated. The results are shown in Fig. 7. For the ultrasonic-stop condition, the yield of the process quickly reaches the maximum level after nucleation and the whole crystallization process could be finished in around 300 minutes. But for the continuous ultrasonic condition, the final yield of the process is lower than the yields of other two conditions although the nucleation could happen much earlier than no ultrasound condition. As for the control group, although its final yield was
approximately equal to the ultrasonic-stop condition, much longer time is needed to reach the same yield due to the long induction time. Table 3 Average Final Protein Concentration and Supersaturation expt no.
time (h)
ultrasonic field
C0 (mg/mL)
S0
Cf (mg/mL)
Sf
1
5
yes
35
11.4
13.1 ± 0.2
4.3 ± 0.06
2
5
only during nucleation
35
11.4
9.0 ± 0.3
3.0 ± 0.1
3
5
no
35
11.4
11.2 ± 0.9
3.7 ± 0.3
4
14
yes
30
9.8
13.4 ± 0.2
4.4 ± 0.06
5
14
only during nucleation
30
9.8
8.5 ± 0.3
2.8 ± 0.1
6
14
no
30
9.8
9.3 ± 1.1
3.0 ± 0.4
The faster process in the ultrasound-stop group could be due to the ultrasound cavitation effect which is able to enhance heat and mass transfer by triggering the micro-turbulence [46]. The cavitation effect can be responsible for increasing of micromixing and nucleation rate and for the generation of plenty of supersaturation locations. The higher yield of this condition could be attributed to the bigger size of the obtained crystals, which could reduce the loss of produce from following centrifuging and washing step. This explanation can be supported by the observation of the crystal morphology and size in the above discussion. For the phenomenon of lower yield in continuous ultrasonic group, the first possible reason is protein denaturation. Long time ultrasound would bring considerable energy to this system. Some studies have demonstrated that long-term ultrasound would damage the protein crystals [51-53] since protein is incredibly sensitive to high temperature which would be triggered by continuous energy input. Furthermore, the second possible explanation is higher solubility and more difficult solid-liquid separation due to smaller crystals. From Fig. 5 and Fig. 6, continuous ultrasound will result in much smaller crystal size. On one hand, particle size could
affect the solubility of the solute. When the particle size is too small (smaller than several microns), it would result in higher solubility and hence lower yield. On the other hand, small crystals can make the solid-liquid separation (centrifuge in this case) difficult. Because some tiny lysozyme crystals will remain in the supernatant and redissolve, the final yield of the crystallization process will be reduced. In addition, prolonging ultrasonic time will consume more electricity energy with no further obvious promotion, which has been proved by literatures [32, 49, 54]. Therefore, compared with protein denaturation and smaller crystal size caused by continuous ultrasonic and the lower efficiency in the control group, the ultrasonic-stop method can produce better lysozyme product with higher yield in a shorter time. Hence, ultrasonic-stop method will be a good strategy for crystallizing proteins such as lysozyme with lower cost and higher efficiency.
80
a
70 60
Yield (%)
50 40 30
ultrasonic ultrasonic-stop control 30 mg/ml lys + 4% NaCl
20 10 0 0
100
200
300
400
500
600
Sample time (min)
700
800
900
80
b
Yield (%)
60
40
ultrasonic ultrasonic-stop control 35 mg/ml lys + 4% NaCl
20
0 0
50
100
150
200
250
300
350
Sample time (min) Fig. 7. The yields at different time in control group, ultrasonic-stop group and continuous ultrasonic group at (a) 30 mg/ml lysozyme with 4% NaCl (T0=19 min) and (b) 35 mg/ml lysozyme with 4% NaCl (T0=15 min)
4. Conclusions This work demonstrated the positive nucleation enhancement effect of ultrasonic in lysozyme crystallization process. When ultrasound was applied, the metastable zone width was evidently narrowed and the induction time was significantly reduced. However, long time continuous ultrasonic would input too much energy which may cause lysozyme denaturation and ultrasound wave could also restrain the growth of crystals. Therefore, the ultrasonic-stop method, in which ultrasound is only applied during the nucleation process, was suggested. It was found that the ultrasonic-stop method can produce better lysozyme crystals with uniform size distribution and intact morphology. The crystallization time was also shortened and hence the efficiency of the whole crystallization process was improved.
Acknowledgements This work was financially supported by the Tianjin Natural Science Foundation (grant number 18JCYBJC40800).
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Highlights
Ultrasound was successfully applied to enhance lysozyme crystallization process.
The effect of ultrasound on induction time, metastable zone and yield was investigated.
The effect of ultrasound on crystal size and morphology was investigated.
The affecting mechanism of ultrasound on lysozyme crystallization was elucidated.
Ultrasonic-stop method was found to be more appropriate strategy.