Forced convection evaporation for bulk protein crystallization - Crystal

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Forced convection evaporation for bulk protein crystallization Michal Kolodziej, Maksymilian Olbrycht, Izabela Poplewska, Wojciech Piatkowski, and Dorota Antos Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.8b00638 • Publication Date (Web): 07 Aug 2018 Downloaded from http://pubs.acs.org on August 13, 2018

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Crystal Growth & Design

Forced convection evaporation for bulk protein crystallization Michał Kołodziej, Maksymilian Olbrycht, Izabela Poplewska, Wojciech Piątkowski, Dorota Antos1 Department of Chemical and Process Engineering, Rzeszów University of Technology, Powstańców Warszawy Ave. 6, 35-959 Rzeszów, Poland

Abstract Forced convection evaporation was adopted for bulk protein crystallization. As a model system ovalbumin in aqueous solutions of ammonium sulfate was used. The crystallization process was performed in a drying chamber, where the protein solution was contacted with a warm air stream at a temperature varied from 35 to 52°C. The slow evaporation of water induced gradual crystallization of the protein. Heat and mass transfer in the water-air system during water evaporation caused the temperature of the protein solution to remain distinctly below the air temperature. This allowed processing the protein under mild conditions. The process design was guided by crystallization phase diagram and kinetic measurements. The supersaturation degree, which determines the nucleation and crystal growth rates, was adjusted by the rate of water evaporation. The latter was precisely controlled by matching the temperature in the chamber to the air humidity. The process could be accomplished in a single crystallization stage in 50 - 95 min., with the protein yield of 72 - 85%. Ovalbumin appeared as clusters of irregular crystals; no amorphous precipitate was formed. An increase in the evaporation rate caused the crystal size to increase.

1. Introduction Over last years, bulk crystallization of proteins has attracted increasing interest in the pharmaceutical, biotechnological, and food industry.1-3 Crystallization process has many advantages over other methods of protein purification, such as:

high recovery of

product, low reagent costs, inexpensive process equipment, small process volumes and 1

Corresponding author: Dorota Antos, Department of Chemical and Process Engineering, Rzeszów

University of Technology, Powstańców Warszawy Ave. 6, 35-959 Rzeszów, Poland; tel.: +48 178651853; fax:+48 178543655; E-mail [email protected]

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Crystal Growth & Design 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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high purification in a single process step.1,4 Moreover, crystallization is an attractive formulation method5 that can be an alternative to costly lyophilization procedures.6 Protein crystals exhibit higher stability,7 activity and purity of the crystalline biomolecule, reduced bulk storage costs, longer shelf life compared to conventional formulation methods that are based on aqueous solutions, amorphous lyophilizates, or spray-dried preparations.1 Crystallization can also be used as a highly selective purification step that has the potential to replace costly chromatography operations.3,8 Industrial protein crystallization was first employed in insulin purification, and human insulin was the first biopharmaceutical molecule produced and commercialized by 1982.3 Since then, the number of successful preparative protein crystallization processes has been increased; some examples are: the crystallizations of lipase,2,9,10 lysozyme,10-12 ovalbumin,13 L-methionine g-lyase,14 an aprotinin variant,15 an intact monoclonal IgG4,16 therapeutic antibodies17,18, and a recombinant interferon gamma (rhINF-γ).19 Bulk crystallization is usually performed batchwise in stirred tank crystallizers, where nucleation and growth of protein crystals is induced by salting out in the presence of a crystallization agent, which typically is an aqueous solution of kosmotropic salts.20 However, a few innovative techniques for the process realization have also been proposed, e.g.: Neugebauer et al.21 developed a tubular crystallizer for continuous production of protein crystals, where the supersaturation profile was controlled using the local temperature along the wall of the crystallizer. Hekmat et. al

22

applied a lab-

scale stirred tank with a cooled tubular reactor in bypass for continuous protein crystallization. Gross and Kind23 developed a bulk protein crystallization process by low pressure evaporation. In this process, the desired supersaturation level was achieved by controlled water removal from the protein solution under mild temperature conditions. That approach was claimed to have a potential for reducing the concentration of crystallization agents in the protein solution to be processed. In this work, we developed another technique for bulk crystallization of proteins. In this technique evaporation of water from the protein solution is forced by convective air flow inside a drying chamber. The parameters of air can be altered to control the concentration of kosmotropic salt in the solution, thus the supersaturation level, as well as the temperature of the protein solution. The model protein was ovalbumin, which was crystallized from aqueous solutions of ammonium sulfate. The process design was based on the crystallization phase diagram, which was determined in our previous 2 ACS Paragon Plus Environment

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work24 and kinetic data acquired from a stirred tank crystallizer in an isocratic mode, i.e., at constant salt concentration in the solution.

2. Materials and methods 2.1. Equipment The protein crystallization was performed in a drying line equipped with a fan, air heater, gauges of temperature, flowrate, humidity sensors, and a weight module (Figs 1A and 1B). The weight module consisted of a shallow wire basket (scale pan) that was fastened to the weighing scale (Fig. 1B). The pan was located in the center of the chamber pipe. The crystallization tank was situated on the pan in such a way that the air stream could flow freely above the water surface in the tank. A magnetic stirrer (Arec X Velp Scientifica, Italy) was placed beneath the chamber, which allowed stirring the solution in the crystallization tank. The air parameters were determined by thermoanemometer Airflow TA465_P (TSI Incorporated, U.S.A) equipped with three probes (type 964) to measure air temperature, its linear velocity and humidity. A photo of the plant is presented in Fig. 1S (Supplementary materials).

Fig. 1. Scheme of the equipment (drying line) used for the crystallization experiments. A) Air flow path; B) position of the stirred tank in the drying chamber; the chamber diameter ID = 10 cm, length = 50 cm.

2.2. Materials Ovalbumin (OVA) with purity higher than 98 w/w% was purchased from Sigma-Aldrich (A5503 Sigma-Aldrich, Poznań, Poland). The protein crystals subjected to light microscopy analysis were stained using Eosin Y (Sigma-Aldrich, USA). Ammonium sulfate (AS), monosodium and disodium phosphate, were purchased from Chempur 3 ACS Paragon Plus Environment


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(Chempur, Poland). A stock solution of OVA was prepared by dissolution of the protein in a 50 mM sodium phosphate buffer, pH = 7.

2.3. Pre-purification of OVA solutions Prior to the crystallization experiments, raw material of OVA was purified to remove high molecular weight aggregates according to the procedure described in our previous study.24 Briefly, stock solutions of OVA with the concentration in the range of 100 - 150 mg mL-1 in phosphate buffer, pH 7, and solutions of the AS salt buffer (pH = 7) with the concentration of 3.5 M AS, were mixed volumetrically to obtain a solution with the following composition: xP = 2.5 – 5 × 10-2 w/w, and xsalt = 0.19 w/w, for the protein and AS respectively, at pH 6.5. Next, the solutions were stirred using a magnetic stirrer (Arec X Velp Scientifica, Italy) at stirring speed, ss = 400 rpm for 24 h, at 22 °C. The liquid and solid phases were separated by centrifugation at ss = 16500 rpm for 15 min, at 22 °C (centrifuge MPW-351RH, MPW MED Instruments, Poland). The monomer content in the purified solution was determined using a SEC-HPLC system (LaChrom, Merck, Germany) equipped with a TSKgel Super SW3000 column 4.6x300 mm, and a guard column with the same resin (TOSOH BIOSCIENCE, Germany).

2.4. Experimental design of the crystallization process The experimental design procedure comprised a few steps, including: -

determination of the phase diagram and the bounds of the operating window for the crystallization,

-

measurement of the crystallization kinetics,

-

crystallization in the drying chamber under operating conditions selected,

-

evaluation of the morphology of the crystalline phase obtained,

-

development of a mathematical tool for supporting the process design.

2.5. Determination of the operation window for the protein crystallization The operating window for the crystallization of OVA was determined in our previous study24 based on the salting out experiments performed at pH 6.5 with different salt and protein concentrations. The following limits for the AS concentration were set: the maximum xsalt = 0.26 w/w, and the minimum xsalt = 0.19 w/w. Above the upper limit, amorphous precipitate and insoluble aggregates were formed, whereas below the lower limit the solubility of the protein increased drastically, which hindered the protein crystallization. 4 ACS Paragon Plus Environment

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2.6. Measurements of the crystallization kinetics The supernatant with the purified OVA was combined with the AS stock solution to obtain solutions with the salt concentration in the range xsalt = 0.23 – 0.26 w/w, and the protein concentration xP = 0.9 - 3.8 × 10-2 w/w. Each of the solutions was divided into portions and transferred into a few Eppendorf tubes (2 mL volume) in volume amount of 0.3 mL per each tube. The tubes were thermostatted in parallel at 22 °C using a low temperature thermostat (Lauda Re110, Lauda-Königshofen, Germany) and stirred with a magnetic stirrer at ss = 200 rpm until the crystallization equilibrium was established. The solutions were withdrawn from the tubes at different time intervals, centrifuged at ss = 16500 rpm for 15 min, at 22°C. Next, samples of liquid phase (0.1 or 0.2 mL) were

subjected to the concentration analysis using a UV detector of the HPLC system at 280 wavelength. The detector signal was corrected with a signal for a blank sample of the solution free of the protein, and converted into the concentration using the detector calibration factor.

2.7. Forced convection crystallization experiments A 2-mL (ID 1.2 cm; height 1.8 cm) or 4-mL (ID 2.8 cm; height 0.64 cm) mini-tank with the protein solution was placed into the drying chamber on the scale pan. The chamber was closed, and air heated by the resistance heater was pumped through the chamber (Fig. 1). The motion of the solution was forced by a magnetic stirring bar (length 10 mm; diameter 1 mm). The values of the linear air velocity, temperature and relative humidity were measured on-line by the sensors (Fig. 1 A), and observed in the control panel.

The temperature and flowrate of air and the duration of evaporation were selected in such a way that the protein and salt concentrations were kept within the operating window, whereas the temperature of the protein solution did not exceed 40°C, i.e., remained within the range 22 - 40°C, depending on the temperature of the air stream. The change in weight of the solution, which corresponded to the evaporation progress, was monitored on-line using the electronic weighting scale. The process was finished after a certain time interval, the tank was withdrawn from the chamber, and the solution was subjected to the concentration analysis (section 2.6). The same procedure was repeated several times for several batches of the same solution, but in each subsequent experiment the residence time of the batch in the chamber was extended until the time set-point for the evaporation was reached. The time set-point was determined by the limit of the salt concentration in the solution. As a result, the kinetic curves for the 5 ACS Paragon Plus Environment


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gradual protein crystallization were obtained. The initial salt concentration in the protein solutions was always xini,salt = 0.19 w/w, while the final one was in the range

xend,salt = 0.25 - 0.26 w/w. The initial protein concentration was changed from xini,P = 3 × 10-2 to 4.3 × 10-2 w/w. The process variables were: the temperature of air, which was varied from Tair = 23 to 50°C; the air linear velocity varied from uair = 0.66 to 1.3 m s-1, and the stirring velocity varied from ss = 0 to 600 rpm. The temperature of air in the front and behind of the empty drying chamber differed with 0.6 degrees, which resulted from heat losses to the environment. The drop in the temperature of air arising from heat transfer during water evaporation was negligible, which stemmed from a large excess of air in relation to the amount of evaporated water.

2.8. Light microscopy analysis The sludge obtained in crystallization experiments was analyzed by light microscopy (Bresser TRM-301, Germany). A small amount of the suspension was mixed with Eosin Y and observed under the microscope at appropriate magnification. Photos of protein crystals were taken using a digital microscopic camera (Bresser MicroCam 10.0 MP, Germany). The CSD analysis was performed using the pictures taken of the sludge using an image processing software (MicroCamLab, BRESSER).

2.9. Resolubilization of OVA crystals Suspension of the crystalline phase was centrifuged at ss =16500 rpm for 5 min. After the phase separation, the crystalline pellets were resolubilized in phosphate buffer at pH 6.5 in a stirred tank.

2.10. DSF measurements of the melting temperature The stability of the protein structure in the liquid solution was tested using NanoDSF measurements with a Prometheus NT.48 (NanoTemper Technologies GmbH, Munich, Germany). Samples of the initial proteins solutions (before crystallization) and those obtained after the resolubilization of the crystalline phase were taken using a thin capillary with a volume of 10 µL. The capillaries were placed into trays of Prometheus NT.48 and subjected to the fluorescence analysis. The emission of fluorescent radiation with the wavelengths of 330 nm and 350 nm was measured with the temperature changes from 20 to 95°C, with the rate of 1°C min-1. The fluorescence curves obtained were 6 ACS Paragon Plus Environment

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differentiated, and the ratio of differentials was used to determine the unfolding transition point, i.e., the so-called melting temperature of the protein.

2.11. Mathematical modelling To calculate time-dependent profiles of the solution temperature, a differential heat balance equation was used, as follows:

ܿ௉ ݉

డ்ೞ డ௧

= ߙ ‫ ܣ‬ሺܶ௔௜௥ − ܶ௦ ሻ −

where: the partial derivative time;

డ௠ డ௧

డ்ೞ డ௧

డ௠ డ௧

ሺ݅௦௧ − ݅௪ ሻ

ሺ1ሻ

determines the temperature change of the solution over

is the ER value; Ts is the temperature of the solution; m is the mass of the

solution; t is the time coordinate; cP is the specific heat capacity of the solution; ist is the enthalpy of steam at the temperature Ts; iw is the enthalpy of liquid water at the temperature Ts, ሺ݅௦௧ − ݅௪ ሻ = ‫ݎ‬, where r is the latent heat of evaporation at Ts; α is the heat transfer coefficient; A is the heat exchange surface area, which includes the wall surface area of the tank as well as liquid-air interface; ܶ௔௜௥ is the air temperature, which is constant provided that heat transported with air is in a large excess compared to that exchanged by water evaporation. The ER value can be determined by the rate of mass transport: ௗ௠ ௗ௧

= ߚ ‫ܣ‬௟௜௤ ሺ‫ݕ‬௦௧ − ‫ݕ‬௔௜௥ ሻ

ሺ2ሻ

where β is the mass transport coefficient; Aliq is liquid-air interface (free liquid surface area); yst is the mass fraction of saturated steam at temperature Ts of the solution; yair is mass fraction of steam in air. The combination of Eq. (1) and (2) at steady state yields:

ሺܶ௔௜௥ − ܶ௦ ሻ =

ఉ ௥ ஺೗೔೜ ఈ஺

ሺ‫ݕ‬௦௧ − ‫ݕ‬௔௜௥ ሻ

ሺ3ሻ

When the heat exchange surface area is equal to the free liquid surface area, i.e., Aliq = A, then Ts is equal to the so-called wet-bulb temperature. This is the minimum temperature of the aqueous solution that can be achieved in a water-air system.

3. Results and discussion 3.1. System characterization and design of forced convection crystallization As mentioned in section 2.4, prior to the realization of crystallization experiments, the experimental system has to be characterized in terms of the equilibrium and kinetics of crystallization as well as kinetics of water evaporation. Therefore, the design procedure 7 ACS Paragon Plus Environment


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includes the following steps: determination of the phase diagram, measurements of the crystallization kinetics for different salt concentrations and supersaturation degrees in the solution, and adjustment of ER accordingly to the crystallization equilibrium and kinetic data.

3.2. Phase diagram of crystallization and kinetic measurements The protein solubility and the operating window for the crystallization of OVA were determined in our previous work.24 The position of the window is illustrated on the binary phase diagram as a function of the protein mass fraction versus the salt mass fraction, which is presented in Fig. 2. As it can be observed, the size of the window is relatively small; the maximum of supersaturation degree, i.e. the ratio of the initial and equilibrium protein concentrations, is about 1.6. The process kinetics were examined for different salt concentrations and supersaturation degrees in the solution. For that purpose, a few operating points were selected near the upper and lower bounds of the operating window, which corresponded to the maximum and minimum of the supersaturation degree (Fig. 2), and the protein solutions were prepared accordingly. The protein concentration in the solutions was analyzed at different time intervals until the equilibrium was established (section 2.6). The time-dependent concentration profiles obtained are shown in Fig. 3. It can be observed that the crystallization kinetics are fast, particularly for the solutions with the highest supersaturation degrees (points 1,3,5 near the upper bound of the operating window in Fig. 2), for which the concentration decay is initiated immediately after the start of the process, and the crystallization equilibrium is established after about 20 min. In the case of the solutions with low supersaturation degrees (points 2,4,6 near the solubility line in Fig. 2), the concentration drop occurs after 15 - 20 min, whereas the equilibrium is reached after about 50 min. The sudden onset of the crystallization at low supersaturation degrees indicates that the nucleation rate of OVA is fast, and the crystallization kinetics is limited by the crystal growth rate. Obviously, this is specific for the protein type and crystallization conditions. Nevertheless, the rate of bulk crystallization is usually markedly higher than that observed in crystallographic analysis, which is performed in a motionless liquid droplet. In bulk crystallizers, the protein solution is stirred or recirculated, which induces collisions of crystals and causes secondary nucleation to occur.25 This accelerates


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crystallization process, which can be realized quickly, i.e., in one or few hours, and at relatively low supersaturation degrees.17,19,26,27

6 5

xp × 10 w/w

2

4 3

*

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Ope r

at i n gw indo Solubil wo ity line f c ry

1 st a l liza

2

2

Undersaturated

1

Precipitate tion

5 1* 4 3*

0 0.19

0.20

0.21

0.22

0.23

3

0.24

6 5*

0.25

0.26

xsalt w/w Fig. 2. Operating window for the crystallization of OVA at pH 6.5 with the operating points selected for the kinetic measurements. Lines: solid – solubility line, dashed – approximation of the upper boundary of the crystallization window, solid symbols – the operating points; the pairs 1,2; 3,4; 5,6 denote the same initial salt concentration, accordingly to their coordinates; the points 1*, 3*, 5* (open symbols) illustrate the equilibrium compositions obtained for the initial solutions 1, 3, 5, respectively. The difference in the equilibrium concentrations for the protein solutions with the same initial salt content (pairs of the curves 1,2; 3,4; 5,6 in Fig. 3) can be attributed to water uptake by protein crystals in the solid phase.24 The depletion of water in the solution induces a change in the salt concentration, and consequently in the protein solubility. The phenomenon is illustrated in Fig. 2, where the points 1*, 3*, 5* indicate the equilibrium concentrations corresponding to the initial solutions denoted by 1, 3, 5. The reported trends in the kinetic data were independent of the salt concentration, therefore, it could be expected that the process was fast for any operating point situated at the upper bound of the operating window.



4.0

xsalt w/w

3.5

0.2334 0.2439 0.2543

3.0

xP × 102 w/w

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2.5

2 1

2.0 1.5

4 3 6 5

1.0 0.5 0

50

100

150

200

250

300

t [min] Fig. 3. Kinetic curves of the protein crystallization corresponding to the operating points presented in Fig. 2. Symbols – experimental data; lines – guide to the eye.

3.3. Forced convection crystallization 3.3.1. Adjustment of the operating variables The water evaporation experiments were performed in the drying line illustrated in Fig.1. The line used is typical for experimental studies on kinetics of the drying process, which is predictable and scalable. The process variables, such as the air parameters (velocity, temperature, humidity), can easily be monitored using a standard equipment that is typically applied in drying technology. The influence of changes in the dimensions of crystallization tanks can be predicted using adequate balance equations and kinetic rate equations for mass and heat transfer. The crystallization kinetics can be altered by the hydrodynamic conditions (stirrer type, stirring speed), which however depend on the crystallization scale. The latter dependency is a typical problem to be handled in scaling up crystallization processes. The crystallization was realized in the 2 or 4-mL stirred-tank reactors, which differed in the ratio of the solution volume to the free liquid surface area and in the heat transfer area (section 2.7). The weight and the temperature of the solution, the temperature of air in the front and behind of the drying chamber were monitored online. The operating variables, which were selected to alter the course of the crystallization were: temperature of air, linear velocity of air, and stirring velocity in the tank. The air temperature determines the driving force for heat transfer (Eqs 1 and 2), hence it affects the evaporation rate, which is a crucial parameter that controls the supersaturation degree. The air velocity affects the mass and heat transfer rates in air. The stirring 10 ACS Paragon Plus Environment

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velocity determines turbidity of the solution, which influences heat and mass transfer kinetics in liquid phase as well as kinetics of nucleation and crystal growth. It also determines the shearing stress to which the protein crystals are exposed, hence it has an impact on the crystal morphology. Additionally, air humidity has to be monitored, since it determines the driving force of mass transfer (Eq. 2). The initial salt concentration in the solution was xini,salt = 0.19, whereas the final one varied from xend,salt = 0.25 to 0.26 w/w. The latter corresponded to the lower bound of the crystallization window (Fig. 2). Relative humidity varied from φ = 10 to 20%. The process was interrupted when the mass of the solution reached the limit determined by the final salt concentration in the solution. Typical changes in the mass and temperature of the solution are depicted in Fig. 4. It can be observed that the temperature of the protein solution (Ts) increases from a room temperature, up to the steady state value, for which the rates of heating the solution by the air stream and cooling by water evaporation are balanced. The steady state of heat and mass transport was established after about 20 min. The time needed to reach the set-point for the weight of the solution, and the corresponding salt concentration depended on the rate of water evaporation. As mentioned above, the equilibrium temperature of the solution was distinctly lower than the temperature of air, which was caused by heat transfer due to water evaporation. Therefore, a mild temperature of the solution could be maintained, which prevented protein denaturation. The temperature of the solution can be altered by the parameters of air. The lowest temperature that can be reached in aqueous solutions by the evaporation of water under ambient pressure is the wet-bulb temperature (Eq. 3). For instance, for air with temperature of 40°C and relative humidity φ =10%, and at A = Aliq (i.e., at the equality of the heat and mass transfer areas, see Eq. 1 and Eq. 2), the wet-bulb temperature is about 17°C.



4.5

44

Tair

40

4.2

TS

T [oC]

36

3.9 32 3.6

28 24

m [g]

3.3

m

20 3.0 0

20

40

60

80

t [min] Fig. 4. Time-dependent profiles of the mass and the temperature of the solution measured in the 4-mL stirred tank crystallizer. The air velocity uair = 0.93 m s-1, the temperature of air, Tair = 43.5°C, the steady state temperature of the solution was Ts = 34°C, relative humidity ϕ = 14%. Circles - experimental profiles of temperature of the solution, Ts, and air, Tair; squares – measured mass, m, of the solution. Lines – simulations using Eqs (1) and (2). The influence of the air parameters on the ER value in the experimental system are depicted in Fig. 5A - 5C. It can be observed that the ER value increases almost linearly with the increasing temperature of air, as the driving force for heat transfer increases (Eq. 1). The influence of flowrate on ER is much less pronouced compared to that of temperature. It results from flowrate dependences of the heat and mass transport coefficients, which are determined by the system hydrodynamics. The influence of the stirring velocity, ss, on ER is also week (Fig. 5C), and vanishes for the values higher than 200 rpm, for which the solution turbidity is suffcient to diminish the contribution of heat and mass trasport resistances in the liquid phase to the overal kinetic rate. A) 12

B) TS

Tair

12

10

2

10

-1

ER [mg × s ]× 10

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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36 43,5 52

8

6

6

exp. points simulation

4 30

35

40

T [°C]

45

50

6

4 0.6

T [°C] 36 43,5 52

10

8

8

25

C) 12

T [oC]

4 0.7

0.8

0.9

1.0

1.1

-1

uair [m s ]

0

100

200

300

400

500

600

ss [rpm]

Fig.5. Evaporation rate (ER) at steady state: A) vs temperature, at the air linear velocity uair = 0.93 m s-1 and ss = 400 rpm, symbols – experimental temperatures of air, Tair (solid circles), and the solution, Ts (open circles), line – simulation of ER and Ts; B) vs air linear velocity at different temperatures of air, ss = 400 rpm, lines - guide to the eye; C) vs stirring velocity at different temperatures of air, uair = 0.84 m s-1, lines - guide to the eye. The stirred tank of 2-mL.


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The results obtained did not depend on the protein presence and its concentration in the solution. The mole fraction of the protein in the solution was very low, i.e., order of magnitude of 10-5, therefore the latent heat of evaporation and vapor preassure remained the same as those for the salt solutions or pure water. The mole fraction of salt was also low enough (the maximum mole fraction for AS was about 0.045) to have negligible impact on water activity. Additionally, to predict ER and the changes in the temperature of the protein solution under different operating conditions, Eqs 1 and 2 were employed, which were solved using a standard numerical routine for ordinary differential equations. The heat transfer coefficient, α, was the only model parameter that was adjusted based on the timedependent profile of the solution temperature measured for a selected set of the experimental data, i.e., for uair = 0.93 m s-1, Tair = 43.5°C (the data in Fig. 4). The heat exchange area (A), was the total area of the tank wall plus the interface (Aliq). The estimated value of the heat transfer coefficient at steady state was α = 26 [W m-2 K-1]. ER =

ௗ௠ ௗ௧

was given from the on-line measurements of the solution weight; the dependency

of mass fraction of saturated steam, yst, on the temperature of the solution was taken from literature data; mass fraction of steam in air (yair) was determined by the air humidity (φ). The value of β was calculated using Eq. (2) for the same set of the experimental data; at the steady state it was equal 1.21 × 10-2 [kg m-2s-1]. The values of both coefficients are characteristic for the experimental setup, as they include the contribution of the system hydrodynamics. Next, the profiles of the solution temperatures were predicted at the same values of α and β for different air temperatures, and different air velocities within the range uair = 0.8 – 1.1, for which a weak flowrate dependence of ER was observed. The predictions were performed for the evaporation course in both 4-mL and 2-mL tanks, which differed in the ratio of the solution volume to the free liquid surface area and in the total heat exchange area, and compared to the experimental data. That comparison served as a model verification. The results of simulations are depicted in Fig. 4 (the reference data for the 4-mL tank) and Fig. 5a (the model predictions for the 2-mL tank). It is evident that a simple set of heat and mass balance equations may be used to support design of the forced convection crystallization process regarding the choice of the temperature of



air and the rate of evaporation as well as the temperature of the protein solution. The latter is crucial for proteins with temperature-sensitive structures.

3.3.2. Crystallization of the protein The crystallization process was performed in the drying chamber in the same way as described above for the water evaporation experiments. To minimize the protein consumption in the experiments, total volume of the solution in the reactors was 2 mL. The process variables, such as the linear velocity and the temperature of air, were selected in such a way that the protein and salt concentrations were kept within the crystallization operating window. Since the crystallization kinetics of the protein were fast, to accelerate ER a relatively high temperature was selected for the air stream (35 50°C) in the final experiments. The temperature of the protein solution at the steady state was maintained in the range Ts = 30 - 38 °C. Two examples of the course of the process are depicted in Figs 6A and 6B. In Fig. 6A the course of a fast crystallization process is shown. The dash-dotted line presents a hypothetical change in the protein concentration during water evaporation, which is not accompanied with the protein crystallization. The slope of that line corresponds to the value of ER that was selected based on the analysis of the crystallization kinetics presented in Fig. 3.

t [min]

A) 6

x*P × 102 w/w

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0

5

10

15

20

25

30

35

40

45

50

55

ER

5

Ope rati ng w

4 3

0 0.19

6

t [min] 0

5

10

20

Ope rati ng

4 indo w

30

40

50

60

70

80

win dow

90 100 110

ER

3

2 1

B)

2

exp. data course of cryst. 0.20

0.21

0.22

1 0.23

xsalt w/w

0.24

0.25

0.26

0 0.19

exp. data course of cryst. 0.20

0.21

0.22

0.23

0.24

0.25

0.26

xsalt w/w

Fig.6. Illustration of the time-course of crystallization on the phase diagram. (A) Fast crystallization from a saturated initial protein solution at ER =14.1x10-2 mg s-1 (Tair = 50 °C, φ = 10.5 %, Ts = 38 °C); (B) slow crystallization from undersaturated solution at ER = 3.9x10-2

mg s-1 (Tair = 35 °C, φ = 16.7 %, Ts = 30 °C).

The line crosses the boundary of the window at about 20 min. After that period nucleation is no longer inhibited, and the crystallization is initiated regardless of the salt concentration and the supersaturation degree of the solution (Fig. 3). The concentration 14 ACS Paragon Plus Environment

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drop is fast; the decrease in the protein concentration becomes insignificant after 30 min., and the equilibrium is established after about 50 min., when the final concentrations of the protein and salt are reached (compare to Fig. 3). This allows the supersaturation level to be kept within the operating window. The process course, which was anticipated based on the analysis of the crystallization kinetics and the phase diagram, was verified experimentally by the measurements of the protein concentrations in the mini-tank crystallizer at different time intervals (section 2.7). The corresponding salt concentrations were calculated according to the ER value and corrected slightly (1-2% for each of the experimental points) by including the water uptake in the crystalline phase (0.18 grams per 1 g of the protein24). The course of the crystallization is illustrated by the solid symbols in Fig. 6. The yield of the operation was determined by the difference between the initial and final protein concentrations in the solution. Since the coordinates of the final solution corresponded to the lower end of the operating window, a high yield of the operation could be achieved. Another example of the process course is shown in Fig. 6B. In that case, a dilute protein solution was used with the initial concentration distinctly below the solubility limit. Moreover, a lower value of ER was used. Therefore, a longer process duration was required to achieve the same final state of the solution in terms of the salt concentration and the supersaturation degree. The process parameters are summarized in Table 1. It can be observed that 72 - 85 % yield of the operation was reached in a single stage of the process. Moreover, the process yield can be further optimized by proper choice of the operating variables.

Table 1. Operating parameters of vapor crystallization corresponding to Fig. 6A (the first row), and Fig. 6B (the second row). xini,p × 102 xend,p × 102 xini,salt xend, salt Y [%] t [min]

4.266

0.629

0.190

0.256

85

50

2.907

0.828

0.190

0.252

72

95

The presence of the crystalline phase was verified by the optical microscopy analysis (section 2.8). Typical photos of crystals obtained for different ER values are shown in Fig. 7. The solid phase acquired in the crystallization experiments was stained by Eosin Y, which allowed us to distinguish protein crystals from amorphous phase.



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The protein crystallized in the form of clusters build from micro crystals of various size, without presence of amorphous precipitate. The morphology varied between clusters built from small, irregular crystals to well defined mono crystals with sharp edges. Since the nucleation of ovalbumin was very fast, it could be expected that a high number of nuclei was formed, which transformed into a high number of small crystals. Moreover, shearing stress caused by stirring might prevent the formation of larger crystals.20 The size of crystals and the number of mono crystals increased with increasing ER, i.e., with increasing the local supersaturation degree, as well as with reduction in the crystallization period (Fig. 7A-C). An increase in the supersaturation degree causes the crystal growth kinetics to accelerate, therefore larger crystals can be formed. Furthermore, a short process duration may be favorable for crystal growth, which can be attributed to the reduction of the stirring period, when the protein crystals are exposed to sheering stress. This also indicates that the process conditions can be adjusted to alter the crystal size distribution. The crystalline phase obtained after each crystallization experiment was subjected to resolubilization by adding phosphate buffer (section 2.9). The solid phase disappeared completely within a short time, which proved that the crystallization process was reversible and no insoluble aggregates were formed. Finally, the DSF measurements were used to determine unfolding transition point of the protein (the so-called meting temperature) in solutions before the crystallization and after the resolubilization of the crystalline phase (section 2.10).

The melting

temperature is a characteristic parameter for the protein and its structure. The melting events recorded for all protein solutions were identical (Fig. S2, Supplementary materials), which confirmed that the stability of the protein was preserved during the operation.


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Fig. 7. Photos of the protein crystalline phase and crystal size distribution (CSD) for different ER values: A) 4.9 x10-2 mg s-1; B) 7.1 x10-2 mg s-1; C) 14.6 x10-2 mg s-1.

4. Conclusions A novel technique for bulk protein crystallization was developed. Ovalbumin was crystallized from aqueous solution of ammonium sulfate in a drying chamber, where the protein solution was contacted with air stream. The process was designed based on the analysis of the phase diagram and kinetic data, and verified experimentally. The crystallization was realized in a single stage at a high yield. The operating variables were selected in such a way that the supersaturation level of the solution was kept inside the operating window of crystallization. The most important parameter was the rate of water evaporation, which could be controlled by the temperature and the flowrate of air. The air temperature determined the driving force of heat transfer. The crystallization kinetics were accelerated by increase in the air temperature, while a mild temperature of the protein solution was maintained. Heat and mass transfer due to water evaporation caused the temperature of the solution to remain distinctly lower than that of the air stream. Additionally, air humidity had to be monitored, as it determined the driving force for mass transfer. The tank geometry, i.e. the free liquid surface area and its ratio to the solution volume, might be also manipulated to optimize the process conditions. 17 ACS Paragon Plus Environment


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A rigorous optimization of the process requires development of a detailed mathematical model of the crystallization kinetics coupled with heat and mass transfer kinetic equations, which will be the subject of another study.

SUPPORTING INFORMATION Photo of the drying line Results of the DSF measurements

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected].; tel.: +48 178651853; Fax: +48 178543655.

Notes The authors declare no competing financial interest.

5.References (1) Hekmat D., Large-Scale Crystallization of Proteins for Purification and Formulation.

Bioprocess Biosyst. Eng. 2015, 38, 1209–1231. (2) Lee T. S.; Vaghjiani, J. D.; Lye G. J.; Turner M. K., A Systematic Approach to the LargeScale Production of Protein Crystals. Enzyme Microb. Technol. 2000, 26, 582−592. (3) Dos Santos R.; Carvalho A. L.; Roque A.C., Renaissance of Protein Crystallization and Precipitation In Biopharmaceuticals Purification. Biotechnol. Adv. 2017, 35, 41–50 (4) Smejkal B.; Helk B.; Rondeau J. M.; Anton S.; Wilke, A.; Scheyerer P.; Fries J.; Hekmat D.; Weuster-Botz D., Protein Crystallization in Stirred Systems - Scale-Up Via The Maximum Local Energy Dissipation. Biotechnol. Bioeng. 2013, 110, 1956–1963. (5) Govardhan C.; Khalaf N.; Jung C.W.; Simeone B.; Higbie A.; Qu S.; Chemmalil L.; Pechenov S.; Basu S.K.; Margolin A.L., Novel Long-Acting Crystal Formulation of Human Growth Hormone. Pharm. Res. 2005, 22, 1461–1470. (6) Webb S.D.; Webb J.N.; Hughes T.G.; Sesin D.F.; Kincaid A.C., Freezing Bulkscale Biopharmaceuticals Using Common Techniques and the Magnitude of FreezeConcentration. BioPharm. Int. 2002, 15, 22–34. (7) Shenoy B.; Wang Y.; Shan W.; Margolin A.L., Stability of Crystalline Proteins.

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Growth Des. 2013, 13, 2499−2506. (11) Judge R.A.; Forsythe E.L.; Pusey M.L., The Effect of Protein Impurities On Lysozyme Crystal Growth. Biotechnol. Bioeng. 1998, 59, 776–785. (12) Lorber B.; Skouri M.; Munch J. P.; Giegé R., The Influence of Impurities on Protein Crystallization; the Case of Lysozyme. J. Cryst. Growth 1993, 128, 1203–1211. (13) Judge, R. A.; Johns, M. R.; White, E. T., Protein Purification by Bulk Crystallization: the Recovery of Ovalbumin. Biotechnol. Bioeng. 1995, 48, 316−323. (14) Takakura T.; Ito T.; Yagi S.; Notsu Y.; Itakura T.; Nakamura T.; Inagaki K.; Esaki N.; Hoffman R. M.; Takimoto A., A High-Level Expression and Bulk Crystallization of Recombinant L-Methionine γ-Lyase, an Anticancer Agent. Appl. Microbiol. Biotechnol.

2006, 70, 183–192. (15) Peters J.; Minuth T.; Schröder W., Implementation of a Crystallization Step into a Purification Process of a Recombinant Protein. Protein Expr. Purif. 2004, 39, 43–53. (16) Zang Y.; Kammerer B.; Eisenkolb M.; Lohr K.; Kiefer H., Towards Protein Crystallization as a Process Step in Downstream Processing of Therapeutic Antibodies: Screening and Optimization at Microbatch Scale. 2011, PloS ONE 6(9):e25282. (17) Smejkal B.; Neeraj J.; Agrawal N. J.; Helk B.; Schulz H.; Giffard M.; Mechelke M.; Ortner F.; Heckmeier, Trout B. L.; Hekmat D., Fast and Scalable Purification of a Therapeutic Full-Length Antibody Based on Process Crystallization. Biotechnol. Bioeng.

2013, 110, 2452 – 2461. (18) Hebel D.; Huber S.; Stanislawski B.; Hekmat D., Stirred Batch Crystallization of a Therapeutic Antibody Fragment. J. Biotechnol. 2013, 166, 206−211. (19) Huettmann H.; Zich S.; Berkemeyer M.; Buchinger W.; Jungbauer A., Design of Industrial Crystallization of Interferon Gamma: Phase Diagrams and Solubility Curves.

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(20) 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. (21) Neugebauer P.; Khinast J. G., Continuous Crystallization of Proteins in a Tubular Plug-Flow Crystallizer. Cryst. Growth Des. 2015, 15, 1089−1095. (22) Hekmat D; Huber M; Lohse C; von den Eichen N; Weuster-Botz D., Continuous Crystallization of Proteins in a Stirred Classified Product Removal Tank with a Tubular Reactor in Bypass. Cryst. Growth Des. 2017, 17, 4162−4169. (23) Groß M.; Kind M., Bulk Crystallization of Proteins by Low-Pressure Water Evaporation. Chem. Eng. Technol. 2016, 39, 1483–1489. (24) Kołodziej M.; Poplewska I.; Piątkowski W.; Antos D., Design of Bulk Protein Crystallization Based on Phase Diagrams Accounting for the Presence of Interfacial Water. Cryst. Growth Des. 2018, 18, 393−401. (25) Agrawal S. G.; Paterson A. H. J., Secondary Nucleation: Mechanisms and Models.

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For Table of Contents Use Only

Forced convection evaporation for bulk protein crystallization Michał Kołodziej, Maksymilian Olbrycht, Izabela Poplewska, Wojciech Piątkowski, Dorota Antos Department of Chemical and Process Engineering, Rzeszów University of Technology, Powstańców Warszawy Ave. 6, 35-959 Rzeszów, Poland

Illustration of the course of crystallization on the phase diagram. The evaporation rate (ER) is selected in such a way that the protein and salt concentrations are kept inside the crystallization operating window; xp, xsalt are mass fractions of the protein and salt, respectively; t is the time coordinate for the crystallization course.


Forced convection evaporation for bulk protein crystallization - Crystal

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