CRYSTAL GROWTH & DESIGN 2002 VOL. 2, NO. 4 263-267
Articles Raman Spectroscopic Monitoring and Control of Aprotinin Supersaturation in Hanging-Drop Crystallization Rosana E. Tamagawa,† Everson A. Miranda,† and Kris A. Berglund*,‡,§ Department of Bioprocesses, School of Chemical Engineering, Campinas State University, Campinas, SP, Brasil, Departments of Chemistry, Chemical Engineering & Materials Science, and Agricultural Engineering, Michigan State University, East Lansing, Michigan 48824, and Division of Chemical Engineering Design, Luleå University of Technology, SE-971 87 Luleå, Sweden Received April 20, 2002
ABSTRACT: Fiber optic Raman spectroscopy is used for in situ monitoring of supersaturation during the hangingdrop crystallization of aprotinin. Schwartz and Berglund (1999) previously demonstrated this technique for lysozyme crystallization and showed it combines two critical elements for protein crystallization studies: real-time monitoring/ control of supersaturation and small amounts of sample. Experiments were carried out using 10 µL of protein solution. A partial-least-squares (PLS) calibration based on Raman spectra of standard solutions allowed an accurate measurement of aprotinin in a range of 2-100 mg/mL with a standard error of 0.54 mg/mL determined by a leaveone-out cross validation. A 10× microscope attached to a Raman fiber optic probe allowed the monitoring of the hanging-drop liquid phase in a noninvasive and real-time mode. Aprotinin solubility determined by measuring the protein concentration of drop solution at equilibrium decreased with increase in NaCl concentration. By continuously collecting Raman spectra of the liquid phase in the drop, the protein concentration was monitored in real time during the whole process. Control of supersaturation by manipulating the evaporation rate of the drop solution allowed the optimization of the process, leading to an increase in the resulting crystal size. Introduction Bovine pancreatic trypsin inhibitor (BPTI), commonly called aprotinin, is a single-chain protein with 58 amino acid residues and a molecular mass of 6511 Da. Aprotinin has been considered an ideal model in crystallographic studies of globular proteins because of its stability against a wide range of thermal, acidic, and basic conditions, as well as its small size. As a pharmaceutical compound, aprotinin has been successfully employed as an anticoagulant to reduce blood loss during cardiac surgeries. Most of the reported processes for purifying aprotinin include ion exchange and affinity chromatography, but none employed crystallization. Several studies reported aprotinin crystallization; however, they were aimed at molecular structure determination.1-3,5-7 In these stud* To whom correspondence should be addressed at the Luleå University of Technology. † Campinas State University. ‡ Michigan State University. § Luleå University of Technology.
ies crystallization is merely a sample preparation tool and has been carried out by trial and error screening methods. This study is a preliminary work to develop aprotinin crystallization as a purification process, with a particular interest in the control of the process. The key to control of crystallization is the ability to monitor and control the supersaturation. The objective of this work was the application of Raman spectroscopy for measuring supersaturation in time and in turn to control it during crystallization. We carried out this study using hanging-drop crystallization, since the ability to deal with small samples is a requirement when dealing with expensive compounds such as aprotinin. Experimental Section Materials. Solutions used in the crystallization experiments were prepared with deionized water and were filtered through 0.45 µm pore size filters. Aprotinin was obtained from Sigma (USA), and all reagents were of analytical grade. Siliconized glass cover slides used for hanging-drop crystallization were purchased from Hampton Research, Laguna Niguel, CA. For distinguishing between salt and protein crystals we used IZIT, an organic reagent by Hampton Research. Raman spectra were
10.1021/cg025524k CCC: $22.00 © 2002 American Chemical Society Published on Web 06/13/2002
264
Crystal Growth & Design, Vol. 2, No. 4, 2002
Figure 1. Raman spectrum of aprotinin/NaCl in aqueous solution at a concentration of 30 mg/mL of protein and 1.0 M of salt in 50 mM sodium acetate buffer, pH 4.5, at 24 °C. collected with a Kaiser Optical Systems, Inc. HoloLab Series 5000 instrument. Methods. (a) Acquisition and Calibration of Raman Spectra. Raman spectra were collected using a HoloLab Series 5000 Kaiser Optical Systems instrument, which employs a 100 mW external cavity stabilized diode laser at 785 nm for sample illumination. A CCD camera, a spectrograph, and a fiber optic probe complete this system. Remote sampling was accomplished by employing a fiber optic probe attached to a 10× microscope objective to focus the incident beam. The calibration model was based on spectra of 20 standard solutions ranging in concentration from 0 to 100 mg/mL of protein and from 0.0 to 2.0 M of (NH4)2SO4. Solutions were prepared in 50 mM sodium acetate buffer, pH 4.5. All spectra were acquired from approximately 10 µL volume and were sums of 10 accumulations collected over 5 s each at 8 cm-1 resolution. A partial-least-squares (PLS) regression model was built using QuantIR, a multivariate regression analysis software by ASIMettler Toledo. A typical spectrum is shown in Figure 1. (b) Hanging-Drop Crystallization. Ten-microliter droplets containing protein and salt were placed on glass cover slides, which were inverted and suspended over vessels with 10 mL of the reservoir solution. The tops of the vessels were greased with silicone in order to ensure an airtight seal. To ensure that the obtained crystals were composed of the protein, 1 µL of an organic dye, IZIT, was added to drops containing the crystals. Protein crystals present a high content of solvent distributed through microscopic channels. These channels allow the organic reagent to permeate through the structure, giving the crystal a blue color. Salt crystals, being more compact, do not absorb the reagent, and they remain transparent.
Results and Discussion Calibration of Raman Spectra. Raman spectra of standard solutions were collected as described and subjected to a PLS calibration using QuantIR. The peak regions selected to correlate spectra (Figure 1) to solution composition were as follows: 410-535, 825-880, 924-955, 1204-1230, 2880-2980, 1025-1050, and 1178-1080 cm-1. These regions correspond to aprotinin vibrational bands. Remaining regions of the spectra containing solvent and glass scattering as well as overlapped peaks caused a poor performance of the calibration, and then they were excluded from the model. Concentrations of standard solutions were from 0 to 100 mg/mL of aprotinin and from 0.0 to 2.0 M of NaCl. The standard error determined by a leave-oneout cross validation was 0,54 mg/mL of aprotinin.
Tamagawa et al.
Figure 2. Aprotinin solubility at 24 °C in the presence of NaCl. The solution was buffered with 50 mM sodium acetate, pH 4.5. The experiment was conducted in triplicate, and the error bars represent the standard deviation.
Solubility Determination. Aprotinin solubility at 24 °C was determined in the presence of NaCl in 50 mM sodium acetate buffer pH 4.5 (Figure 2). Droplets (10 µL) of protein and salt solution were suspended above vessels with 10 mL of the respective crystallizing solution. Drops contained initially 10, 15, 25, 50, and 70 mg/mL of aprotinin and 1.0 M of salt, and the respective reservoirs contained 2.6, 2.3, 2.0, 1.7, and 1.4 M of salt. The drop-reservoir systems were left at room temperature for 3 weeks to achieve equilibrium. Raman spectra from the liquid phase were then collected in order to determine protein concentration. The solubility of aprotinin in the presence of NaCl was in agreement with the data reported by Lafont et al.3 They have reported 44, 15, 5, and 3 mg/mL of aprotinin at equilibrium in the presence of 1.4, 1.7, 2.0, and 2.3 M of NaCl, respectively. In Figure 2 these values are 44, 17, 7, and 6 mg/mL, respectively. In agreement with Lafont et al.,3 the aprotinin solubility decreased with an increase in salt concentration. Effect of Evaporation Rate on Aprotinin/NaCl Crystallization. Two drops identified as drops A and B, each containing 14 mg/mL of aprotinin and 1.0 M NaCl, were suspended above reservoirs with 3.0 and 2.0 M NaCl, respectively. Drop and reservoir solutions were buffered with 50 mM sodium acetate, pH 4.5. By continuous collection of Raman spectra from the liquid phase, the aprotinin concentration in drops A and B was monitored over time (Figure 3). While the changes are relatively small as compared to the error shown in Figure 2, it should be noted that these are trend analyses, not absolute measurements. The reproducibility within the trend is much higher than in the absolute measurements. These results permitted differences from the evaporation stage to the crystal formation stage to be delineated. The increase of protein concentration shown in Figure 2 reflects the evaporation caused by mass transfer from the droplet to the well, whereas the decrease represents the crystallization of aprotinin and its transfer to the solid phase. As expected, the higher the difference of vapor pressure between drop and reservoir, the higher the evaporation rate and the earlier the onset of crystallization. Crystal growth in drop A started at about 400 min, whereas in drop B it started at about 600 min. The presence of a
Aprotinin Supersaturation
Figure 3. Aprotinin concentration over time for in two hanging drops (2, drop A; 0, drop B) submitted to different evaporation rates in the presence of NaCl at 24 °C. NaCl concentrations in the reservoir were 3.0 M (drop A) and 2.0 M (drop B). Solutions were buffered with 50 mM sodium acetate, pH 4.5.
Figure 4. Monitoring of aprotinin supersaturation during hanging-drop crystallization of two drops (2, drop A; 0, drop B) submitted to diferent evaporation rates in the presence of NaCl. Solutions were buffered at 50 mM sodium acetate, pH 4.5. Reservoir solutions contained 3.0 and 2.0 M NaCl for drops A and B, respectively.
crystal in the laser path causes an increase in scattering that is easily distinguished from the solution scattering. In this event, the laser position was changed to avoid the crystal. The supersaturation in the drops was monitored and is graphically represented in the solubility diagram in Figure 4. In this diagram, protein and salt concentrations of drops A and B were plotted with the solubility curve (solid line). In the course of evaporation, salt and protein concentrations increased gradually as the solution became supersaturated. Supersaturation is attained when concentration profiles crossed the solubility limit shown in the solubility diagram. Due to the difference of evaporation rates, drops A and B reached different supersaturation degrees before crystallization started. The point of crystallization is represented in Figure 4 by the dashed arrow, which reflects the depletion of protein from liquid to solid phase, returning the solution to equilibrium (solubility limit). The crystallization stage is also visualized in Figure 3 through the decrease of concentration over time.
Crystal Growth & Design, Vol. 2, No. 4, 2002 265
Figure 5. Aprotinin crystals in drop A as described in Figures 2 and 3. The drop initial composition was 14 mg/mL aprotinin and 1.4 M NaCl. The reservoir composition was 3.0 M NaCl. Solutions were buffered with 50 mM sodium acetate, pH 4.5.
Figure 6. Aprotinin crystals in drop B as described for Figures 2 and 3. The drop initial composition was 14 mg/mL aprotinin and 1.4 M NaCl. The reservoir composition was 2.0 M NaCl. Solutions were buffered with 50 mM sodium acetate, pH 4.5.
The crystals obtained in drops A and B are presented in Figures 5 and 6, respectively. Drop A resulted in a higher number of crystals than drop B; however, the crystals obtained in both drops were about the same size, approximately 200 µm. The dimensions of crystals in drops A and B were approximately the same for different reasons. In drop A the size is fixed by high supersaturation and corresponding excessive nucleation. On the other hand, in drop B the size was the consequence of low supersaturation (and corresponding lower nucleation) and lack of solute resources in the drop solution. The maximum supersaturation attained in drop B when crystallization started was quite close to the solubility limit. Solution was then desupersaturated, returning the solution to equilibrium, and crystal growth was stopped due to depletion of solute from the liquid phase. Control of Crystallization through the Manipulation of Evaporation. Supersaturation monitoring provides the ability to dynamically control the crystallization in response to events occurring in the solution (i.e., to lower the supersaturation when nucleation starts or to increase it when the solution is completely desupersaturated). In this study the evaporation rate in a hanging drop was manipulated to control the
266
Crystal Growth & Design, Vol. 2, No. 4, 2002
Figure 7. Aprotinin crystallization with supersaturation control: (9) aprotinin concentration in the drop (×10-6); (s) NaCl reservoir concentration. The drop and solutions were buffered with 50 mM sodium acetate, pH 4.5. The experiment conducted at 24 °C.
supersaturation and optimize the process in terms of crystal size. Evaporation was controlled by changing the composition of the reservoir solution: salt concentration was increased in order to increase evaporation from the drop, thereby increasing the drop concentration, and decreased in order to decrease evaporation, thus holding the drop concentration. A drop with 14 mg/mL of aprotinin and 1.4 M NaCl was placed on a siliconized glass cover, which was inverted and sealed over the top of a vessel containing 10 mL of 3.0 M NaCl solution. Drop and reservoir solutions were buffered with 50 mM sodium acetate at pH 4.5. The vessel was equipped with inlet and outlet ports that permitted changes in the reservoir solutions without removing the glass cover or disturbing the solution from which the protein was crystallizing. By continuously collecting Raman spectra from the liquid phase, protein concentration in the drop solution was monitored and is shown in Figure 6. This figure also presents the profile of salt concentration in the reservoir, which was changed according to events occurring in the drop. The first of these changes occurred at 600 min when crystallization started. The start of crystallization was observed through the decrease in the protein concentration. The reservoir liquid was then replaced by a solution with the same salt concentration present in the drop, 1.8 M NaCl (estimated value). When the salt concentrations in the drop and reservoir were made the same, evaporation was stopped due to equilibrium of the vapor pressure in the drop-reservoir system. When the evaporation was interrupted, the increase in supersaturation was stopped, which favored the growth of existing crystals instead of new nucleation. As crystals grew, the drop solution was then desupersaturated until equilibrium (solubility limit) was achieved. The decrease of protein concentration toward the equilibrium is presented in Figure 8. At this time, the reservoir liquid was changed again to 3.0 M NaCl, as shown in Figure 7. Through evaporation, the drop solution became supersaturated again, as shown in Figure 8. To avoid the formation of new nuclei and favor crystal growth, supersaturation was kept close to the solubility limit. To interrupt evaporation and avoid excessive supersaturation, the reservoir solution was
Tamagawa et al.
Figure 8. Control of supersaturation during aprotinin crystallization at 24 °C in the presence of NaCl. The initial drop composition was 14 mg/mL aprotinin and 1.4 M NaCl. Reservoir solutions varied from 1.8 to 3.0 M NaCl, as shown in Figure 9. Solutions were buffered at 50 mM sodium acetate, pH 4.5.
Figure 9. Aprotinin crystals obtained with supersaturation control. The drop initial composition was 14 mg/mL aprotinin and 1.4 M NaCl. The reservoir composition varied from 1.8 to 3.0 M NaCl. Solutions were buffered with 50 mM sodium acetate, pH 4.5.
changed to 1.9 M NaCl, which was the salt concentration estimated in the drop at this instance. The existing crystals were then allowed to grow until the solution was completely desupersaturated. Once more, the salt concentration in the reservoir was increased to 3.0 M in order to supersaturate the drop solution. Again, precaution was taken to avoid excessive supersaturation. To interrupt evaporation, the reservoir solution was changed to 2.0 M NaCl, the same salt concentration present in the drop. The crystals were then subjected to a third growth stage in which the solution returned to equilibrium. Figure 9 is a photomicrograph of the crystals obtained in this controlled crystallization. The crystals achieved dimensions of approximately 600 µm, 3 times larger than crystals obtained in the noncontrolled experiments (Figures 5 and 6). Conclusions Aprotinin solubility was measured using Raman spectroscopy to determine the concentration of solution at equilibrium with crystals in 10 µL drops. In agreement with the literature, the aprotinin solubility in the
Aprotinin Supersaturation
presence of NaCl decreases with an increase in salt concentration. Moreover, Raman spectroscopy was used to measure the aprotinin supersaturation with high accuracy during the hanging-drop crystallization. When the compositional change of the hanging drop was monitored during vapor diffusion, supersaturation, which is known to affect nucleation and growth, was determined in real time. Subsequently, the Raman data were used to manipulate the evaporation in order to control supersaturation and optimize aprotinin crystallization in terms of crystal size. Aprotinin crystals obtained with supersaturation control achieved dimensions of approximately 600 µm, 3 times larger than crystals generated in noncontrolled crystallization under the same conditions. Acknowledgment. The authors wish to thank CAPES of Brazil for financial support of R.E.T. Additional support from the Center for New Plant Products and Processes at Michigan State University is also appreciated. Kaiser Optical Systems, Inc. is also thanked for supplying the Raman spectrometer used in this study.
Crystal Growth & Design, Vol. 2, No. 4, 2002 267
References (1) Hamiaux, C.; Pe´rez, J.; Prange´, T.; Veesler, S.; Rie`s-Kautt; Vachette, P. The BPTI decamer observed in acidic pH crystal forms preexists as a stable species in solution. J. Mol. Biol. 2000, 297, 697-712. (2) Lafont, S.; Veesler, S.; Astier, J. P.; Boistelle, R. Comparison of solubilities and molecular interactions of BPTI molecules giving different polymorphs. J. Cryst. Growth 1997, 173, 132-140. (3) Lafont, S.; Veesler, S.; Astier, J. P.; Boistelle, R. Solubility and prenucleation of aprotinin (BPTI) molecules in sodium chloride solutions. J. Cryst. Growth 1994, 143, 249-255. (4) Schwartz, A.; Berglund, K. A. The use of Raman spectroscopy for in situ monitoring of lysozyme concentration during crystallization in a hanging drop. J. Cryst. Growth 1999, 203, 599-603. (5) Veesler, S.; Lafont, S.; Marrcq, S.; Astier, J. P.; Boistelle, R. Prenucleation, crystal growth and polymorphism of some proteins. J. Cryst. Growth 1996, 168, 124-129. (6) Walter, J.; Huber, R. Pancreatic trypsin inhibitor, a new crystal form and its analysis. J. Mol. Biol. 1983, 167, 911917. (7) Wlodawer, A.; Nachman, J.; Gilliland, G. L.; Gallagher, W.; Woodward, C. Structure of form III crystals of bovine pancreatic trypsin inhibitor. J. Mol. Biol. 1987, 198, 469-480.
CG025524K
