ARTICLE pubs.acs.org/IECR
Design of Protein Crystallization Processes Guided by Phase Diagrams Sze Kee Tam, Hok Chung Chan, and Ka Ming Ng* Department of Chemical and Biomolecular Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong
Christianto Wibowo ClearWaterBay Technology, Inc., 4000 W. Valley Boulevard, Suite 100, Pomona, California 91789, United States ABSTRACT: Many proteins including pharmaceuticals and industrial enzymes, after separation and purification, are high-valueadded products. In this study, a general framework based on solid liquid equilibrium (SLE) phase diagrams for the conceptual design of protein crystallization processes is proposed. First, the coordinates for phase diagrams involving proteins are defined. Second, relevant regions of the phase diagram are determined by experiments. Finally, process synthesis is performed by visualizing the process paths on the phase diagram. This approach is demonstrated with two case studies. One is the design of a lysozyme recovery process in which salt-lean lysozyme solids are recovered from a lysozymesodium chloride solution. The other is the design of a process for separating lysozyme and ovalbumin in the presence of ammonium sulfate. The former design is in agreement with patent literature, and the latter design was verified with experiments. Thus, the feasibility of designing protein crystallization processes based on a phase diagram with ionic coordinates and color mapping is demonstrated.
’ INTRODUCTION Many purified proteins, including industrial enzymes and biopharmaceuticals, are important high-value-added products. Biopharmaceuticals, for instance, constituted approximately 10% of the global prescription drug market in 2007.1 Chromatography and filtration common in industrial protein purification accounted for as much as 80% of the product cost.2 Thus, alternatives including ultrafiltration, microfiltration, and crystallization are of high interest.3 In particular, it is highly desirable to separate and purify proteins by crystallization because of its relatively low operating cost. Salting-out is one of the most important crystallization techniques for the recovery and purification of proteins.4 This is achieved by adjusting the pH, ionic strength, and temperature of the solution to alter protein solubility. Interestingly, while there is voluminous literature on the precipitation of a single protein, relatively few basic studies have been devoted to the separation of two or more proteins by crystallization.5 A notable achievement is the recovery of ovalbumin from a solution containing conalbumin and lysozyme, with the product ovalbumin crystal purity greater than 99 wt %.6 Phase diagrams are deemed essential for protein crystallization because of the presence of various solid phases in a typical protein system.7 This article presents a framework for designing protein crystallization processes guided by phase diagrams. Representation of Protein Phase Behavior. Protein is a polymer of amino acids, which are ampholytes due to their ability to donate and accept protons. Thus, its solubility varies with pH and salt concentration.8,9 A framework for the determination and representation of SLE phase diagram for systems involving ampholytes has been developed in earlier publications.1012 The representation method can be readily extended to protein systems as discussed below. r 2011 American Chemical Society
Coordinate System for Describing a System with Proteins. Consider a system containing a single protein, water, and two electrolytes, MOH and HX, where Mþ and X represent monovalent cation and anion, respectively, although the development below can be easily extended to multivalent species. Water can be considered as an electrolyte as well.10 A protein with n acid groups and m basic groups can donate and receive proton(s), and the neutral protein is denoted by HnP. Pn is the protein molecule after donating n protons and (HnP 3 mH)mþ is the protein molecule after receiving m protons. If the protein loses i protons from its acidic groups and receives j protons on its basic groups, it is represented as (Hn-iP 3 jHþ)iþj. If it receives an additional proton, we have
ðHni P 3 jHþ Þi þ j þ Hþ S ðHni P 3 ðj þ 1ÞHþ Þi þ j þ 1
ð1Þ
If it donates an additional proton, we have ðHni P 3 jHþ Þi þ j S ðHni1 P 3 jHþ Þi þ j 1 þ Hþ
ð2Þ
At complete ionization, we have ðHni P 3 jHþ Þi þ j S ðn i þ jÞHþ þ Pn
ð3Þ
where i = 0, 1, 2, ..., n and j = 0, 1, 2, ..., m. The protein can associate with Mþ and X from MOH and HX to form protein-related salts Mi(Hn-iP)(HX)j, where i = 0, 1, 2, ..., n and j = 0, 1, 2, ..., m. Received: February 6, 2011 Accepted: May 20, 2011 Revised: May 4, 2011 Published: May 20, 2011 8163
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This condition is the basis for the coordinate system. The number of coordinates is determined by the degrees of freedom as follows. There are two simple cations, Hþ and Mþ, three simple anions, OH, X and Pn, [(m þ 1)(n þ 1) 2] complex ions from the protein, four electrolytes, MOH, HX, MX, H2O, and (m þ 1)(n þ 1) protein-related salts (including HnP). The total number of components in this system is (2mn þ 2m þ 2n þ 9). For each of the electrolytes and complex ions, there is one dissociation equation. Hence, the total number of independent reactions is [(m þ 1)(n þ 1) 2] þ 4 þ [(m þ 1)(n þ 1)] = 2mn þ 2m þ 2n þ 4. Based on Gibbs phase rule, the number of degrees of freedom for the isobaricisothermal protein system is (2mn þ 2m þ 2n þ 9) (2mn þ 2m þ 2n þ 4) 2 = 3. The general expressions for the ionic coordinates have been discussed elsewhere.12 Among the five cations and anions, Mþ, X and Pn are chosen to represent the protein system. If concentrations are expressed in terms of molality, the coordinates are RðMþ Þ ¼
RðX Þ ¼
RðPn Þ ¼
½Mþ
ð4Þ
½Mþ þ ½Hþ ½X
½X þ ½OH þ n½Pn n½Pn ½X þ ½OH þ n½Pn
ð5Þ
ð6Þ
To reduce the dimensionality, a projection can be obtained by normalization. By choosing the Mþ and X as reference components in the projection, the coordinates are given as SðMþ Þ ¼
SðX Þ ¼
½Mþ ½Mþ þ ½X þ n½Pn
ð7Þ
½X
ð8Þ
½Mþ þ ½X þ n½Pn
However, since the molecular weight of protein is much larger than that of its counterions, when concentrations are expressed in terms of molality, all saturation surfaces in the phase diagram cluster near the water vertex. For this reason, mass fraction is used in place of molality: TðMþ Þ ¼
x Mþ xMþ þ xX þ xPn
TðX Þ ¼
x Mþ
xX þ xX þ xPn
∑ xM
i¼1
zi þ i
þ
∑
j¼1
x
zj Xj
þ
ð11Þ
p
∑x
k¼1
∑ xM
n Pkk
zi þ i
þ
a1
∑
j¼1
x
zj Xj
þ
p
∑x
k¼1
n Pk k
xPnl
ð13Þ
l
c1
∑ xM
i¼1
i
a1
TðPnl l Þ ¼
ð12Þ
j
c1 i¼1
ð10Þ
xMzi þ c1
xX zj
z
TðXj j Þ ¼
ð9Þ
For a system with additional proteins or ions, the corresponding phase diagram becomes more complex and the dimensionality increases. Consider a system having c cations, a anions (not counting protein ions) and p proteins. There are c 1 coordinates representing the cations, a 1 representing the anions, and p 1 representing the protein ions: TðMizi þ Þ ¼
Figure 1. Hypothetical isobaricisothermal SLE phase diagram of protein system with single protein, single cation, and single anion: (a) complete picture; (b) zoomed image.
zi þ i
þ
a1
∑
j¼1
xX zj þ j
p
∑ xP
k¼1
nk k
where i = 1, 2, ..., c 1; j = 1, 2, ..., a 1; and l = 1, 2, ..., p 1. The total number of coordinates is thus c þ a þ p 3.
’ PROTEIN PHASE BEHAVIOR Single Protein System. Consider a system consisting of water, HnP, MX, MOH and HX. The system can be described by two cations, Hþ and Mþ, and three anions, OH, X and Pn. Using the ionic coordinate representation discussed previously, the phase behavior of the protein system can be depicted in the 8164
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Figure 2. Solventless projection of hypothetical isobaricisothermal SLE phase diagram for protein system: (a) showing saturation solubility surfaces; (b) showing solubility data as contour curves (in mass fraction of water); and (c) a cut showing the solubility curve along the line connecting HnP and MX.
phase diagram in Figure 1. All solubility surfaces in the phase diagram, that for pure protein, MOH, MX, and protein salts, tend to cluster around the H2O vertex and a solventless projection in term of mass fractions is thus suggested. There is no saturation region near the HX vertex because the acid is usually a liquid in the temperature range under consideration and such a low pH region is normally irrelevant for process design. The gradient color area in the middle part represents the composite solubility surface of protein salts Mi(Hn-iP)(HX)j, where i = 0, 1, 2, ..., n and j = 0, 1, 2, ..., m. This representation is more practical because there are usually a large number of protein salts due to the large values of m and n, and it is difficult to clearly demarcate the boundaries between the solubility surfaces of individual salts with different solid compositions. Therefore, it is convenient to consider that the protein salts exhibit the solid solution behavior, in which the
solid composition is a continuous function of the liquid composition. Readers interested in solid solution phase behavior are referred to earlier publications.13,14 The values of m and n represent the maximum number of protons that protein can receive and donate. Such information can be obtained from the protein titration curve, which tracks the net surface charge of protein as a function of pH. In this study, m is defined as the number of protons that the protein molecule can donate at a sufficiently high pH value. Conversely, n is defined as the number of protons that the protein molecule can receive at a sufficiently low pH. A corresponding solventless projection is shown in Figure 2a. This projection offers no information on the amount of water at any point. One convenient way to present the solubility information is by drawing contour lines representing saturated solutions containing the same amount of water in mass fraction or the sum 8165
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Figure 3. Solventless projection of hypothetical isobaricisothermal SLE phase diagram for protein system with two proteins, single cation and single anion system: (a) complete picture; (b) cut with fixed protein ratio; and (c) cut with fixed cation to anion ratio.
of the mass fraction of hydroxide and hydrogen ions. The contour plot in terms of the mass fraction of water is shown in Figure 2b. Note that a smaller number corresponds to a higher solubility. It is interesting to observe how the solubility changes along a certain cut at a constant ratio of Mþ to X, such as the curve shown in Figure 2c. This cut, connecting HnP and MX in Figure 2b, shows the solubility behavior of a system containing the protein and an equimolar mixture of Mþ and X. Binary Protein System. Consider a system with two proteins, A and B, denoted by HnAPA and HnBPB, as well as MOH and HX. There are two cations, Mþ and Hþ, two anions, OH, X apart from the protein ions and 2 protein ions PnAA and PnBB. Therefore, the projection of its phase diagram is three-dimensional (Figure 3a). The red dotted lines indicate a cut at a constant ratio of protein A to protein B, which is shown in detail in Figure 3b. This cut features the
saturation regions for MOH and MX, and a composite region representing a mixture of protein salts M(iþk)(H(nA-i)PA)(H(nB-k)PB)(HX)j. No distinction is made between the regions for protein A salts and protein B salts, as protein mixtures tend to precipitate in the form of solid solution. To observe the effect of counterions on the system, a different cut taken at a fixed ratio of MOH and HX (indicated by the blue dotted lines in Figure 3a) is more useful. As shown in Figure 3c, such a cut highlights the saturation regions of the two pure proteins as well as that of the mixed protein salts.
’ APPLICATION IN PROCESS SYNTHESIS In this section, two examples are presented to illustrate the usefulness of phase diagram representation in synthesizing protein 8166
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Industrial & Engineering Chemistry Research separation or purification processes. The first example deals with the isolation of a single protein (lysozyme), while the second one addresses the issue of separating a mixture of two proteins (lysozyme and ovalbumin). Because sufficient phase behavior data are not available from the literature, SLE experiments were performed. The materials and experimental methods are described below. Materials and Analytical Methods. Materials. A commercial hen egg white lysozyme, Delvozyme, from DSM was used as the source of lysozyme. According to the specifications, Delvozyme in the form of lysozyme chlorhydrate has a purity equal to or greater than 95 wt %. Ovalbumin powder was obtained from Sigma Aldrich. Ovalbumin and lysozyme used as standards were purchased from Sigma. Sodium chloride, ammonium hydroxide solution, sodium hydroxide, and trifluoroacetic acid (TFA) were obtained from Riedel-de Ha€en, hydrochloric acid and sulfuric acid from Mallinckrodt Chemical Works, ammonium sulfate from USB Corp. and acetonitrile from Burdick & Jackson. Deionized water was purified with Millipore Milli-Q-UF plus to a resistivity greater than 18 MΩ cm. Analytical Methods. The concentration of lysozyme, by itself, was determined by a Shimadzu PharmaSpec UV-1700 UVvis spectrophotometer. The absorbance of lysozyme in the sample was measured at a wavelength of 280 nm. The concentration of lysozyme was calculated by converting the absorbance to concentration based on the calibration curves constructed using a pure protein standard. The concentrations of a binary mixture of ovalbumin and lysozyme were determined using an Alltech 250 mm long 4.6 mm inner diameter (ID) Prosphere C18 300A 5u column in an Agilent 1100 Series HPLC. The mobile phase A is Milli-Q water with 0.1% TFA and mobile phase B is acetonitrile with 0.1% TFA. Five mL of sample was eluted at a flow rate of 1 mL/ min. The eluent changed from 15% B to 65% B linearly in 20 min and the concentration of B stayed at 65% for another 15 min. The absorbance at 280 nm was detected on G1315B photodiode absorbance detector. The concentration of ovalbumin and lysozyme were calculated by converting the area to concentration based on the calibration curves constructed using the ovalbumin and lysozyme standards. The concentrations of the cation, sodium, and ammonium ions were measured using a Dionex CS12A cation column with CSRS 300 4 mm autosuppressor in DX500 ion chromatography system eluting with 20 mM methanesulfonic acid. The same chromatography system with a Dionex AS14A anion column and ASRS Ultra II 4 mm auto suppressor eluted by 3.5 mM Na2CO3/ 1.0 mM NaHCO3 was used to determine the concentration of chloride and sulfate ions in the sample. Example 1. Lysozyme Purification. Lysozyme is an enzyme capable of damaging bacterial cell walls by attacking peptidoglycans. A large number of studies on lysozyme solubility in ammonium sulfate and sodium chloride solution are documented in the literature.15,16 In this example, hen egg white lysozyme in aqueous NaCl solution was used as the starting material. NaOH and HCl were selected for adjusting the pH as they have common ions with the existing salt. From the titration curve of hen egg white lysozyme,17 the net charge of the protein is 13 as pH approaching 14. Therefore, the value of n is taken as 13 and pure lysozyme is denoted by H13Lys. On the basis of our degree of freedom analysis, the degree of freedom of this isobaric isothermal lysozymesodiumchloride aqueous system is 3.
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Figure 4. (a) Sketch of solventless projection of isothermalisobaric phase diagram for lysozymesodium chloridehydrogen chloridesodium hydroxide system; red dotted lines indicate iso-pH cuts at pH 8.4, 6.4, and 4.3 (from left to right), and blue dotted line indicates a cut along the line connecting H13Lys and NaCl. (b) Change of solubility along a cut joining lysozyme and NaCl in a solventless projection of isothermalisobaric lysozyme system, along with literature data at different pH values.
(Readers interested in more details of this analysis and system are referred to ref 18.) Choosing Naþ and Cl as the independent coordinates, the coordinate expressions for the solventless phase diagram of the system are given as xNaþ ð14Þ TðNaþ Þ ¼ xNaþ þ xCl þ xLys13 TðCl Þ ¼
xCl xNaþ þ xCl þ xLys13
ð15Þ
The conceptual solventless projection for lysozyme system is shown in Figure 4a. Note that the diagram is not to scale. It is expected that there are saturation zones representing pure lysozyme (H13Lys), NaCl, and lysozyme with counterions, Nai(H13iLys)(HCl)j. The latter is represented by gradient color to reflect the fact that the solids contain different amounts of counterions Cl, OH, Hþ, and Naþ. The blank regions close to the NaOH and HCl vertices are not of interest because the extreme pH conditions would destroy the protein anyway. Most literature data on lysozyme/salt systems are reported in terms of salt concentration (ionic strength) and pH, which is 8167
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Table 1. Summary of Experimental Results for Lysozyme Precipitation Experiments liquid phase (mg/g sample) 13
Lys
Cl
þ
Na
solid phase (mg/g sample) 13
Lys
Cl
Naþ 67.67
0.03
110.69
71.05
46.88
100.56
0.47
63.38
40.46
327.39
39.81
23.86
0.10 0.31
101.11 91.89
65.22 59.33
66.93 54.78
106.58 90.87
60.60 53.96
0.68
78.92
50.55
251.19
59.62
36.83
0.05
88.40
57.35
75.01
86.90
52.01
0.02
90.75
58.98
75.35
97.54
58.59
0.09
90.78
59.01
70.43
88.77
52.49
0.16
78.92
51.55
84.09
75.02
44.62
0.37
81.88
53.31
281.38
60.10
38.17
0.58 0.75
93.09 65.92
60.59 42.67
224.59 287.02
85.59 42.29
49.28 26.14
0.46
86.64
56.64
261.88
67.69
40.40
0.76
76.43
49.96
353.77
52.06
30.53
0.73
88.96
58.36
205.49
66.91
41.52
0.44
65.62
43.20
295.27
44.38
23.85
0.61
51.68
34.18
292.76
38.89
21.93
1.05
52.74
34.81
342.68
13.66
22.11
0.71 0.97
40.55 24.16
27.19 16.19
268.00 229.76
27.75 14.04
15.61 7.60
0.73
33.39
23.21
337.13
27.76
14.95
1.13
23.78
16.38
355.78
20.81
8.30
5.48
65.11
42.86
348.08
7.92
25.61
1.42
12.31
8.34
301.76
7.95
4.48
1.86
12.36
8.44
379.09
11.42
4.22
7.50
41.05
27.26
279.54
11.64
18.94
9.65 3.80
34.51 12.95
22.51 8.59
241.13 385.91
28.12 12.64
16.03 4.31
18.47
18.61
11.30
371.08
6.45
7.11
27.38
13.34
7.80
479.19
5.29
3.36
29.87
13.19
7.81
525.52
6.30
2.81
42.25
13.09
7.90
519.35
17.66
2.51
45.63
13.24
7.83
467.63
17.74
2.96
104.12
3.56
3.35
498.98
0.79
1.97
156.36
4.31
5.11
501.37
7.21
1.93
adjusted by the addition of electrolytes. Three sets of such data, taken at three different pH values (4.3, 6.4, and 8.4), are shown in Figure 4b. Since no information is available on the solubility as a function of the amount of cations and anions, the essentially nonlinear cuts at constant pH can only be illustrated qualitatively by the dotted red curves in Figure 4a. At high pH, the iso-pH cut is closer to the NaOH vertex; while at low pH, it is closer to the HCl vertex. Note that the data points at xNaCl = 0 do not show the formation of pure lysozyme. This is because these iso-pH cuts do not lie within the saturation region of pure lysozyme (H13Lys), but in that of the lysozyme salts (Nai(H13iLys)(HCl)j) instead. Note that in Figure 4b, as pH increases toward the isoelectric point of lysozyme (pI = 10.7), the mass fraction of water increases. In addition, precipitation experiments were conducted to obtain the data for a cut at an equimolar ratio of Naþ/Cl (along the dotted blue line in Figure 4a). These solubility data are also plotted in Figure 4b.
All these data show that the solubility decreases then increases with increasing salt concentration, which is consistent with the conceptual picture in Figure 2c. The experimental solubilities with a water mass fraction in the range of 0.640.98 are much higher than the solubility of desalted isoelectric lysozyme of less than 85 mg/L (which is equivalent to a water mass fraction of 0.999 as indicated by the diamond symbol in Figure 4b).19 Theoretically, there should be a saturation region of sodium chloride near the right end of Figure 4b. The fact that this region cannot be observed from the experimental data implies that it would be difficult to use crystallization to remove NaCl in a protein purification process, since an extremely low protein concentration would be required to access the NaCl saturation region. Therefore, an alternative method such as ultrafiltration would be preferable to remove NaCl from the system. As precipitation of lysozyme from hen egg white in sodium chloride solution usually results in NaCl-containing lysozyme crystals, the feed composition must be located inside the protein salt saturation region, in which a solid solution-like phase behavior is expected. For this reason, solidliquid equilibrium data are especially important in designing the process. Since solidliquid equilibrium data in terms of ion concentrations for lysozymesodium chloride system were not available, the following experiments were performed. A stock solution with 30 wt % concentrated lysozyme chlorhydrate and a stock solution with 20 wt % sodium chloride solution were prepared and filtered with low protein binding Millex-GP 0.22-μm syringe filters. A desired amount of lysozyme stock solution and DDI water were mixed with a magnetic stirrer. Then, the sodium chloride solution at a specified amount was slowly added into the diluted lysozyme solution to avoid high local concentration. The lysozyme solution became cloudy and precipitants were formed. The pH of the solution was adjusted by 1 N hydrochloric acid and 1 N sodium hydroxide solution. After 24 h, liquid phase samples were obtained by collecting the supernatant, while solid samples were recovered by filtration. The amounts of sodium ion, chloride ion, and lysozyme in the samples were determined using the aforementioned analytical methods. The experimental results are summarized in Table 1. The liquid compositions are plotted in solventless coordinates using eq 14 and eq 15 (Figure 5). The solubility data plotted as contour lines are in mass fraction of the sum of hydrogen and hydroxide ions. Data interpolation was performed using MATLAB griddata. Representation of the solid equilibrium data, that is, composition from the solid phase sample, is more involved. A gradient color map is first constructed. Red and blue are used to represent the values of T(Cl) and T(Naþ), respectively, with a darker color indicating a higher value of the corresponding ion. For a given point in the phase diagram, the experimental solid composition is converted to obtain T(Cl) and T(Naþ) values. The corresponding color in the gradient color map is plotted at the same given point in the phase diagram. Thus, conversely, by matching the color in a phase diagram with that in the gradient color map, the solid composition that is in equilibrium with the liquid composition can be identified. No attempt was made to fit the data using a thermodynamically consistent solubility model. The data indicate that lysozyme salts with low Naþ and Cl content can be obtained. The solubility tends to decrease with the addition of NaOH. The region becomes darker when the regions get closed to the NaCl, showing that lysozyme salts with 8168
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Figure 5. Experimental results for the isothermalisobaric lysozymesodium chloride-hydrogen chloridesodium hydroxide system, with the contour lines representing the solubility data and the gradient color representing the distribution of solid phase composition.
high Naþ and Cl content are more likely to be obtained in the region with high NaCl content. Consider the synthesis of a process for recovering lysozyme salts with T(Cl) < 0.1 and T(Naþ) < 0.1. Instead of the gradient colors, let us define three regions in Figure 6a. Lysozyme salts with low Naþ and Cl content can be obtained in the yellow region (T(Naþ) < 0.1 and T(Cl) < 0.1). The pink color (T(Naþ) < 0.1 and T(Cl) > 0.1) represents a region with higher Cl but still low Naþ content, while the green color (T(Naþ) > 0.1 and T(Cl) > 0.1) represents a lysozyme with high Naþ and Cl content. With this information, it is possible to synthesize a continuous process for lysozyme purification. The process paths are shown in Figure 6a, and the conceptual phase diagram is depicted in Figure 6b. The crude lysozyme solution, which is a dilute lysozyme solution with sodium chloride (point F), is mixed with a recycle stream (point 4) to give point 10 (not shown in flowsheet). By adding NaOH to reach point 1, supersaturation is generated because of decreasing solubility in this direction. As a result, the mixture splits into a solid phase (lysozyme with low NaCl content) and a liquid phase with a composition represented at point 2. HCl is then added to this liquid phase to give point 3. Knowing that the saturation region of NaCl is very small and crystallization for the removal of NaCl is impossible, ultrafiltration is used to remove NaCl from the system. The retentate, the composition of which is given by point 4, is recycled back to the feed stream. To prevent accumulation of impurities, a portion of stream 2 is
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Figure 6. Process for the recovery of lysozyme: (a) simplified solventless projection with process paths; (b) process flow sheet.
Table 2. Material Balance Summary for the Lysozyme Purification Process (Example 1) ionic mass fraction (%) þ
stream
Na
Cl
Lys13
OHþ Hþ
total flow (kg/h)
F
1.41
2.18
0.52
95.89
1000.0
10
1.51
2.33
0.29
95.88
2711.2
1
1.58
2.32
0.29
95.81
2714.7
2 20
1.57 1.57
2.31 2.31
0.16 0.16
95.96 95.96
2710.5 2439.4
purge
1.57
2.31
0.16
95.96
271.0
3
1.56
2.41
0.16
95.86
2444.8
4
1.56
2.41
0.23
95.79
1712.5
5
1.57
2.42
0.00
96.01
732.2
P
10.00
10.00
80.00
0.00
4.2
purged. Table 2 summarizes the material balances for this process, using 1000 kg/h of crude lysozyme feed as a basis. The product yield is defined as the percentage ratio of target component in the product stream to that in the feed stream. On the basis of the mass balance in Table 2, the lysozyme yield was calculated to be 64.6%. The first step for recovering lysozyme with the addition of NaOH is consistent with a patented process for recovering lysozyme20 in which NaOH is added to generate supersaturation. 8169
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Figure 7. Sketch of solventless projection of isobaricisothermal SLE phase diagram for ovalbuminlysozymeammonium hydroxidehydrogen sulfateammonium sulfate system: (a) complete picture; (b) cut along a fixed lysozyme to ovalbumin ratio; and (c) cut along a fixed ammonium to sulfate ion ratio.
To increase the yield of lysozyme, the patented process involves lowering the temperature after adding NaOH to further decrease the solubility. While it is possible to generate such a process alternative involving temperature swing, more experimental data have to be taken to generate the phase diagram at a lower temperature. Therefore, this alternative is not considered here. The recovery of lysozyme and the removal of NaCl from lysozymesodium chloride solution using Pellicon XL 50 Ultrafiltration Cassettes were demonstrated in our experiments. Example 2. OvalbuminLysozyme Separation. Ovalbumin naturally coexists with lysozyme in hen egg white, and has been successfully purified using bulk crystallization.4 The presence of a salt such as ammonium sulfate can affect the interaction between ovalbumin and lysozyme, making it possible to separate them by changing pH and salt concentration.21 An extended study of the phase behavior of ovalbuminlysozyme system with different salts, especially ammonium sulfate, is also available.22 For this reason, the ovalbuminlysozyme ammonium sulfate system is selected to illustrate the application of the phase diagram approach in synthesizing a protein separation process. Again, detailed description of this system can be found in ref 18.
According to the literature, the net charge of the ovalbumin is 36 in high alkaline condition.23 Therefore, ovalbumin and its corresponding ion are denoted by H36Ova and Ova36. Considering the complete dissociation of water and the proteins, there are two cations, Hþ and NH4þ, two anions, SO42 and OH apart from protein ions, and two protein ions, Ova36 and Lys13. The isobaricisothermal aqueous system has 4 degrees of freedom and the solventless projection is threedimensional. With a choice of SO42, NH4þ, and Lys13 as the reference components, the coordinates in the solventless projection are xSO4 2 ð16Þ TðSO4 2 Þ ¼ xNH4 þ þ xSO4 2 þ xOva36 þ xLys13 TðNH4 þ Þ ¼
xNH4 þ xNH4 þ þ xSO2 þ xOva36 þ xLys13 4
ð17Þ
TðLys13 Þ ¼
xLys13 xNHþ4 þ xSO2 þ xOva36 þ xLys13 4
ð18Þ
Figure 7a shows the solventless projection of the ovalbumin lysozyme binary protein system. Pure lysozyme and ovalbumin can 8170
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Table 3. Summary of Experimental Results for Lysozyme Ovalbumin Precipitation Experiments liquid phase (mg/g sample) 13
LYS
36
OVA
NH4þ
SO42
solid phase (mg/g sample) 13
LYS
OVA36 NH4þ SO42
0.9
18.7
61.4
114.5
224.0
12.6
39.9
108.9
1.2
14.6
58.9
142.2
164.6
26.8
41.6
121.6
1.2 0.5
15.9 16.8
68.0 71.9
142.3 142.6
53.4 8.9
67.8 113.2
47.6 51.5
115.8 110.3
1.6
13.2
58.9
118.0
258.7
67.0
41.3
92.3
3.6
13.2
61.5
117.7
211.9
42.8
35.9
94.9
5.4
19.8
55.7
114.2
36.6
69.2
44.2
98.2
1.4
16.1
64.3
144.6
88.9
44.9
53.3
120.6
6.6
18.2
57.8
116.2
87.2
62.2
48.5
109.0
9.0
26.6
37.9
93.4
235.4
10.4
24.4
70.3
21.1 1.6
24.9 32.9
33.4 45.0
91.3 108.7
68.1 16.6
53.6 84.0
52.6 47.7
118.3 126.8
1.6
17.3
27.6
97.7
64.6
33.8
39.7
113.3
7.0
28.5
51.5
69.9
22.9
68.4
18.8
144.4
2.3
36.5
50.8
87.6
5.9
59.7
42.2
119.4
11.4
26.7
51.4
132.4
37.2
80.5
57.5
149.1
12.9
28.1
40.2
63.4
17.3
74.7
60.3
145.9
21.6
32.5
42.7
79.2
19.7
60.9
43.0
138.6
5.8 9.1
36.4 35.4
43.4 66.7
128.0 116.2
223.4 195.8
66.1 81.6
69.5 78.5
119.7 136.5
15.5
34.8
67.7
97.9
214.7
52.3
89.1
124.3
11.4
33.1
74.6
108.9
103.1
57.2
54.6
101.1
16.6
32.3
52.4
76.7
103.7
43.5
58.1
75.8
14.9
47.0
28.1
32.8
28.5
72.4
30.7
32.1
2.1
33.1
90.8
143.4
414.2
28.8
71.7
124.2
2.4
29.1
78.3
141.9
320.6
38.3
80.2
122.0
1.9 1.0
37.4 42.4
82.2 78.0
132.9 142.4
121.4 47.6
65.0 88.2
23.3 68.9
112.6 104.2
6.0
27.6
80.6
118.5
476.3
49.7
52.5
101.9
11.8
33.0
77.5
117.2
287.7
50.0
72.2
102.1
7.0
40.8
77.7
116.8
96.4
62.5
77.3
105.9
3.5
53.3
57.3
115.9
23.3
80.4
75.8
99.7
13.3
25.0
62.3
80.4
389.8
41.6
45.7
70.2
20.5
27.5
56.0
79.0
133.5
33.6
40.1
75.3
14.0 8.7
53.5 64.0
60.8 59.8
77.1 77.4
56.2 33.7
55.7 82.5
60.6 71.0
73.5 81.2
21.7
33.0
45.9
57.5
426.2
64.6
50.2
55.8
20.2
27.0
39.8
53.7
151.9
48.6
43.8
50.5
12.3
39.7
43.9
54.8
50.7
65.1
32.9
52.9
7.5
53.2
43.0
53.5
46.2
97.1
57.6
64.4
22.1
26.6
28.8
32.5
220.0
43.8
31.5
31.1
17.8
23.6
25.4
32.4
87.2
47.6
40.4
34.7
11.3 2.8
48.3 58.3
32.5 21.2
30.4 30.7
56.3 54.9
69.0 93.7
34.2 36.8
39.0 41.4
be crystallized at the regions close to the pure protein vertices. The saturation regions of ammonium sulfate and ammonium bisulfate may be present in the phase diagram. However, since ammonium bisulfate is not expected to crystallize out under the conditions of interest, its saturation region is not shown in the conceptual phase diagram. The large region in the middle is the composite saturation regions of protein salts containing counterions.
For easier visualization of the phase behavior, two types of triangular cut that are especially useful for process design are made. Figure 7b shows a cut at a constant ovalbumin to lysozyme ratio, which allows for better observation of the effect of ammonium and sulfate ions on the protein solubility. The gradient color in the composite saturation region of protein salts represents the solid composition that is in equilibrium with the corresponding liquid composition. Figure 7c shows another cut taken at a constant sulfuric acid to ammonium hydroxide ratio. Saturation regions for pure lysozyme, pure ovalbumin, and ammonium sulfate can be identified in this cut. As in the other cut, solidliquid equilibrium data in the protein salt composite saturation region is shown using a color gradient map. As the source of lysozyme was in the form of lysozyme chlorhydrate, before performing separation experiments, the chloride ion was removed as follows. Stock solutions of ovalbumin and lysozyme were first prepared. In the preparation of the ovalbumin stock solution, an excess amount of ovalbumin powder was added to DDI water to form a slurry. The slurry was adjusted to the desired pH value (4.5, 7, and 9.5) with 1 N ammonium hydroxide solution and 1 N sulfuric acid, and stirred with a magnetic stirrer for 24 h to attain equilibrium. After that, the slurry was centrifuged at 20 °C at 10000g. The concentrated ovalbumin stock solution was obtained by collecting the supernatant. The lysozyme powder was mixed in DDI water to prepare a lysozyme solution of mass fraction of 0.1. Saturated ammonium sulfate solution was then added to the lysozyme solution until reaching 5% mass fraction of ammonium sulfate. (The ammonium sulfate solution was in turn prepared by mixing 40 g of ammonium sulfate solution and 60 g of DDI water.) Then, the pH of the solution was adjusted to 10 using a 1 N ammonium hydroxide solution. The solution was stirred with a magnetic stirrer at 4 °C for 24 h. After that, the lysozyme solids were filtrated out and redissolved in DDI water, which was then adjusted to the desired pH values (4.5, 7 and 9.5) with diluted ammonium hydroxide solution and sulfuric acid. All solutions were filtered using low protein binding Millex-GP 0.22-μm syringe filters. To determine the phase diagram, ovalbumin, lysozyme, ammonium sulfate stock solutions and DDI water were mixed together to generate a supersaturated solution. The desired amount of ovalbumin and lysozyme solution were mixed with DDI water using a magnetic stirrer. A known amount of ammonium sulfate solution was slowly added into the mixture to avoid high local concentration. The mixture became cloudy and precipitants were formed. The mixture was kept at 20 °C and stirred gently in order to attain solidliquid equilibrium. After 24 h, the slurry was centrifuged at 20 °C and 15000g. A liquid phase sample was obtained by collecting the supernatant, and the solid phase sample was dissolved in a known amount of DDI water. The amount of ammonium ion, sulfate ion, ovalbumin, and lysozyme in those samples were measured. The results are summarized in Table 3. Interpolation of the data was performed using MATLAB griddata3. Figure 8 shows cuts of the solventless projection with fixed lysozyme to ovalbumin (mass) ratio (such as Figure 7b) at 20:80 and 15:85. All data points obtained from the experiments lie on the composite saturation region of mixed ovalbumin lysozyme salts (NH4)iþk(H13-iLys)(H36-kOva)(H2SO4)j. Solubility data are represented by the mass fraction of the sum of hydrogen ion and hydroxide ion, as indicated by the number on 8171
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Figure 9. Cut along a fixed ratio of ammonium and sulfate ions, xNH4þ/ xSO42 = 6:4.
Figure 8. Cut along fixed ratios of lysozyme and ovalbumin: (a) xLys13/ xOva36 = 20:80; (b) xLys13/xOva36 = 15:85. The contour line represents the solubility data, and the color index represents the relative amount of lysozyme and ovalbumin in the solid.
the contour lines. Solid equilibrium data are represented using a gradient color index in terms of the lysozyme to total protein mass ratio in the solid phase, Θ¼
xLys13 xLys13 þ xOva36
ð19Þ
which lies between 0 (pure ovalbumin represented by yellow) and 1 (pure lysozyme represented by red). Two observations can be made regarding these two cuts. At a given value of (T(Lys13) þ T(Ova36)), there is more lysozyme-rich solids, indicated by the reddish color, at a relatively high concentration of ammonium ion compared to sulfate ion, that is, on the left of the colored region. As the excess ammonium ion must come from ammonium hydroxide, pH is high in this region as well. As the relative amount of sulfate ion increases, pH
decreases and the solid composition gradually shifts from lysozyme-rich to ovalbumin-rich (represented by the yellowish color). This is consistent with the fact that the isoelectric point of lysozyme is 10.7 and that of the ovalbumin is 4.57; thus, a lysozyme-rich solid is obtained at a high pH and ovalbumin-rich solid crystallizes at a low pH. It can also be observed that there is more ovalbumin-rich solid in the colored region, which is consistent with the results obtained by Blanch et al.24 The effect of lysozyme to ovalbumin ratio on the solid composition can be observed in the cut along a fixed ammonium to sulfate ratio (Figure 9). Again, all data points lie within the composite region of protein salts. Not surprisingly, lysozyme-rich solids are obtained at a high lysozyme to ovalbumin ratio while the ovalbumin-rich solids are obtained at a low ratio. Consider the synthesis of a process for separating a mixture of lysozyme and ovalbumin into lysozyme-rich solids and ovalbumin-rich solids. The process paths are shown in Figure 10, and the flowsheet is depicted in Figure 11. Figure 10a shows the process paths of the entire process. The cuts at constant lysozyme to ovalbumin ratio, 20:80 and 15:85, with process paths are shown in Figure 10bd. For easy visualization, the regions in which lysozyme-rich (Θ > 0.5) and ovalbumin-rich (Θ < 0.5) solids are precipitated are colored pink and yellow, respectively, in Figure 10bd. The feed F, which contains a mixture of lysozyme and ovalbumin, is first mixed with a recycle stream (point 5) to give point 10 . Then, an ammonium sulfate and ammonium hydroxide solution is added to move the composition to point 1, which is located in the region where a lysozyme-rich solid can be precipitated (Figure 10b). A lysozyme-rich solid (point P1) and supernatant (point 2), connected by the tie-line passing through point 1, are obtained in the first crystallizer. Since these two phases have different lysozyme to ovalbumin ratios compared to point 1, they are located on different cuts. Specifically, point 2 is on the 15:85 cut. Sulfuric acid is added to the supernatant to give point 3, which is located in the region where ovalbumin-rich solid can be precipitated (Figure 10c). Hence, ovalbumin-rich solid (point P2) and a supernatant (point 4) are obtained in a second crystallizer. Note that point 4 is on the 20:80 cut. Ammonium sulfate and water in the supernatant are partially 8172
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Figure 10. Process path on the solventless projection in the ovalbuminlysozyme system; (a) complete process flow sheet; (b) process point 1, 10 , 2, and P1 on fixed LysOva cut at xLys13/xOva36 = 20:80; (c) process point 2, 3, 4, and P2 on fix LysOva cut at xLys13/xOva36 = 15:85; (d) process point 4, 5, and 10 on fix LysOva cut at xLys13/xOva36 = 20:80 [b, process points lying on the cut of xLys13/xOva36 = 20:80; O, process points lying on xLys13/ xOva36 = 15:85; 0 product streams].
Figure 11. Process flow sheet for the purification of lysozymeovalbumin from egg-white.
removed using ultrafiltration, giving the composition indicated by point 5 (Figure 10d). Since the protein ratio does not change during ultrafiltration, points 4 and 5 are located on the same cut. After partial purging to prevent accumulation of impurities, stream 50 is recycled. Table 4 summarizes the material balances
for this process, using 100 kg/h of lysozymeovalbumin mixture feed as a basis. The calculated yield of the lysozyme product is 65.2% and that of the ovalbumin product is 79.6%. From the feed stream containing 20.0% lysozyme (based on the total protein mass), two protein products containing 63.9% lysozyme and 93% ovalbumin (both based on total protein mass), respectively, were obtained. The yield can be further improved by increasing the recycle. However, a larger recycle would lead to a higher demand on the ultrafiltration unit, and a higher level of impurity in the products. To apply the conceptual design on a real protein system, an experiment separating lysozyme and ovalbumin from hen eggwhite was performed. Hen egg-white consists of 88 wt % of water, 9 wt % of proteins and some minerals, and among those proteins, 54 wt % of ovalbumin and 3.4 wt % of lysozyme.25 A 1000 g sample of egg white was obtained from fresh egg. After centrifugation at 20 °C and 15000g, the insoluble proteins, a gel-like solid, in egg white were removed. The supernatant, with a composition indicated as point F in Table 5, served as the feed to the process. A solution with 40 wt % ammonium sulfate with 8173
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Table 4. Material Balance Summary for Lysozyme Ovalbumin Separation Process (Example 2) ionic mass fraction (%) stream NH4
þ
SO4
LYS13 OVA36 OH-þ Hþ total flow (kg/h)
2-
F
0.00
0.00
2.17
8.69
89.14
100.0
10
3.77
8.77
0.95
3.82
82.69
453.5
1 2
6.17 5.88
11.86 11.00
0.72 0.49
2.88 2.80
78.37 79.83
601.4 590.4
3
5.23
12.16
0.44
2.49
79.69
664.3
4
4.89
11.40
0.37
1.48
81.86
646.6
5
4.83
11.26
0.61
2.44
80.86
392.8
50
4.83
11.26
0.61
2.44
80.86
353.5
Purge
4.83
11.26
0.61
2.44
80.86
39.3
6
4.98
11.61
0.00
0.00
83.41
253.9
P1 P2
21.78 17.66
58.09 40.11
12.86 2.94
7.26 39.29
0.00 0.00
11.0 17.6
Table 5. Summary of Experimental Results for Lysozyme and Ovalbumin Separation from Egg White ionic mass concentration (mg/g sample) 13
LYS
OVA36
NH4þ
SO42-
point F
3.4
86.0
0.8
0.9
point 2
2.6
38.2
59.0
109.3
point 4
1.9
16.3
63.8
157.7
Lys-rich solid
33.3
19.4
59.0
154.6
Ova-rich solid
8.3
107.4
49.2
113.2
5 wt % ammonium hydroxide solution was slowly added into an equal mass of centrifuged egg-white to form a supersaturated solution, the composition of which lies in the same region as point 1, and the pH reached 8.2. The system was stirred gently with a magnetic stirrer at 20 °C for 24 h. Then, the sample was centrifuged to separate the residue and the supernatant. The residue, a lysozyme-rich solid, could be obtained as product and the supernatant was labeled as point 2. Then, the supernatant was mixed with a 20 wt % of ammonium sulfate and 1 wt % sulfuric acid solution to produce a composition similar to point 3. The pH reached 4.40. The system was stirred gently with a magnetic stirrer at 20 °C for 24 h. Finally, solids were separated out by centrifugation and ovalbumin-rich solids were obtained. The supernatant (point 4) was collected. The amounts of ammonium ion, sulfate ion ovalbumin, and lysozyme in all samples were measured. The experimental results are summarized in Table 5. On the basis of these results, it was confirmed that lysozyme and ovalbumin could be purified from their mixture via the crystallization process as conceptualized using the experimental phase diagram.
’ CONCLUSIONS Salt precipitation and isoelectric point precipitation are common methods to crystallize proteins. These methods rely on the reduction of protein solubility by changing the salt concentration and pH. The traditional solubility plot representing protein solubility data as a function of pH and salt concentration does not provide information on the effect of cation and anion on the
protein solubility. To avoid this problem, ionic mass coordinates are proposed for representing protein systems. With proper reduction in dimensions, process paths and thus effective separation processes can be conveniently represented. The design of crystallization process requires the compositions of the solids and liquid in equilibrium. For solid solutions, the traditional representation method does not provide both of solid and liquid compositions in two-dimensional projections of a multicomponent system. A color mapping method was developed to solve this problem. Composition data of the solid phase are converted according to a gradient color map or color index and overlay at the corresponding liquid phase composition point on the phase diagram. The distribution of the solid phase composition can then be seen in its totality. With the aid of ionic coordinates, color mapping technique, one can come up with designs for separating or purifying protein(s) as demonstrated by two case studies. Thermodynamic modeling of protein phase behavior, which is beyond the scope of this article, can be readily incorporated into the framework discussed here. Indeed, various protein solubility models have been discussed in the literature. For example, Agena et al.26 proposed a model which represents the transfer of protein between the two phases by the solubility product and describes the deviation from ideal solution behavior by the UNIQUAC activity coefficient model. Interaction parameters in such a model can be fitted from experimental data, and the model can be used to calculate the relevant phase diagrams that are useful for process synthesis.
’ AUTHOR INFORMATION Corresponding Author
*Tel.: þ852 2358 7238. Fax: þ852 2358 0054. E-mail: kekmng@ ust.hk.
’ ACKNOWLEDGMENT Research support of the Research Grants Council (Grant HKUST 605208) is gratefully acknowledged. The authors would also like to thank Grace Poon for her assistance in the experimental work. ’ REFERENCES (1) Van Arunum, P. Moving to the next level in biomanufacturing. Pharm. Tech. 2010, 34 (4), 64. (2) Boswell, C. Bioseparation to clean up in protein therapeutics. Chem. Market Rep. 2002, 262 (5), FR12. (3) Thommes, J.; Etzel, M. Alternatives to chromatographic separations. Biotechnol. Prog. 2007, 23, 42. (4) Foster, P. R.; Dunnill, P.; Lilly, M. D. Salting-out of enzymes with ammonium sulphate. Biotechnol. Bioeng. 1971, 13 (5), 713. (5) Dixon, M.; Webb, E. C. Enzyme fractionation by salting-out: A theoretical note. Adv. Protein Chem. 1962, 16, 197. (6) Judge, R. A.; Johns, M. R.; White, E. T. Protein purification by bulk crystallization: The recovery of ovalbumin. Biotechnol. Bioeng. 1995, 48 (4), 316. (7) Asherie, N. Protein crystallization and phase diagrams. Methods 2004, 34 (3), 266. (8) Shih, Y. C.; Prausnitz, J. M.; Blanch, H. W. Some characteristics of protein precipitation by salts. Biotechnol. Bioeng. 1992, 40 (10), 1155. (9) Scopes, R. K. Separation by Precipitation. In Protein Purification: Principles and Practice, 3rd ed.; Springer-Verlag: New York, 1994; pp 71101. 8174
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