33
Biotechnol. frog. 1990, 6,33-40
Affinity Cross-Flow Filtration: Some New Aspects David C. Herak’ and Edward W. Merrill Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
A variety of affinity cross-flow filtration (ACFF) experiments were conducted to evaluate the technique as a means of effectively separating biomolecules. Agarose particles, which contained a specific affinity ligand for the targeted protein, were used as “affinity escorts” in the ACFF process. Both conventional agarose particles (40-150 pm) and small agarose particles (Superose, 11-15 pm) were used as the basis for affinity escorts. Batch ACFF washing experiments were conducted in both constant-volume and volume-reduction modes and compared with model predictions. The different affinity ligand-adsorbate systems studied include Cibacron Blue and human serum albumin, Cibacron Blue and lysozyme, protein A and immunoglobulin G, and concanavalin A and horseradish peroxidase.
Introduction As a result of recent advances in biotechnology, it is possible to produce many therapeutic proteins by using genetically modified organisms. The purification of these desired proteins, which are typically produced in low concentrations and in broths containing numerous contaminants, represents a dominant part (40-9070)of the overall process cost (1). Innovative purification techniques that possess high resolution, high yield, and ease of scaleup are needed to reduce the expense of downstream processing. There has been recent interest in combining biospecific adsorption with membrane separations for protein purification (2-5). Affinity chromatography is a widely used laboratory technique that utilizes biospecific interactions for highly selective protein purification but is not especially well suited for large-scale operations. By contrast, membrane separations, such as cross-flow ultrafiltration and microfiltration, are amenable to large-scale protein separations but are limited by their inability to separate proteins that are relatively similar in size (within a molecular weight factor of 10) (6). Both the high specificity of biospecific adsorption and the scalability of cross-flow filtration can be utilized in affinity cross-flow filtration (ACFF). When an affinity ligand, which is specific for the targeted protein (adsorbate), is attached to a high molecular weight polymer or microparticle, an “affinity escort” is created. Figure 1 shows how the affinity escorts are used in the ACFF process for protein purification. The affinity escorts can selectively bind the desired protein, and the complex can be retained by a membrane while the contaminants are free to pass through the membrane pores. After the contaminants are eliminated, the buffer can be changed to release the bound protein and a second filtration step can isolate the product from the escorts. ACFF has been demonstrated both in batch and continuous modes with a few different affinity pairs and escort types ( 2 , 3 , 5 ) . Our previous work showed that Cibacron Blue-agarose particles can be used successfully to recover human serum albumin (HSA) in ACFF experiments and that process performance can be predicted with a simple model (5). This paper presents an extension of our
* Address correspondence to David C. Herak, Hercules Incorporated, Research Center Wilmington, DE 19894.
earlier work. New affinity systems are investigated, and two different sizes of agarose particles are utilized as affinity escorts. Both conventional agarose particles (40-150 pm) and small agarose particles (Superose, 11-15 pm) were used as the basis for affinity escorts. In addition to the constant-volume ACFF washing procedure used in our earlier work, a volume-reduction ACFF washing step is also explored.
Theoretical Considerations Protein Adsorption to Porous Affinity Particles. To understand and model the ACFF process, it is first necessary to understand the interaction of proteins with the porous agarose affinity particles. Assuming that all binding sites are identical and the reaction between the adsorbate and the affinity ligand is reversible, a secondorder rate expression can describe protein adsorption kinetics: dq/dt = k,C(qm - 4) - kzq (1) where C is the concentration of the free adsorbate, q is the concentration of bound adsorbate, qm is the maximum concentration of bound adsorbate, and k , and k , are the forward and reverse rate constants, respectively. At equilibrium, eq 1 leads to the Langmuir isotherm
qmC* q* = -
K,j + C* where the superscript asterisk denotes an equilibrium value and Kd is the dissociation constant ( k 2 / k 1 ) . Numerous investigators have shown that the equilibrium relationship between free and bound adsorbate can be described well by a Langmuir isotherm. To model the protein adsorption rate to porous affinity particles, it is necessary to consider the three sequential steps necessary for adsorption. First, the adsorbate must transport from the bulk solution across a film (boundary layer) to the external surface of the particle. Next, the adsorbate molecules must travel through the pores of the particle to reach a binding site. Finally, there must be a binding reaction between the adsorbate and the immobilized ligand. Arve and Liapis have developed a comprehensive biospecific adsorption model that can account for all three adsorption mechanisms (7, 8). Such a complex model is usually not necessary, and several simpler protein adsorption models can be derived
8756-7938/90/3006-0033$02.50/0 0 1990 American Chemical Society and American Institute of Chemical Engineers
Biotechnol. Prog., 1990, Vol. 6, No. 1
34
RECYCLE
BUFFER RECCNDlTlCNlffi
RETENT ATE DESORPTION
I
1
I
0 .
I
CRUDE EXTRACT
1
I
1
I
TANK
FILTRATE
Figure 2. Schematicdiagram of the ACFF washing step. The affinity escorts (stars) bind the desired biomolecules (circles) so that they are retained by the membrane, while contaminants (squares)are free to pass into the filtrate stream. IMPVRlTlES
Figure 1. Overall schematic diagram of the ACFF process. The affinity escorts (stars) bind the desired biomolecule (circles)for separation from the contaminants (squares). with the appropriate approximations. I t has been found that the protein adsorption to particles greater than 50 pm is generally limited by pore diffusion, so that a shrinking core model often gives a good description of protein adsorption rates (9-11). The shrinking-coremodel assumes that the adsorption of the protein to the ligand is instantaneous and irreversible, which predicts a protein-saturated outer shell that encroaches upon an unreacted core. Arnold makes the additional assumption that film diffusion is negligible compared with pore diffusion, which allows for an analytical ~ o l u t i o n .On ~ the basis of Arnold's equations, a computer program was written to find the best value of the pore diffusion coefficient, D,, to model our experimental adsorption data. Chase has used a lumped kinetic model that combines diffusional resistances with intrinsic rate constants (12). With this simplification, the mass transfer of the adsorbate to the immobilized phase can be described by a pseudokinetic rate equation that is the same as eq 1,except for the substitution of the lumped rate constants, k,, and k2,, for the intrinsic rate constants. Although Chase admits that his model is a gross oversimplification, he also points out that the model is useful and avoids the need for unknown diffusional parameters (13). On the basis of the lumped kinetic model, a computer program was written to optimize the value of klL for each set of adsorption data. Modeling of the ACFF Washing Step. The adsorption step is followed by the washing step, where the actual separation of the adsorbate from contaminants occurs. Figure 2 shows a schematic of a batch washing step. For a constant-volume wash, makeup buffer is added to compensate for the fluid lost in the filtrate stream. Assuming that the washing step begins after the adsorption step has come to equilibrium, there will be a tendency for the bound proteins to release from the affinity escorts during the washing step because the free adsorbate concentration decreases as the washing proceeds. We have developed a general model for the ACFF constant-volume washing step, which is summarized below (5). A mass balance on the volume of fluid contained in the stirred tank plus the retentate side of the filtration unit gives
2
dC V - = -Q&2 - V0+ (3) dt where V,, is the system volume, Qfil is the filtrate flow
rate, and 4 is the volume fraction of affinity escorts. The membrane rejection factor, f, depends on the ability of a species to pass through the membrane. (The value o f f can range from 0 to 1 depending on the ability of a protein to pass through the membrane. For all of our work, the value off = 1 because the proteins could easily pass through the large pores of the microfiltration membranes.) The two terms on the right-hand side of eq 3 account for the adsorbate loss in the filtrate stream and adsorbate release from the affinity escorts. The dq/dt in the adsorbate release term, which depends on the mass-transfer resistances and kinetic interactions, was modeled with use of the lumped kinetic model with the klL values determined from the adsorption experiments. Thus, the washing step can be defined by two coupled differential equations (eq 1with lumped rate constants and eq 3), but they cannot be solved analytically. Therefore, a computer program was written, which employed a fourth-order Runge-Kutta numerical solution, to solve the equations and predict the concentration versus time profile for a constant-volume wash. Analytical solutions have been derived for the two limiting cases. (1)Equilibrium limit: Reaction and diffusion are rapid enough to continually maintain equilibrium. (2) No-release limit: Reaction and diffusion are so slow that there is no release of the bound adsorbate from the affinity escorts (5). For a volume-reduction washing procedure that operates with no addition of makeup buffer, a similar set of equations can be derived. For the limiting case where equilibrium is maintained, it is possible to derive
where v is the volume of affinity escorts. Although eq 4 is not particularly simple, for the case of no membrane rejection of adsorbate (f = l),which was true for all experiments in our work, one can see that the free adsorbate concentration becomes constant. This result means that there is no driving force for the bound adsorbate to release from the affinity escorts.
Experimental Section Materials. All proteins used were purchased from Sigma. The agarose particles purchased, both with and without affinity ligands, are summarized in Table I. The Sepharose CL-6B and Superose 6 particles were modified with Cibacron Blue by using the method of Bohme and co-workers14and are identified later as Blue Sepharose and Blue Superose, respectively. All other chemicals used
35
Biotechnol. hog., 1990, Vol. 6, No. 1
Table I. Agarose Gels Purchased for Study in This Paper, Along with the Manufacturers' Specifications
FILTRATION
1
RETENTATE RECYCLE
WIT
L
I
size, name manufacturer pm Sepharose CL-6B Pharmacia 45-165 Pharmacia 11-15 Superose 6 Pharmacia 45-165 Con A-Sepharose 4B
protein A-agarose O
Pierce
agarose affinity typea ligand 6% CL none 6% CL none 4% concanavalin A 45-165 6% CL protein A
4
FILTRATE
Loop
CL = cross-linked.
were of analytical grade. Sodium azide (0.02%) was added to all aqueous buffers as a bacteriostat. Protein Adsorption to Agarose Affinity Particles. The affinity particles, adsorbing proteins, and buffers used for protein adsorption and ACFF experiments are shown in Table 11. Equilibrium experiments were conducted in 15-mL centrifuge tubes by mixing 5-10 mL of protein solutions with samples of the affinity gel (0.2-0.7 mL). The protein concentration in the supernatant was measured periodically with ultraviolet (UV) absorbance a t 280 nm &So) until equilibrium was established. After the adsorption equilibrium was reached, the supernatant was removed and fresh buffer was added to the centrifuge tube to see the effect of dilution on the equilibrium. Protein adsorption rate experiments with agarose affinity particles were conducted in a magnetically stirred 125mL Erlenmeyer flask that was maintained a t a constant temperature (25 "C) with a water bath. A UV detection loop with a chart recorder was used for continuous monitoring of protein concentration. A 2-pm sintered metal filter was used to prevent the agarose particles from entering the detection loop. For experiments involving horseradish peroxidase (HRP), the concentration was based on A403readings of samples that were periodically collected. Closely related desorption experiments were performed with the same apparatus. After adsorption was complete, the protein solution was diluted or an eluting compound (e.g., NaCl) was added and the protein deorption from the affinity particle was monitored. Affinity Cross-Flow Filtration Experiments. The apparatus used for all ACFF experiments is shown in Figure 3. The protein and affinity escort mixture was contained in a stirred 500-mL Erlenmeyer flask. A Masterflex peristaltic pump (up to 1L/min) was used to pump the mixture to a Millipore Minitan cross-flow filtration unit, which was equipped with either one or two membrane plates (Durapore 0.2-pm mean pore size membranes with 60 cm2 of surface area). When the mixture passes through the Minitan, most of it is retained by the membrane and is recycled back to the stirred flask, but a portion of the fluid passes into the filtrate stream. The liquid volume in the system was maintained constant in most experiments by addition of makeup buffer to the stirred flask. Rotameters, which were equipped with control valves, were used to control and monitor the retentate and filtrate flow rates. Simultaneous to the actual ACFF experiment, continuous monitoring of the protein concentration was possible with the UV detection loop. Many of the ACFF experiments were conducted with just the adsorbing protein present and without any contaminants, so that A280 could be used to monitor adsorbate concentration.
Results and Discussion Protein Adsorption Equilibria. Protein adsorption equilibrium data for each affinity system were fit to the
MAKE-LP
WFER I _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ - - - - - - - - - - - - - - - - - - - - - ~
Figure 3. Schematic diagram of the apparatus used for filtration and ACFF experiments. 3 0 ~ a
20
10
0 0.00
1.oo
0.50
1.50
FREE PROTEIN CONC. (mg/ml)
b .c
0
E
\
E"
Y
zw
20
c
Ba 0
[
LYSOZYr\/E m
HSA
10
z
3
0 m
FREE PROTEIN CONC. (mglml)
Figure 4. (a) Isotherms for HSA and lysozyme with Blue Sepharose no. 2. (b) Isotherms for HSA and lysozyme with Blue Superose no. 2. The curves represent the fit of the Langmuir isotherm.
Langmuir isotherm with a nonlinear regression. The results for the equilibrium parameters, K , and ,Q are presented in Table I11 along with an estimation of the percentage of ligands that are bound to an adsorbate molecule when the particles are saturated. (It is important to note that a few batches of Blue Sepharose and Blue Superose with different Cibacron Blue contents were explored in the protein adsorption studies. The dye contents of each gel are listed in Table 111.) Experimental data generally conformed well to a Langmuir isotherm, although there was a slight trend in most sets of equilibrium data that shows evidence that the binding sites have a range of affinities due steric hindrance and interactions between bound proteins. Figure 4 shows some typical adsorption isotherms, which show that the Blue Sepharose no. 2 adsorbed significant
Biotechnol. Prog., 1990, Vol. 6,No. 1
36 Table 11. Affinity Systems affinity escort Blue Sepharoseb Blue Superoseb protein A-agarose Con A-Sepharose4B
Studied for Protein Adsorption and ACFF Experiments. adsorbate(s) adsorption buffer HSA, lysozyme 10 mM Tris, pH 7.5 HSA, lysozyme 10 mM Tris, pH 7.5 10 mM Tris, pH 7.5 IgG 20 mM Tris, 0.1 M NaCl, 1 mM Caz+, HRP I 1 mM Mn2+, 1 mM Mg2+, pH 7.2
eluting agent 2.0 M NaCl 2.0 M NaCl 0.10 M glycine and HC1, pH 2.5 0.2 M methyl a-D-glucopyranoside
a The adsorption buffer was modified with the eluting agent listed to make the elution buffer. Blue Sepharoses and Blue Superoses with various amounts of Cibacron Blue were studied. The amount of dye on each gel is included in Table 111.
Table 111. Affinity Systems Studied for Protein Adsorption Rates and Equilibria particle type (ligand content, mg/mL) adsorbate Kd,mg/mL Blue Sepharose no. 1 (0.83) HSA 0.102 f 0.012 lysozyme 0.00402 f 0.00088 Blue Sepharose no. 2 (3.67) HSA 0.166 f 0.020 Blue Superose no. 2 (5.76) Blue Superose no. 3 (3.11) protein A-agarose (1.5) Con A-Sepharose 4B (14)
lysozyme HSA HSA lysozyme HSA IgG HRP I” HRP I
0.110 f 0.013 0.00166 f 0.00024 0.124 f 0.009 0.294 f 0.054 0.00797 f 0.00075 0.189 f 0.021 0.0756 f 0.0058 0.140 f 0.022 0.150 f 0.042
q m mg/mL
% ligand utilized
12.5 f 0.3 12.1 f 0.3 21.9 f 0.7 20.9 f 0.6 23.2 f 0.4 6.62 f 0.13 5.53 f 0.37 35.0 f 0.6 7.03 f 0.24 7.35 f 0.14 13.2 f 0.5 12.8 f 1.1
19 86 7.5 7.2 38 1.5 1.2 37 2.9 137
” HRP grade I contains only about 30% actual HRP. The numbers are based on the amount of HRP I used in the experiments, which is not indicative of the amount of true HRP. Therefore, the percent ligand utilization is not applicable. amounts of both HSA (MW = 66 400) and lysozyme (MW = 14 000), but the Blue Superose no. 2 adsorbed a significant amount of only lysozyme. This result is attributed to the exclusion of the larger HSA molecules by the small pores of the dense, highly cross-linked Superose particle. Equilibrium results from two different batches of Blue Sepharose with dye contents that differed by a factor of 4 showed, as expected, that more protein adsorbed to the agarose with more Cibacron Blue. However, the fractional utilization of the dye ligands by both HSA and lysozyme was much higher for the Blue Sepharose with the lower dye content, which indicates that multiple-site attachment and steric blocking by adsorbed proteins is more prevalent with higher ligand content. The lysozyme molecules utilized about 5 times more of the binding sites than the HSA for both Blue Sepharoses, which can be attributed to the relative sizes of the protein molecules. From the protein A-agarose/IgG isotherm, a result of more than one bound IgG molecule for each protein A on the agarose gel was found. Since each protein A molecule has four to five potential IgG binding sites, this finding is reasonable (15, 16). The results of the Con A-Sepharose and HRP grade I adsorption isotherm are a little difficult to interpret, since HRP I contains only about 30% of actual HRP. (The RZ value (A,03/A,,,) is a measure of HRP purity. For HRP I, RZ = 1.1 compared to RZ i= 3.2 for pure HRP. The HRP grade I was used in this work because of the very high cost of pure HRP.) HRP binds to concanavalin A (Con A) because it is glycosylated and the Con A has an affinity for the carbohydrate region of the HRP. If some of the contaminants in the HRP grade I preparation are also glycosylated, then the measured affinity constants would be affected. Protein Release from Agarose Affinity Particles. After protein adsorption to affinity particles attained equilibrium, release of the bound protein was studied by using two methods: dilution and buffer change. For a reversible protein adsorption, dilution of the free adsorbate concentration should result in a release of bound adsorbate to reestablish the equilibrium. Dilution experiments for
all the affinity systems showed that protein adsorption was only partially reversible. There was some release of bound protein but far less than that necessary to establish an equilibrium that was in agreement with the initial adsorption isotherm. The reason for this irreversibility is not well understood, but studies on protein adsorption to surfaces suggest that protein adsorption can be followed by conformational changes of the protein structure that increase the binding affinity (17,18). (The term “irreversible”is used in this paper to describe adsorbed proteins that do not release upon dilution. However, irreversible binding does not mean that proteins will not release upon a change of buffer.) Buffer changes to affect protein desorption were much more effective than dilution. For all affinity systems studied, a change to the elution buffer resulted in a very rapid desorption of almost all bound protein. Protein Adsorption Rates and Comparisons with Models. Protein adsorption rate data were collected for all affinity systems and were analyzed with use of the different models discussed above. Table IV presents the k,, values from the lumped kinetic model and the D, values from the shrinking core model. The parameter values in Table IV are more meaningful in conjunction with the graphs showing the adsorption data and the model curves, of which a few representative graphs are presented. Figure 5 shows the rate of lysozyme adsorption to Blue Sepharose no. 2 for two different volume fractions of the affinity gel. The solid and dashed curves represent the best fits of lumped kinetic and shrinking core models, respectively. There is a trend for the lumped kinetic model to fall above the data initially and below it at longer times, which can be seen in Figure 5 and was also found with most other sets of adsorption data involving conventional agarose particles. This trend is attributed to the inability of the lumped kinetic parameters to properly account for the increasing pore diffusion limitation as adsorption proceeds. The shrinking core model generally fits the adsorption data a little better since pore diffusion is usually the rate-limiting step. Despite the simplicity of both the lumped kinetic and shrinking core mod-
37
Biotechnol. Prog., 1990, Vol. 6, No. 1
Table IV. Adsorption Parameter Values Based on Analysis of Protein Adsorption Rate Experiments with the Lumped Model of Chase and the Shrinking Core (DJ Model of Arnold Kinetic &)
affinity particle & adsorbate Blue SeDharose no. 1 & HSA Blue Sepharose no. 2 & HSA Blue Superose no. 2 & HSA Blue Sepharose no. 1 & lysozyme
run no.
6
1 2 3 4 5 6 7 8 9 10 11 12 13
0.023 0.033 0.034 0.0078 0.027 0.0075 0.026 0.0080 0.0080 0.015 0.033 0.050 0.013
Blue Sepharose no. 2 & lysozyme Blue Superose no. 2 & lysozyme Con A-Sepharose & HRP I protein A-agarose & IgG 0.40
0.1 0
I
/,,-;,,,;I
*%--_
___-----Shrinking Core
--------________ -----_______
@ = 0.026
0.00
5
0
1s
10 TIME (minutes)
Figure 5. Results of lysozyme adsorption to Blue Sepharose no. 2 (runs no. 6 and no. 7) along with model curves.
.
0.40
I
I
1
! 0.30
0
1
2
3
4
5
TIME (minutes)
Figure 6. Results of lysozyme adsorption to Blue Superose no. 2 (run no. 10) along with the model curves.
els, they provide reasonably good fits to all experimental data and the best fit parameter values are fairly consistent for different experiments with the same affinity system. The more complex, multiparameter Arve and Liapis model was also used to analyze the adsorption rate data, but the analysis is not presented here (19). In short, the additional parameters allow for a slight improvement in fitting adsorption data, but the parameters obtained vary considerably and are not that statistically significant. Figure 6 shows the adsorption of lysozyme to Blue Superose no. 2. For the Blue Superose adsorption data, the lumped kinetic and shrinking core models both fit the data quite well, so it is difficult to draw any conclusions as to the most important adsorption mechanism. However, the reduced diffusion limitation of the small Superose particles is reflected by the fact that the k , , values are about 2-5 times higher for both lysozyme and HSA adsorption to the Blue Superose compared with the Blue Sepharose.
C,, mg/mL 0.400 0.515 0.572 0.398 0.398 0.394 0.388 0.194 0.359 0.359 0.387 0.597 0.0944
k,,, mL/mg-min 0.0574 0.0416 0.187 2.25 2.24 0.820 0.921 4.47 4.52 4.35 0.414 0.491 0.304
iOsD,, cm2/s 1.27 1.21 0.0402 40.6 39.4 26.0 24.8 6.27 7.16 5.56 5.70 5.40 4.08
ACFF Constant-Volume Washing Experiments. Table V summarizes the results of the various constantvolume washing experiments (the schematic of the washing step was shown in Figure 2). It includes the experimental conditions and the resulting loss of adsorbate, along with theoretical estimates based on the kinetic model and the two limiting cases. The results of the washing experiments and their relationship to model predictions are easier to evaluate in graphical form. Figure 7 shows the adsorbate concentration profiles for three different ACFF constant-volume washing experiments. I t should be noted that the washing step begins with an equilibrium mixture of free and bound adsorbate, so that as the washing proceeds and the free adsorbate concentration decreases, some bound adsorbate will release from the adsorbent in an effort to reestablish equilibrium. The three curves in each plot are based on different assumptions regarding the interaction of the adsorbate with the affinity escort. The upper curve represents a concentration profile based on the assumption that reaction and diffusion are infinitely fast so that the equilibrium values of bound and free adsorbate concentrations are always maintained. The lower curve represents the other limiting case when the desorption rate of the adsorbate from the affinity escorts is infinitely slow so that no protein is released from the affinity escorts during the washing step. The curve in the middle tries to account for the actual rate of adsorbate release and is a numerical solution of the system mass balance (eq 3) and the lumped kinetic model (eq 1). The experimental data falls within the two limiting cases in the three plots shown in Figure 7 and also for all other washing experiments conducted. It should be emphasized that the theoretical curves are not empirical and have no adjustable parameters but are instead predicted based on the previously measured affinity parameters. Figure 7a shows a washing run with Blue Sepharose no. 2 and HSA (run no. 1). Because the k,, value for Blue Sepharose no. 2 and HSA is low, the kinetic control curve is quite close to the no-release limit. The data show little release of HSA as the data points fall near both the kinetic control and no-release curves. However, the data points do fall a little below the kinetic model for VI V,, > 2. The total amount of HSA lost during this washing step was 61%, which would be intolerably high for a commercial operation. However, the loss of adsorbate during the ACFF washing step can be reduced by using a higher fraction of affinity escorts and a lower adsorbate load (5). Figure 7b shows another washing run with Blue Sepharose no. 2 as the affinity escort but with lysozyme as the adsorbate (run no. 3). These data agree well with
38
Biofechnol.Prog., 1990, Vol. 6, No. 1
Table V. Results of Various Batch ACFF Washing Experiments for Different Affinity Systems and Operating Conditions Along with Theoretical Predictions Based on Different Assumptions
affinity escort & adsorbate Blue Sepharose no. 2 & HSA Blue Sepharose no. 2 & Lysozyme
mg/mL
V,, mL
mL/min
exptl
1 2
0.024 0.020 0.032 0.017 0.010 0.010 0.051 0.050 0.063 0.042 0.051
0.97 0.78 0.70 0.72 0.51 0.51 0.21 0.10 0.20 0.33 0.7
250 250 250 250 250 250 250 250 300 300 370
49 49 49 49 57 16 49 49 125 41 47
61 43 4.0 29 38 39 29 33
