Studies on Scale-up Parameters of an Immunoglobulin Separation System Using Protein A Affinity Chromatography Kyung A. Kang and Dewey D. Y. Ryu' Department of Chemical Engineering, University of California at Davis, Davis, California 95616 Effecta of operational and system parameters on process scale-up of murine immunoglobulin ( 1 g G 2 ~purification ) using Protein A affinity chromatography are investigated. Parameters studied are those related to sample application, elution, ligand concentration on support, and column size change. Between sample application velocities of 0.004 and 0.030 cm/s (16-108 cm/h), the product concentration profiles in eluate did not show significant differences. With given system parameters, the retention time and bandwidth of the peak could be predicted by moment theory. The mean equilibrium constant during elution showed a strong effect of pH between 2 and 4. Within the range of protein A concentrations studied, 0.15-1.22 mg/mL of gel, the column capacity shows a linear relationship with the concentration of protein A immobilized. Dimensional scale-up of the column in the radial direction increased the total purification capacity linearly as the cross-sectional area increased without increasing pressure drop; however, the product concentration was diluted. Scale-up of the column dimension in the axial direction enables higher concentrations of product in the eluate, although the retention time increases linearly as the gel height increases. Introduction In many affinity separation systems both ligands and products are biomolecules of high molecular weight. Thus, their three-dimensional structures and activities are very susceptible to the change of many environmental conditions such as temperature, pH, and/or ionic strength. During the immobilization of ligands on gel matrices, the natural conformation of the ligands changes and the degree of the change differs in each system or each molecule in a given matrix system. The degree of their susceptibility also varies with different support matrices and the methods of immobilization on the support. The gel matrix used for each separation has a varying degree of tolerance to the hydrostatic pressure due to the gel matrix itself and to the pressure generated by the flow of the liquid phase. Because of these characteristics, which make it difficult to generalize the performance of affinity chromatography, the best scale-up criteria selected for one system may not be applicable to the others. However, a systematic study on scale-up-related variables will be critically important because it will enable one to predict some important aspects of system behavior or, at least, a trend of system performancerelevant to certain aspects of scaleUP. Process scale-up may be achieved by manipulation of operational and system parameters as well as by changing column size. In other words, scale-up can be expressed as an increase in system capacity to process raw material in a unit time. For better understanding of the problems involved in the characterization and the scale-upof affinity separation of murine IgGaa., some operational and system parameters of major importance were selected and their effects on the system performance were studied by using laboratoryscale columns. The following parameters were studied. (A)Operational Parameters: (1)Samplevolume with constant mass of murine IgGSa, and sample application velocity during adsorption stage and their effects on protein
* Author to whom correspondence should be addressed. 875&7938/91/3007-0205$02.50/0
elution concentration profile and process time and (2) flow rate and pH during the elution step and their effects on purification process time. (B)System Parameters: (1)Concentration of ligand immobilized on the surface of the gel matrix and its effects on the purification capacity and elution equilibrium and (2) length and diameter of the column and their effects on the system purification capacity and product concentration in eluate. The main objective was to study the effects of these operational and system parameters on the performance of an affinity purification system (IgG purification using protein A-Actigel) and to relate the results to characterization and scale-up of a more general affinity separation system. Materials and Methods Materials. I. IgG2a,: Protein To Be Purified. Because monoclonal antibodies have been used for diagnostic, analytical, and therapeutic purposes and, especially, for immunoaffinity purification, the demand for this biomaterial has been increasing very rapidly. At the same time, the developmentof hybridomacell culture has made mass production of monoclonal antibodies practical, and as a consequence, purification of monoclonal antibody has been a problem of practical importance. Therefore, one of the most frequently used murine immunoglobulins, IgGZa,, was chosen as the protein to be purified. The molecular weight of IgG2a, is approximately 150 OOO. Murine IgG2a, was purchased from Sigma (St. Louis, MO). II. Protein A: Ligand. Protein A (SPA)has very high affinity for the Fc region of immunoglobulins. This protein may be obtained from the cell wall of Staphylococcus aureus and its purified form is commercially available. The molecular weight of SpA is around 42 OOO. Because the active site of immunoglobulin binding to SpA is far from those of the antigenic active sites, the separation of immunoglobulin using SpA does not affect the activity of the immunoglobulin to its antigen. SpA has five binding sites to immunoglobulins; however, not all the sites are
0 1991 American Chemical Society and American Institute of Chemical Engineers
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reactive at the same time and most of the time the reaction is not stoichiometric (Langone, 1982a-c; Lindmark et al., 1982). Protein A was purchased from Sigma (St. Louis, MO). III. Actigel: Support Matrix. Actigel (purchased from Sterogen, San Gabriel, CA) was used as the support matrix for the affinity purification of immunoglobulin. The information on the particle size of Actigel provided by the manufacturer gave a range of 50-150 rm. The actual size distribution for this gel matrix was not available from the manufacturer. The measurement from a particle size measuring device, Matersizer (Malvern Instruments, Malvern, England), indicated that the swollen particle size was in the range between 35 and 170pm with a square mean diameter (d,) of 100 pm. The void fraction (a)of Actigel, which was measured by using blue dextran, was 0.27. The entire porosity (&,ratio of the volume of pores to the entire volume of a gel particle) of gel measured by the manufacturer was 0.9. The density (p,) of the wet gel is assumed to be 1g/cm3. The porosity measured by using immunoglobulin (8,ratio of the volume of pores that IgG can pass through to the entire volume of a gel particle) was 0.73. The pore size of Actigel is 20 million daltons. Actigel is a cross-linked agarose (4%) with a short arm of five atoms between gel matrix and ligand. The ligandcoupling chemistry of Actigel is by the irreversible reduction of the Schiff base of the amino group of ligands with sodium cyanoborohydride. Methods. A. Column Packing. After the immobilization of SPA, the gel was degassed under vacuum for 30 min. The column to be used was filled with degassed adsorption buffer with no air bubble at the outlet or on the wall of a column. With a Pasteur pipet, the column was filled with the degassed gel from the bottom very slowly up to 1-2 cm higher than the height desired for an experiment (and with Tris buffer 3-5 cm above the top surface of the gel). Then, the column was placed vertically until there is no more reduction in gel height (approximately 1-2 h). Once the gel was settled, the column was connected to the pump and Tris buffer was pumped into it slowly [with a velocity of 0.005 cm/s (18 cm/h)] for approximately 30 min and then the final gel height was adjusted. Once the gel height was fixed, the top surface of the packed gel was kept evenly flat before any experiment. Whenever there was a change in gel height by more than 2 mm from the predetermined height or any perturbation a t the top of gel packing during the experiment, the column was repacked and the experiment was repeated. Thus, experiments were conducted while the gel height did not change any more than 2 mm within the range of superficial velocity used in the experiments. For the experiment with higher flow rates, the gel height was less than 8 cm and the column diameter was 0.7 cm. Since the entire purification process time was relatively short under the condition of higher flow rate (less than 20 min) and the column height remained nearly constant, it was safe to assume that there was no significant change in the column performance. B. AffinityPurification Experiments. The overall experimental procedure of immunoglobulin purification using SPA-Actigel affinity chromatographywas as follows. A predetermined amount of SPA was immobilized on Actigel by the method that the manufacturer provided. Immobilized SpA concentration on the gel matrix was determined by subtracting the amount of unbound SpA in the supernatant. IgGBa, in Tris buffer (pH 8.0) was then adsorbed on SPA-Actigel. After the column was washed with 5-10 times the gel volume of Tris buffer or until the chart recorder showed a base line, the immunoglobulin was eluted by 0.02 M glycine buffer at pH 3.0.
Blotechnol. Rog., 1991, Vol. 7, No. 3
In order to avoid mixing of buffers in the headspace of the column, whenever adsorption buffer had to be changed to elution buffer or vice versa, one buffer above the gel surface was drained completely and the other buffer was applied slowly without disturbing the gel surface while the valve at the outlet was closed. For the cases where this operation was not possible (columns of larger diameters), adapters were used. The concentration of IgG in the eluate was measured by an UV detector at 280 nm, and the eluate was collected by a fraction collector (Pharmacia, Piscataway, NJ). Glass columns and adapters of varying diameters used for the experiment were from Bio-Rad (Richmond, CA). Ail results represent duplicate experiments, and statistically meaningful results are reported. The detailed descriptions of each set of experiments are as follows. Z. Effect of Sample Application. The objective of this set of experiments was to see if there was any change in adsorption characteristics with the change in conditions of sample application by observing the concentration profile in the eluate. For this set of experiments, SpA concentration on the gel surface was approximately 0.8 mg/mL of gel. The diameter of the column for these experiment was 0.7 cm, and the gel height was 1.5 cm (matrix volume, 0.58 mL). The amount of IgG used for experiments was small enough to form a thin band at the top of gel. The experimental variables tested were as follows: (1)Effect of concentration of IgG (i.e., constant mass of IgG in various sample volumes) on the IgG elution concentration profile. Samples of 74 pg of IgG in a total adsorption buffer volume ranging between 80 and 800 pL were applied to the columns. After the washing step, IgG was eluted by elution buffers and elution profiles were studied. (2) IgG adsorption characteristics with changes in the sample application velocity. The rationale for this experiment was that if there was reaction or diffusion limitation in the separation system and a small amount of sample was applied at higher velocity, the unadsorbed or undiffused IgG would go down to the lower part of the column and would form a broader band because of insufficient residence time. Therefore, the elution profile of IgG would have a lower peak and a broader bandwidth. Samples of IgG of mass 184 pg in a total volume of 2 mL were applied to the column with various velocities in the range of 0.011-0.152 cm/s (39-547 cm/h). After the washing step, IgG adsorbed was eluted by elution buffer. The elution buffer velocity was 0.011 cm/s (39 cm/h) for all experiments. From these experiments,the retention time and bandwidth of the concentration profile of the eluate were measured. IZ. Effect of Elution-Related Parameters. The experiments for this section were, basically, pulse tests of adsorbed IgG2aNby elution buffer. For these experiments, the amount of IgG was small enough to form a very thin band at the top of the gel surface but large enough to give a response at the end of the column. From several tests using various sample masses, it was found that 74 pg (80 pL) of IgG was the smallest amount that provides, on the chart recorder, a signal strong enough to be differentiated from noise and shows a defined peak and bandwidth in the elution profile. One of the objectives of this study was to estimate some of the system parameters (equilibrium constant and dispersion coefficient) by using moment theory. These variables obtained from small-scale experiments will help us understand the performance of larger scale affinity columns. The concentration of immobilized SpA used in this set of experiments was the same as that used in experiments described in section I. The diameter of the column used for these experiments was 0.7 cm, and the gel height was
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varied for different experiments between 3 and 8 cm (gel volume, 1.15-3.1 mL). The protein was elutedat different superficial velocities between 0.004 and 0.303 cm/s (16108 cm/h). For the study of the effect of pH change on the mean equilibrium constant, the adsorption and washing steps were performed in the same way as in the experiments outlined in section I. For the elution stage, glycine buffers of pH 2.0,2.5,3.0,3.5,and 4.0 were used. The mean equilibrium constants were computed from the retention time measured. III. Immobilized Ligand Concentration. In order to observe the effect of the ligand concentration on the gel matrix, a series of different amounts of SpA were immobilized to a constant amount of Actigel. The final amounts of SpA immobilized were 0.15,0.45,0.75,0.9,and 1.22 mg of SpA/mL of gel. Columns of diameter 0.7 cm and height 1.5 cm (gel volume, 0.6 mL) were prepared with the gel matrices of varying SPA concentration. Breakthrough experiments with IgG concentration of 0.092 mg/mL of buffer were performed in the columns prepared. For this experiment, a flow rate of 0.1 mL/min [superficialvelocity, 0.004 cm/s (16 cm/h)] was used. IV. Dimensional Scale-Up of Column. Experiments were performed to observe the relationship between the change of column dimension and IgG concentration profile. Immobilized SPA concentration for this part of the experiments was approximately 2.0 mg/mL of gel. For the first set of experiments four columns of different diameters, 0.7, 1.0, 1.5, and 2.5 cm, were filled with gel matrix to a gel height of 1.5 cm (gel volume of 0.6,1.2,2.7, and 7.4 mL, respectively). A constant mass of IgG (184 p g / 2 mL of Tris buffer) was applied to each column, and the product concentration profiles in the eluate from these four columns were studied. For the second set of experiments, three different diameter columns (0.7,1.0, and 1.5 cm) were filled with the same amount of gel matrix (2.65 mL) and elution profiles were studied. The superficial velocity for all experiments was 0.011 cm/s (39 cm/h).
Theory When the radial dispersion is small enough to be neglected and local equilibrium between the concentrations of the biomolecule in bulk liquid and in the liquid phases of pores is assumed,the partial differential equation for concentration in an affinity separation column can be expressed by the following (Kucera, 1965; Arnold, 1985b; Jaulmes and Vidal-Madjar, 1989): a
a = D, at
az2
- u---
az
R,
(1 - a ) k p[C - C,(R,)] (1)
affinity chromatography may not be valid for all systems. However, in pulse tests, the amount of biomolecules used for adsorption/desorption is very small compared to the total capacity of the entire column; therefore, the assumption may be applicable. For the pulse test of this system, the initial and boundary conditions can be expressed as follows:
< 0.0) = 0.0
(4)
Ca(Z,t < 0.0) = 0.0
(5)
co6(t)
(6)
=J)= finite
(7)
Ci (r = 0 ) = finite
(9)
C(z,t
and C(Z
C(z
-
= 0) =
= 0.0 dr o where COis the protein concentration at t = 0.0 at the inlet of the column and s ( t ) is the Dirac delta function. The three important values to be predicted for the protein elution profile are (a) retention time, (b) bandwidth, and (c) skewness of the peak. Although it is very difficult to solve eqs 1-3, their moments can be obtained by transferring the equations to the Laplace domain. For the system described by the equations and initial and boundary conditions, the normalized first moment (retention time) is derived and given as
where Ap'1 is the first moment, Z is the entire gel height, and
When the system parameters, CY, 8, and pp and system variables Z and u are known, then by plotting experimentally measured retention time, Ap'1 vs aZ/u,the value of 1 + 60 can be obtained from the slope of the plot (eq 11). By using the relationship of eq 12 with the value obtained for 60,the mean equilibrium constant, K,of the affinity system can be estimated. The second centralizedmoment for the system expressed by eqs 1-10 can be derived as
where
aCa = k,(C, - c a / K ) az
(3)
where a is void fraction, B porosity, C concentration of biomolecules in the liquid phase, Ca the concentration on the solid phase, Ci the concentration in pores of the gel particles, t time, r radial position in a gel particle, R, the radius of a gel particle, z axial position in the column, D1 dispersion coefficient, Di diffusion coefficient of the biomolecule, pp density of gel particle, u the superficial velocity of buffer in the column, k, adsorption coefficient, k, mass transfer coefficient, and K the mean equilibrium constant between the liquid phase in the pores and solid phase of the gel particles. The assumption of linearity in
The term 61 is the contribution of adsorption, diffusion, and mass transfer at the boundary layer to the second moment (or total dispersion). By rearranging, eq 13 can be expressed as
and (16) Since the dispersion coefficient,DI,is always correlated
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Table I. Effect of IgG Concentration the Product Elution Profile sample volume, p L 80 240 400 560 640 800
peak height, scale 9.0 8.0 8.0 8.5 7.0 8.0
(a) in the Sample on
dimensionless retention timeb 16.2 16.5 15.2 16.0 17.3 15.4
dimensionless bandwidthCat half peak height 2.7 2.7 2.7 2.8 2.9 2.4
a Peak height, retention time, and bandwidth at half peak height with changingsample volume are shown. Sample mass, 74 pg of IgG; column diameter, 0.7 cm; gel height, 1.5 cm; superficial velocity of elution buffer, 0.011 cm/s (39 cm/h). Dimensionless retention time = retention time/mean residence time of buffer in the column. c Dimensionless bandwidth = bandwidth/mean residence time of buffer in the column.
*
with velocity, u, the dispersion term, Dl/v is usually used instead of DI only. The second centralized moment, Ap2, can be expressed by using the bandwidth at the half-peak, w0.s:
Table 11. Effect of Sample Application Velocity on Peak Height, Retention Time, and Bandwidth at Half Peak Height. sample peak dimensionleea application height, dimensionless bandwidth a t velocity, cm/s scale retention time half peak height 0.011 7 16.0 2.14 0.024 8 15.4 2.40 0.043 7 14.7 2.40 0.108 7 14.0 2.40 0.152 7 16.0 2.70 a IgG mass in sample, 184 pg; total sample volume, 2 mL; column diameter, 0.7 cm; gel height, 1.5 cm; superficial velocity of elution buffer, 0.011 cm/s (39 cm/h).
contact with the SpA molecules. The results presented in Table I1 support this conclusion and show that the sample application flow rate does not affect the final elution concentration profile significantly within the range of sample application velocity evaluated. Therefore, the process time during the adsorption step, T ~ may ~ , be expressed in terms of the effect of sample application parameters:
(17) Therefore, the second moment can be estimated from the measurement of bandwidth from the concentration profile of product elution. By plotting the values of Ap2u vs (u/Z)Ap’1 2, values of 2Dl/v and 2 4 can be estimated from the slope and the intercept of plots, respectively. Therefore, the system characteristics of equilibrium and dispersion can be estimated from two experimentally measured values, retention time and bandwidth of elution profiles.
Results and Discussion I. Sample Application. Table I shows the effect of change in sample volume (from 80 to 800 pL), when the total IgG mass in each sample was kept constant (74 pg), on the elution concentration profile of product. From this result, it was found that the product elution profile did not depend on the IgG concentration in the sample, CO, when the total product mass in the raw material remained constant. In this affinity purification system, the adsorption of product to the ligand occurs fast; therefore, the samples can be applied in a relatively high concentration to reduce the sample application time. However, this may be valid only when there is no other major difference in the material properties, such as viscosity, between concentrated and dilute samples. In the range of sample application velocity from 0.011 to 0.152 cm/s (39-547 cm/h), there was no significant difference in the peak height or bandwidth although bandwidth increased slightly when the sample application velocitywas0.152 cm/s (547 cm/h) (Table 11). The finding that there is no significant change in retention time and bandwidth, despite the 10-fold change in sample application velocity, also indicates that there is no significant diffusion or adsorption limitation in the adsorption conditions. As previously noted, from the measurements, the swollen particle size was in the range between 35 and 170 pm with a square mean diameter (d,) of 100 pm. The void fraction (a)of Actigel is 0.27 and hence the interstitial velocities were in the range of 0.04-0.56 cm/s (144-2016 cm/h). By taking the square mean particle size of 100 gm, the actual contact time between IgG and SpA can be estimated as 0.018-0.25 s, which means that IgG diffuses and molecules are adsorbed very rapidly as they come in
where Ci, is the product concentration in the source material and umax is the maximum sample application superficial velocity without gel matrix damage. By applying concentrated sample with a relatively high velocity, the process time can be reduced. It may be noted that for the type of soft gel (crosslinked agarose) used in the current research, the manufacturers recommend that the superficial velocity should not exceed 0.05 cm/s (in the manufacturer’s manual, it is specified as 170 mL cm-2 h-l) to prevent any damage to the gel. The maximum superficial velocity of 0.152 cm/s (547 cm/h) used in the present experiments was a factor of 3 higher than that recommended by the manufacturer. Since the column diameter used for this experiment was relatively small, the bed height is low, the sample volume applied was small (2 mL), and the superficial velocity for the sample application higher than 0.05 cm/s was applied for less than 2 min, gel compression was never observed. 11. Elution of Product. (A) Elution Buffer Flow Rate. Moment theory is used very frequently for the prediction of chromatography behavior of a linear system. Since the amount of IgG used for the pulse test is small enough to form a very thin band, it was assumed that the adsorption/desorption is in the linear range. Sometimes, during experiments, a thin band was actually visible. The normalized first moment and centralized second moment are used for the prediction of retention time and the bandwidth of the product concentration profile in the eluate (see Theory). By plotting the values of term Ap’1 vs aZ/u with some of the system and operational parameters known, the value of 1+ 60can be obtained. From the value 1+ 60, the mean equilibrium constant can be estimated (eqs 11 and 12). Figure 1shows this relationship for the elution superficial velocity range between 0.004 and 0.030 cm/s (16-108 cm/ h). The slope of the figure represents the value 1 + 60, which was 13.65; the equilibrium constant of elution for the purification system was computed from this value and the value for the system was 3.7. Although a minor difference in K was noticeable whenever a new batch of gel with SPA was prepared, this value did not fluctuate much when the pH of the elution buffer was kept constant. From the plot, the regression coefficient, 98%, and the
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W t M . Rog., 1991, Vol. 7, No. 3
50001
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300
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a z/v (sec) Figure 1. Determination of mean equilibrium constant, K,
+
during elution by pulse test. Slope (1 60) = 13.65;RC = 0.98 [gel height 3.8-4.2 cm and superficial velocity of elution buffer 0.011-0.0303cm/s (39-108cm/h)].
-7 an
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velocity of elution buffer. Slope (2D1 u ) = 0.0041 and RC 0.8. [gel height 3.5-8.0 cm and superficia/ velocity of elution buffer 0.004-0.030cm/s (16-108cm/h)].
elution characteristics of this purification system appear to give a linear equilibrium relation between SpA and IgG. Moment theory seems to be applicable to this affinity system. In order to determine the dispersion characteristics of the affinity purification system, the second moment expression of moment theory was applied (see Theory). From only seven values, a, /3, pp, 2,u, the retention time, and the bandwidth of concentration profiles of IgG, which can be obtained easily, the dispersion of a purification system can be characterized to a certain extent. Figure 2 shows the relationship between Apzu and (u/Z)Ap'l (eq 16), and the slope of the correlation gives the value 2Dl/u. From the experiments with Actigel-SpA this value was determined as 0.0041 cm (i.e., Dl/u = 0.00205 cm). The intercept value, 2~x61,indicates the effect of adsorption/ desorption, diffusion, and mass transfer effect combined on dispersion. By comparing the term D p ( 1 + 6 0 ) ~ / u ~ to 61, the order of magnitude of fluid mechanical dispersion to that of other causes can be obtained. For this affinity separation system, in order for the combined dispersion to be of the same order of magnitude as fluid mechanical dispersion, the intercept value, 21x61, should be 3.4 for a velocity of 0.03 cm/s (108 cm/h) and 25.8 for 0.004 cm/s (16 cm/h). Although the experimental values in Figure 2 are somewhat scattered, it appears that the dispersion in the system is mainly fluid mechanical (the intercept is approximately 4.1 from the linear fitting in the figure) except for the upper range of the experiments. ( B )piY Effmt on Mean Equilibrium Constant for Elution Process. Affinity chromatography has a unique characteristic that the ligands have extremely high specificity and affinity to the product with only a few
I
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3
K = 5.6 ( ~ H J- 41.2 (pH)* + 101 (pH)
- 80.4
I
I 2.5
3.5
4.5
pH of Elution Buffer
Figure 3. Effect of change in pH of elution buffer on mean equilibrium constants during elution (gel height 1.5-4.2 cm; superficial velocity of elution buffer 0.011 cm/s (39cm/h)].
exceptions. In conventional chromatographic separation, the resolution, which is defined to be the retention time difference between the product and unwanted materials divided by the sum of the two bandwidths of the peaks, has been used as an index of the product separation capability of a column from unwanted material in the eluate. Therefore, in order to increase the resolution,various elution schemes that separate the product apart from contaminants (such as a gradient, a series of steps, etc.) are applied during the elution step. However, in most affinity separations, the purity of a product is determined by the amount of remaining unwanted materials after the washing stage. As a consequence, most elutions for affinity chromatographyare by stepwise change of buffer condition for faster process time. For the purification of the immunoglobulin, a step change of buffer pH from adsorption to elution was used. The pH for the product adsorption was 8.0 but the elution of the product was performed at lower pH. A small reduction of pH from the optimum adsorption can reduce the value of the mean equilibrium constant greatly (Figure 3). The experimental results showed the following correlation between the pH of elution buffer and the mean equilibrium constant, K:
K = 5 . 6 ( ~ H-) ~41.2(pH)'
+ 10l(pH)- 80.4
2 S p H I 4 (19) The mean equilibrium constant changes by a factor of (pH)3, and therefore, by reducing elution pH far from the optimum adsorption pH, the process time can be reduced tremendously (see equation 19). Although lower pH gave faster elution, extremely low pH may denature the product irreversibly; therefore, it is necessary to perform the elution in the pH range where the purified protein is stable and maintains its activity or to neutralize the eluate immediately after the elution. The results also showed that immediate neutralization of eluate after elution at pH 3 retained full activity of IgG. 111. Effect of Immobilized Ligand Concentration (BreakthroughCurves). A breakthrough experiment is one of the best methods for the estimation of the product adsorption capacity of a purification system. Theoretical and some experimental studies on breakthrough curves for the scale-up of affinity chromatography have been reported by Arnold et al. (1985a). If the breakthrough curves are symmetric to the center point (the point at C/Ch = 0.5 in Figure 41, the breakthrough volume (or the mass of the protein to be separated in the volume) at C/Ch = 0.5 represents the column capacity. In the range of the experiments, SpA concen-
Biotechnol. Frog., 1991, vol. 7, NO. 3
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Figure 4. Breakthrough curves for varying concentration of immobilizedligand [superficialvelocity of adsorption buffer 0.004 cm/s (16cm/h); IgG concentration in sample 0.092 mg/mL of buffer; column diameter 0.7 cm; gel height 1.5 cm).
Figure 7. Breakthrough curves normalized with respect to the SPAconcentrationon gel matrix (columnconditions are the same as in Figure 4). Table 111. Effect of Ligand Concentration on Retention Time and Mean Equilibrium Constant. concentration of dimensionless mean equilibrium ligand, mg/mL of gel retention time constant for elution 0.15 8.08 2.7 0.45 8.67 2.85 0.75 8.61 2.8 0.9 8.61 2.8 1.05 8.0 2.6 1.25 8.67 2.84 a IgG mass in sample, 74 pg; column diameter, 0.7 cm; gel height, 1.5 cm; superficial velocity of elution buffer, 0.011 cm/s (39 cm/h).
Ligand Concentration on Gel (mg-SpA/ml-Gel)
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Reaction Time (min)
Figure 6. Batch adsorption kinetics of IgG; adsorption of IgG on SpA-Actigel (gel volume 0.5 mL; initial concentration of IgG 1 mg of IgG/2 mL of buffer; SpA concentration on gel matrix 0.76 mg/mL of gel). tration 0.15-1.22mg/mL, the column capacity showed a linear increase with increase of SpA concentration on the gel surface (Figure 5). The binding ratio of SPA to IgG was 1:0.41 by their masses. This means that after the immobilization only 10% of SpA retains the affinity to IgG, under the assumption that only one active site of SpA has affinity to IgG. For a comparison, a batch adsorption experiment was performed with an excessamount of IgG (Figure 6). When 0.5 mL of Actigel matrix containing 0.78 mg of SpA was reacted with 1 mg of IgG (in 2 mL of buffer), more than 70% of the gel saturation capacity (approximately 590 pg
of IgG) was adsorbed within 5 min. The saturation level was found to be 0.59 mg of IgG in 0.5 mL of SpA-Actigel matrix having 0.78 mg of SpA/mL of gel. Approximately 43 % of the SPA retained ita affinity to IgG, which is about 4 times higher than in column separation. By normalizing the sample volume with the immobilized SPA concentration, it was possible to see the change in profiles of breakthrough curves with changes in the concentration of SPA immobilized (Figure 7). If the resistance to diffusion increases in a gel particle as the SpA concentration on the matrix increases, the normalized breakthrough curves would become shallower because IgG molecules will take more time to reach SpA molecules. Actual profiles of breakthrough curves did not change significantly with changes in SpA in the range of the experiments, although the curve from the SPA concentration of 0.15 mg/mL of gel deviates from the rest of the curves. This deviation is also shown in Figure 5. The first point in Figure 5 is rather far away from the regression line. This is probably due to the experimental error in the measurement of unbound SPA concentration in the liquid phase during SpA immobilization. The phenomenon that the normalized breakthrough curves can be transposed with each other suggests that the diffusion limitation does not increase significantly as immobilized SPA concentration increases, within the SpA concentration range of the experiments. The change of ligand concentration does not affect the mean equilibrium constant for elution (Table 111),which means that the concentration change does not appear to change properties related to the equilibrium or mass transport in the gel matrix, within the range of the experiments. IV. Column Dimension Effect. ( A ) Column Size Change in Radial Direction. Elution concentration profiles from columns of four different diameters, 0.7,1.0, 1.5, and 2.5 cm, are studied (Figure 8). These profiles are
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211 Column Diameter D I0.7 0 D.1.0 A D-1.5 A D=L5
0 0
0 0
6
0.61 0.4
E
8 z
0.2
0.0
0
5
10
15
20
25
Dimensionless Time
Figure 8. Normalized IgG elution profiles with respectto change of column diameter [elution buffer superficial velocity 0.011 cm/s (39 cm/h); gel height 1.5 cm]. Table IV. Effect of Column Dimension, Column Diameter, and Gel Height on the Elution Profile column gel peak dimensionless diameter, cm height, cm height, scale retention time 0.7 6.9 20 68.53 1.0 3.4 11 33.3 1.5 1.5 4.2 13.3 a IgG elution profiles are from columns of different diameters filled with the same volume of gel matrix, 2.65 mL. IgG mass in sample, 74 wg; superficial velocity of elution buffer, 0.011 cm/s (39 cm/h).
to reduce the yield. Therefore, it is imperative to obtain the product in high concentration during the elution stage in the shortest possible process time. For the protein, which can be concentrated after the elution step without difficulties, purification with columns of large diameter may reduce the total process time. The column scale-up in the axial direction has an advantage of obtaining higher product concentration in eluate (Table IV). However, there are other problems due to the mechanical weakness of commonly used affinity gel matrices (agarose, Sepharose, etc.). One of the most serious problems in affinity chromatography separation is gel matrix damage that could be caused by the hydrostatic pressure. The hydrostatic pressure, Phy, due to the gel height is expressed as where pp is density of the swollen gel matrix used, g is the gravitational acceleration constant, and Zn is the gel height of column n. Since pp and g are constants and with a constant amount of gel volume the gel height is inversely proportional to the cross-sectional area, the pressure may be written as a function of Z n and Dn: Gel matrices that could tolerate high pressure (Le., cellulose, silica,or synthetic polymer) do not require special consideration of this pressure effect, although they have other problems such as low efficiency of ligand coupling, nonspecific binding of product to the surface of the gel matrix, etc. Besides the increase in hydrostatic pressure, axial dimensional scale-up of column requires longer purification time when the same superficial velocity of sample or buffer is applied, as can be seen from eqs 11and 23:
normalized by the highest peak value, which was obtained from the elution profile of the column with diameter 0.7 cm. When a constant amount of product is introduced to the columns of different diameters, the peak heights of the product concentration profiles are inversely proportional to the cross-sectional areas (A or diameter square term, D2) of the columns (Figure 8) when the same superficial elution velocity is applied to all columns. The larger column has capacity left to have the same elution concentration as the smaller ones, and that capacity where rl;,is the retention time of the protein in the column increases with the ratio of cross-sectional area of column, of diameter D,. without increasing pressure on the gel matrix. However, The final decisions on the direction of dimensionalscaleif the gel is not to be used with its full capacity, use of a up should be made on the basis of (1)the characteristics larger diameter column will dilute the concentration of and stability of the product related to the separation eluted product. conditions and process time and (2) the tolerance of gel (B) Effect of Constant Amount of Gel Filled in matrix to the pressure. Different Diameter Columns. Effects of changes in axial and radial dimensions of the column can be seen by Conclusions observing the purification performance of columns of Based on the results of the study on characterization different diameters filled with the same gel volume (Table and scale-up of affinity chromatography of IgG, the IV). Since the gel volume remained constant, the ratio of conclusions are as follows. gel heights Z1:Z2:Z3 becomes (1/D~)2:(1/D~)2:(1/D~)2. I. Sample Application (Adsorption). In IgG sepTherefore, for the same amount of the gel (2.65 mL) filled aration using SPA-Actigel affinity chromatography, the in 0.7-, 1.0-, and 1.5-cm-diameter columns the gel heights affinity between ligands and products is very high and the were6.9,3.4, and 1.5cm, respectively. The concentration adsorption rate is very fast. Therefore, the sample of the product in eluate appears to be linearly proportional application flow rate is mainly limited to the maximum to the gel height ratio, Z1:Z2:Z3,which is the same as (1/ flow rate at which the gel matrices can function effectively D1)2:(l/D2)2:(l/D3)2when the same superficial velocity without damage. One may maximize the process time was applied to all columns (Table IV): and obtain higher production rates by preconcentrating the source material to reduce the sample application Cen0: l / A n 0: 1/D: (20) volume and by applying the highest flow rate that the gel matrix can tolerate. where C,, is the product concentration in the eluate from 11. Elution. In the range of superficial velocity of eluthe column of cross-sectional area An. tion in the experiments [0.004-0.03 cm/s (16-108 cm/h)], Many bioproducts separated by affinity chromatography have following characteristics: (1)They are expensive. the effects of flow rate on scale-up could be predicted by (2) They are present in low concentration in the source the moment theory. It is shown that the equilibrium and material. (3) Their biological activity is susceptible to the dispersion of an affinity purification system may be change of environmental conditions and, consequently, characterized by the measurement of two variables, e.g., could be damaged by steps of different purification or retention time and bandwidth. concentration processes. Furthermore, each and all of the Since the resolution of affinity chromatography is concentration steps increase total process time and tend determined during the washing stage, which means that
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only the product is to be obtained during the elution step, stepwise elution can be used to reduce the process time. In the affinity system used in the present work, the mean equilibrium constant (in the pH range between 2 and 4)changed as a function of (pH)3. A slight reduction in pH value from the optimum pH for the adsorption will reduce the value of K and, as a result, shorten the process time tremendously. Retaining the activity of product and ligand is the determining factor of the degree of step change from the optimum adsorption to the elution conditions. 111. Ligand Concentration on Gel Matrix. Within the range of SpA concentrations on the gel matrix used in the experiment (0.15-1.22mg of SpA/mL of gel), the column capacity was linearly dependent on the concentration of ligand on gel matrix. Nevertheless, the elution mean equilibrium constant did not change with changes in SpA concentration. IV. Column Size Change in Radial and Axial Directions. When the column size increases in the radial direction, the capacity of the affinity column increases with the ratio of the cross-sectional area of the column. Therefore, an increase in column diameter may increase the capacity linearly without increasing hydrostatic pressure or retention time. However, if the column is not operated with its full capacity, use of a larger diameter will cause lowering of the product concentration in the eluate. When the column size increases in the axial direction, the column capacity changes linearly with the gel height. However, the retention time of product output and the gel compression increase linearly with the increase in gel height. With the same volume of gel matrix and the same superficial velocity, the concentration of eluted product decreases with the increase in column diameter, the retention time is inversely proportional to the crosssectional area of column, and the gel compression with constant superficial velocity increases with the decrease in column diameter.
Notation cross-sectional area of column, cm2 C, C,, Ci concentration in liquid, solid, and interparticle, respectively,of biomolecules to be purified, mg/ cm3 concentration of biomolecule in eluate and input, mg/cm3 concentration of biomolecule in source material, mg/cm3 column diameter, cm effective intraparticle diffusion coefficient,cm2/s dispersion coefficient, cm2/s square mean of gel particle diameter, cm gravitational acceleration constant, cm/s2 immunoglobulin G mean adsorption equilibrium coefficient adsorption coefficient mass transfer coefficient monoclonal antibody of nth column hydrostatic pressure, g/cm3 flow rate, cm3/s radial position in a gel particle, cm
A
column radius, cm regression coefficient radius of a gel particle, cm protein A time, s effective elution velocity or interstitial velocity ( Q / (a A), cm/s, cm/h superficial velocity, cm/s, cm/h sample application velocity, cm/s, cm/h maximum sample application velocity without damaging gel matrix, cm/s, cm/h bandwidth at half of the peak height, s axial position of the column, cm total column length, cm Greek Symbols a void fraction ratio of the volume that IgG can pass through to P the entire volume of a gel particle ratio of the volume of pores to entire volume of a PP gel particle 60 [(I - a)/a18(1 + K pp/B) = -
AN’I APZ PP TPI TI
Dirac delta function normalized first moment, s second centralized moment, s2 density of gel particle, g/cm3 process time during adsorption step, s retention time, s
Acknowledgment This research was supported by the National Science Foundation (Grant NSF BCS-89-02499). Literature Cited Arnold, F.; Chalmers, J.; Saunders, M.; Croughan, H.; Blanch, H.; Wilke, C. A Rational Approach to the Scale-up of Affinity Chromatography. ACS Symp. Ser. 1985a,271, 113-122. Arnold, F.; Blanch, H.; Wilke, C. Analysis of Affinity Separations 11: The Characterization of Affinity Columns by Pulse Techniques. Chem. Eng. J. 1985b,30,B25-B36. Chase, H. Prediction of the Performance of PreparativeAffinity Chromatography. J. Chromatogr. 1984,297,179-202. Jaulmes,A.; Vidal-Madjar,C. Theoretical Aspectaof Quantitative Affinity Chromatography: an Overview. In Advanced Chromatography; Giddings,J., Grushka, E., Brown, P., Eds.; Marcel Dekker: New York, 1989;Vol. 28,pp 144. Kucera,E. Contribution to the Theory of ChromatographyLinear Non-Equilibrium Elution Chromatography. J.Chromatogr. 1965,19,237-248.
Langone, J. Use of Labeled Protein A in Quantitative Immunochemical Analysis of Antigens and Antibodies. J. Immunol. Methods 1982a,51, 3-22.
Langone, J. Applications of Immobilized Protein A in ImmunochemicalTechniques. J.Immunol.Methods 1982b,55,277296.
Langone, J. Protein A of Staphylococcus aurew and Related Immunoglobulin Receptors Produced by Streptococci and Pneumococci. Adv. Immunol. 1982c,32, 157-252. Lindmark, R.; Thoren-Tolling, K.; Sjoquist, J. Binding of Immunoglobulins to Protein A and Immunoglobulin Levels in Mammalian Sera. J. Immunol. Methods 1982,62,1-13. Accepted February 4,1991.
