Productivity Model for Separation of Proteins Using Ceramic Monoliths

Article pubs.acs.org/IECR

Productivity Model for Separation of Proteins Using Ceramic Monoliths As a Stationary Phase Milena A. Vega,† Eva M. Martín del Valle,*,† Ramón L. Cerro,‡ and Miguel A. Galán† †

Department of Chemical Engineering, University of Salamanca, P/Los Caídos S/N, 37008, Spain Department of Chemical and Materials Engineering, University of Alabama in Huntsville, Huntsville, Alabama 35899, United States

‡

ABSTRACT: The compact geometry and outstanding mechanical properties of ceramic monoliths are valuable features when designing reusable standard affinity chromatography columns. High throughput protein separations are becoming standard industrial practice. Column reuse is an important consideration for the development of separation processes when production rate per unit volume of column is taken into account. Due to the cost and downtime needed to regenerate chromatographic columns, throughput depends on the use of the same column for several adsorption−desorption cycles without a significant loss in adsorption. An adsorption−desorption dynamic model was developed and used to estimate the optimum reuse of ceramic monolith columns in affinity chromatography. The model experimental metal chelate affinity chromatography (IMAC) system is a ceramic monolith covered with agarose type D-5 activated with 1.4 butanediol diglycidyl ether as spacer arm, iminodiacetic acid as chelating agent, and Cu2+ as ligand. The effect of performing several adsorption/elution cycles on column performance was evaluated using five monoliths at different flow rates, for a total of five cycles each. The strategy used to interpret experimental results leads to a theoretical model for successive monolith reuses that can be readily used to determine the optimal reuse of monoliths on the basis of maximum production rate per volume of chromatographic columns.

1. INTRODUCTION Metal chelate affinity chromatography (IMAC) has been used extensively in the separation and purification of proteins.1−3 Amino acids are selectively attached to metal ions as Cu(II) > Ni(II) > Zn(II) > Co(II).4 When these amino acids are present on the surface of an protein such as histidine (His)5−7 a highly selective and reversible adsorption process takes place.1 The traditional approach to packing of affinity chromatography columns is a random bed of solid particles.8 Random particle beds demand large pressure gradients due to enhanced tortuosity and small void fractions. In addition, tortuosity is also responsible for dead-pockets and slow mass transfer rates. Membranes and monoliths have been proposed as chromatography supports providing a number of advantages over traditional affinity chromatography with porous-bead packed columns.9,10 Our group pioneered the use of ceramic monoliths, similar to the monoliths used in the manufacture of catalytic converters in the automotive industry.11 Monolithic materials have quickly become well-established stationary phases for biological separation and purification. The simplicity of their in situ preparation methods and the large variety of readily available reaction schemes make the monolithic separation media an attractive alternative to liquid chromatography columns, packed with particulate and membrane materials.12 Ceramic monoliths are a natural choice of support for IMAC columns because of the large surface/ volume ratios, low pressure drop during flow, strong mechanical properties, and simple methods used for preparation and handling of active chromatographic columns. In addition, monolith columns are relatively inexpensive and can be easily regenerated. Lately, high degrees of specificity have been achieved with IMAC showing degrees of purification and resolution © 2014 American Chemical Society

comparable to those obtained with highly biospecific adsorbents.13 The apparent affinity of a protein for a metal chelate depends strongly on the metal ion involved in coordination. When imminodiacetic acid (IDA) is used as a chelator immobilized on an inert support such as agarose, transition divalent metals, particularly Cu(II), 4,14 form coordination complexes known as polydentate chelates, depending on the number of occupied coordination bonds.15 Our experiments were designed to demonstrate the feasibility of using and reusing several cycles of ceramic monoliths coated with agarose D5, activated with iminodiacetic acid (IDA) and Cu(II) as the metal−ligand, with the purpose of defining a production policy that will maximize throughput per unit volume of column. The protein used for the adsorption/elution experiments was bovine serum albumin (BSA). BSA was purposely chosen for these experiments since it does not show high adsorption efficiency on Cu(II). Relatively slow adsorption/elution rates result on cycle duration times more comparable with the time needed to regenerate the column. This property allowed us to perform experiments for a relatively small number of reuse cycles and still have a basis of comparison against the use of new columns. Adsorption column reuse is an important consideration for the development of industrial separation processes, since the economics of these processes depend on the continued use of the same column without a significant loss in adsorption capacity. Thus, practical evaluation of a separation process to be Special Issue: Alı ́rio Rodrigues Festschrift Received: Revised: Accepted: Published: 15456

January 14, 2014 March 12, 2014 March 13, 2014 March 13, 2014 dx.doi.org/10.1021/ie500184s | Ind. Eng. Chem. Res. 2014, 53, 15456−15466

Industrial & Engineering Chemistry Research

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a mixture of agarose type D5, phosphate buffer at pH 4.5, and water. The mixture was heated at 70 °C and subjected to vigorous agitation to obtain a homogeneous solution. The column cores, previously weighed (dry), were immersed a few minutes into the agarose. Immersion was carried out twice. After the second immersion, the monolith was left to rest and dry for 2 h and then weighed to calculate the amount of agarose remaining on the walls. Next, the column cores were introduced into a reactor containing a mixture of 100 cm3 of a solution 0.4 M of NaOH, 1.13g of NaBH4, 38 cm3 of acetone, and 19 cm3 of 1.4 butanediol diglycidyl ether. The reactor was agitated for 6 h at a speed of 118 rpm and additional amounts of 19 cm3 of 1−4 butanodiol diglycidyl ether were added after 2 and 4 h of reaction time. respectively. Once the reaction is completed, the monolith is washed with ion-free water. 2.3. Procedure to Attach Iminodiacetic Acid (IDA) and Bind the Metal Ligand. Column sections previously rinsed after the coating process were introduced into a reactor agitated at 119 rpm containing a mixture of 100 cm3 of a 0.1 M solution of Na2CO3 and 9 g iminodiacetic acid (IDA). After 12 h of reaction time, the column sections were removed from the reactor and rinsed with ion-free water. The column sections were fitted into a laboratory column support XK16 and 100 cm3 of a solution of 5 mg/cm3 of CuCl2 was circulated through the column for 2 h at a rate of 1.6 cm3/ min. During this preparation step, the column sections turned, from their original yellow color to a light blue color, typical of the presence of Cu2+. Excess copper solution remaining inside the monolith cells were removed from the column by circulating 100 cm3 of ion-free water at a flow rate of 1.6 cm3/min for 30 min. The Cu2+ not specifically bound to the support was eluted by circulating through of column 100 cm3 of a phosphate solution pH 4.5 at a flow rate 1.6 cm3/min for 30 min. Before the first adsorption step, a 50 mMTris-buffer (tris(hydroxymethyl)-aminomethane) at pH 8.0 was circulated through the column for 2 h, until approximately 0.02 absorbance at 280 nm. Without this intermediate step adsorption of the protein was very low. There is evidence16 that Tris exhibits copper-complexing ability with free cupric ions. Arguably, washing with Tris removes copper not attached to the matrix. To test the effect of Tris buffer on adsorption sites, a solution of BSA was circulated through a new monolith without previous exposure to Tris buffer. In the circulating solution, clear evidence of the copper−BSA complex was found at the 600 nm peak. The amount of protein adsorbed, however, was smaller by about 50%, from the amount that would have been adsorbed after treating the monolith with the Tris-buffer. Preliminary studies focused on the adsorption process without

scaled up for industrial use, involves determination of the useful lifetime of the adsorbent, knowledge of process adsorption kinetics, and calculation of efficiency factors to predict the overall performance of the column after reuse.

2. MATERIALS AND METHODS 2.1. Materials. Agarose type D5 Conda Laboratory, sodium hydroxide (pellets), and acetone (extra pure) were supplied by Table 1. Physical Properties of Affinity Support property/monolith

1

2

3

4

5

weight (g) overall volume, VM (cm3) coating load (g) coating average thickness (urn) total surface area of coating (cm2)

4.54 11.76 2.25 108

4.53 11.76 1.79 86

4.53 11.76 3.07 152

4.58 11.88 3.04 147

4.45 11.56 2.03 98

211

210

204

209

209

SharlauChemie S.A., sodium borohydride 98%, iminodiacetic acid (disodium salt), potassium phosphate dibasic 98%, and BSA were supplied by Sigma. 1.4 butanedioldiglycidyl ether 95% was supplied by FlukaChimie S.A. Anhydrous sodium carbonate, and potassium phosphate dihydrogen for analysis were supplied by Panreac. Copper chloride(II) was supplied by Riedel-de Haen. The 400/6 standard automotive monolith (400 cells/in.2 and 0.006 in. wall thickness) from Corning N.Y. is made of cordierite and contains traces of iron, titanium, potassium, and calcium. Monolith columns are cut to fit inside a standard column container from standard automotive monoliths and are approximately 0.013 m in diameter and 0.075 m long. Because of the small diameter of the column, some of the cells located on the periphery of the column, cannot be accessed by the circulating solutions. Thus, instead of using average geometrical parameters, we counted the number of square cells that remained open for flow and use this count to compute the interfacial area of coating. The percentage error, nevertheless, is not small due to the small size of the columns used in our experiments. The 400/6 monoliths have bulk density of ρM = 0.385 g/cm3, a value corresponding to the ceramic monolith, before coating with agarose and an internal specific cell area of aM = 27.56 cm2/cm3. Additionally, to count the number of cells in the matrix, an enlarged photograph of the final part of the monolith was taken, and the number of cells was counted manually.11 Main physical properties of the 400/6 ceramic monolith are shown in Table 1. 2.2. Coating Column Sections with Agarose. The column inserts, cut out of a ceramic monolith, were coated with

Figure 1. Schematic representation of a multicycle IMAC process. Showing the successive steps for a complete cycle. 15457

dx.doi.org/10.1021/ie500184s | Ind. Eng. Chem. Res. 2014, 53, 15456−15466


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4. ADSORPTION/ELUTION RESULTS 4.1. Protein Surface Concentration. There are several facts to learn about affinity chromatography when using coated

using Cu(II) were carried out, where it was found that the adsorption of the protein is not performed. Therefore, retention without Cu(II) protein assumes zero. Additional washing and reconditioning steps are ubiquitous in preparation of adsorbing matrices. In certain applications,17 the same buffer used to wash and equilibrate the matrix previous to adsorption is later used as the elution media at a different pH. In other applications,18 there is a step where the matrix is treated with a strong acid salt (NaCl) at pH 4.0, followed by washing with a mixture of sodium phosphate and sodium chloride at pH 7.0. Elution takes place later with either sodium acetate or sodium phosphate but at a much lower pH. It is possible to conclude that the effect of matrix treatment with a buffer/scavenging solution goes beyond the simple removal of unbounded metal ions. Most likely, washing with Tris buffer affects electron exchange properties of the chelating compounds.

Figure 2. Schematic representation of the protein adsorption process showing the random surface coverage by large protein molecules. The large red blobs represent the protein molecules, while the smaller red dots are active surface adsorption sites. Notice the large amount of available surface and active sites that cannot be covered by adsorbed protein molecules.

3. EXPERIMENTAL SECTION 3.1. Adsorption/Elution Experiments. In order to evaluate the efficiency of monolith column sections after several cycles of adsorption/elution processes, experiments involved five consecutive cycles (one first use and four reuses) at five different flow rates: 1.50 cm3/min for the first monolith, 5.50 cm3/min for the second monolith, 8.40 cm3/min for the third monolith, 10.60 cm3/min for the fourth, and finally 14.00 cm3/min for the fifth monolith. Please note that we define the first cycle, n = 1, as the first use of the column (fresh adsorbent), the next cycle for the first reuse is indicated by n = 2, the following cycles, n = 3, 4, and 5, correspond to the second, third, and fourth reuses, respectively (Figure 1). To carry out adsorption experiments, 50 cm3 of a BSA solution [0.7 mg/cm3] in buffer phosphate at pH 4.5 and 25 °C was circulated continuously, through an affinity column XK-16 Pharmacy. The initial concentration used in all adsorption experiments was C0 = 1.06 × 10−5 mmol/cm3. During experiments, BSA adsorption in the column was measured by taking an aliquot of 1 cm3 every 20 min and protein absorbance was measured using a UVICON 922 spectrophotometer at a wavelength of 280 nm, until the concentration of BSA in the sample reached a constant value, indicating that the adsorption process has, for practical purposes, stopped. Although sample taking affected the total amount of solution circulated, adsorption during the first 60 min of the experiment where the volume circulated varies little and took place at a much faster rate than the rest of the experiment. If a volume correction is introduced for the later parts of the experiment, the total amount of protein adsorbed would be slightly smaller than the amount computed assuming a constant volume of solution, but this correction is well within experimental errors. Traces of BSA solution remaining in the monolith channels were washed out with buffer phosphate at pH 4.5 and the column was prepared for the elution process. Intermediate washing steps will add 0.5 h each to the operating cycle while the elution process is estimated to take 2 h, followed by a tris-buffer rinse of another 2 h. If we define the adsorption time as tads, a whole adsorption/elution cycle can be completed in an amount of time equal to tcycle = tads + 5 h. Indeed, the choice of a relatively slow BSA−Cu system, allows experimental cycle times of the same order of magnitude as the regeneration process.

ceramic monoliths as supports.11 First, since agarose has negligible microporosity, all proteins adsorbed on the matrix are attached to sites located on the external walls of the agarose film coating the monolith walls. Second, proteins are much larger than the active adsorption sites on the agarose surface. Thus, when a protein adsorbs on one site, it covers and renders nonaccessible many other active sites. Third, protein adsorption is a random process where proteins sometimes overlap slightly but leave many intermediate active sites and the agarose surface uncovered; see Figure 2. Since adsorption is a random process involving a very large number of molecules, we can consider the percentage of overlaps as well as the percentage of uncovered surface, as statistically constant from one cycle to another. According to the values obtained when the chromatography matrix is reused at the same flow rate, the adsorption capacity decreases, because the specific area available for carrying out the adsorption process is reduced. This behavior is due to fouling, since the BSA protein molecules cannot be removed in the elution process, blocking adsorption sites. Additionally, the residual imidazole which cannot be removed after the adsorption process of occupied Cu(II) sites will not be available in the next adsorption cycle. Adsorption equilibrium profiles versus time are shown in Figure 3 for initial and four reuses. From Figure 3, it can be seen that even though the initial concentration of protein in solution C0 was the same for all cycles, CE|n, the adsorption capacity of the monolithic column decreases, when the number of reuses are increased for the same flow rate. This behavior is repeated in the five flow rates studied. This behavior is due to fouling caused by the retained protein in the adsorbent surface which cannot be removed. Indeed the residual imidazole deposited on the adsorbent surface after elution was not possible to remove with tris-HCl buffer pH 8.0. This behavior implies that the actual available adsorption process area is an important factor in the adsorption process, which influences the adsorption capacity of the monolithic column. Additionally there is a pointed tendency that the adsorption capacity increases with increasing flow rate 15458



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Figure 3. Protein adsorption equilibrium curves, 50 mM phosphate solution, pH 4.5, 298 K, for various flow rates studied.

of the adsorption process. In order to justify this behavior it should be taken into account that equilibrium concentrations are also affected by the operating flow rate used for circulating the protein solution. Larger flow rates show lower equilibrium protein concentration levels, suggesting that larger intake pressures open up channels that are partially obstructed during wash-coating. This is perhaps the only drawback of using monoliths with parallel channels where individual channel flow rate depends strongly on the hydraulic radius of the channel; that is, partially obstructed channels will show a much slower flow rate. There may be other alternative reasons for variation of equilibrium concentrations with flow rate, related to mass transfer rates near the adsorbing surface, but we believe channel opening is the leading one. However, regardless of the cause, variations are small and monotonic. Thus, to account for these variations we have averaged the results for a given monolith during its first use and reuses, on the basis of the solid surface available for adsorption, and the results are plotted in Figure 4.

Figure 4. Averaged protein adsorption equilibrium curves in phosphate solution pH 4.5 for five flow rates studied with corresponding reuses.

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Table 2. Comparison between and ρTAS and ρBE,n for Five Flow Rates Studied: Q (cm3/min) ρBE,n × 10−7 (mmol/cm2) n n n n n

= = = = =

1 2 3 4 5

Q = 1.5

Q = 5.50

Q = 8.40

Q = 10.60

Q = 14.00

ρTAS × 10−6 (mmol/cm2)

6.17 5.99 5.33 3.78 3.15

7.16 6.71 5.49 4.86 4.16

7.70 6.23 5.02 3.85 3.13

9.20 6.95 5.04 3.71 2.68

9.36 8.17 7.11 6.28 5.11

3.12

Table 3. Adsorption/Elution Efficiencies for Reused Monoliths Q (mL/min) number of cycles of use (n) m (cm3 of solution/area cm) ρRAS (mmol/cm2) × 10−7 ηA0 (dimensionless) ηA (dimensionless) ηD (dimensionless)

1.50

5.50

8.40

10.60

14.00

5 0.24 7.33 0.23 0.84 0.80

5 0.24 8.02 0.26 0.89 0.85

5 0.24 8.64 0.28 0.89 0.77

5 0.24 10.39 0.33 0.88 0.70

5 0.237 10.29 0.33 0.91 0.85

The time required to reach equilibrium is, within experimental error, very close to the same for all runs, regardless of circulating flow rates and regardless of the number of reuse cycles. There may be a minimum flow rate such that circulation rates become rate limiting, but it is well below the range of flow rates to efficiently open most of the monolith channels. Also, it is an important fact that superficial velocities,

Figure 5. Representative figure Cu2+ sites occupied by imidazole.

Each adsorption equilibrium line represents an average of experimental results for the different flow rates.

Figure 6. Protein concentration logarithm of per unit area experimental vs number of uses (n − 1) for the protein BSA in pH 4.5 phosphate solution for the five flow studied, 298 K. 15460



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Figure 7. Extrapolation of adsorbed surface protein concentration per unit of support surface ρBE,n vs cycle number n, to determine the real protein surface adsorption concentration, ρRAS. Flow rates are Q = 1.5, 5.5, 8.4, 10.6, and 14.0 mL/min.

defined as the volumetric flow rate per unit cross sectional area of column, are much larger in monolith supported columns than the typical velocities attained in equivalent packed bead columns. Adsorption curves can be modeled using a simple first order kinetic equation where the rate of adsorption is equal to the rate of removal of available sites, and it is proportional to the concentration of available sites, considering film control.11 4.2. Adsorption Coverage Efficiency. Nonspecifically bound BSA proteins in the matrix are removed using phosphate solution pH 4.5. This value being about 5%, the total protein adsorbed in the matrix could be verified experimentally. To simplify calculations, we estimated that the correction factor value for all cycles is ϕ 0.95. The BSA protein bound specifically to the ligands was removed using 0.2 M imidazole solution in phosphate buffered saline (PBS) pH 7.4. The elution process was terminated when there was no measurable concentration of BSA in the aliquot samples taken from the solution. After desorption and previous to the next adsorption cycle, the column is washed again with a Tris-HCl buffer solution at pH 8.0 for 2 h and a flow rate of 1.5 cm3/min to remove imidazole remaining in the adsorbent. This step is essential to carry out after each imidazole elution process in order to condition the column to the next adsorption cycle. Otherwise, the imidazole bound to the Cu(II) will not be able to adsorb protein in the next adsorption cycle. Additionaly, rinsing the monolith walls with Tris-HCl buffer serves two purposes: one is to eliminate free copper ions from the matrix and the second is to condition the electron-exchanging ability

of the chelating complex. Thus, washing with the Tris buffer is an integral part of the adsorption/elution cycle. The amount of protein eluted with imidazole is the protein that is specifically bound to the matrix. Assuming that one molecule of imidazole displaces a protein molecule specifically adsorbed; the number of imidazole moles used to elute the protein would be equal to the number of protein moles adsorbed specifically. Additionally, because the imidazole displaces the protein bound to Cu(II), it takes the place left by the protein. Therefore, to reuse the adsorbent, it is necessary to wash the matriz with tris-HCl buffer pH 8.0 for removing the imidazole bound to Cu(II). Although the amount of copper is 2−3 orders of magnitude greater with respect to the BSA protein, the imidazole concentration used in this study is high (0.2 M). Therefore, it seems possible that imidazole occupies directly free sites of Cu(II) which were not occupied before by the protein. For this reason, eliminating the imidazole from the matrix is necessary. Thus, imidazole adsorption only reduces the number of active sites by a small percentage. This situation, in turn, eliminates concerns about polydentate chelates resulting in coordination complexes involving two or more coordination bonds. The theoretical amount of protein that can be deposited over the adsorbing surface is the ratio of the interfacial adsorption area to the area of a single molecule of protein: T ρAS (mmol/cm 2) =

1014(nm 2/cm 2) δ E(nm) × 10−6 × N (molecules/mol) (1)

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Where, N is Avogadro’s number and δE is the footprint of a molecule of protein that is the area occupied by one protein molecule deposited on the adsorption surface. An order of magnitude estimate of the footprint of BSA is the square of the diameter of BSA, D = 7.28 nm.19 Our computed value of ρTAS is similar to the value reported by Foose et al.;20 although Foose et al. defined the footprint as the area of a circle and, as a consequence, obtained a slightly smaller footprint. Obviously, one cannot cover the entire surface using circles, but both methods give comparable order of magnitude results. Consequently, as the values in Table 2 show, the surface concentration of protein, that is the number of moles of protein adsorbed per unit surface area determined experimentally, is smaller than the theoretical maximum. This lack of total coverage is due not only to the fact that a collection of irregular shapes cannot totally cover a given surface but also to the overlapping and interference during the adsorption process, mentioned in section 2. On the basis of these assumptions, we can argue that the number of moles of protein adsorbed on a given surface is limited by the interfacial area of the agarose-solution system. In fact, the number of moles of protein that binds on a surface can be calculated as a surface concentration, that is, in units of moles of protein per unit adsorption area. The surface concentration, ρBE,n (mmol/m2) can be computed as the product of the difference between the initial concentrations and saturation concentration (when equilibrium is reached), multiplied by the ratio of the volume of protein solution to the interfacial area of the column, m, and by the fraction of the protein that is actually adsorbed, not simply occluded, ϕ: ρBE, n (t ) = [C0 − C E(t )]mϕ

ηA =

ηD =

=

[C0 − C E|1]ϕm T ηA0ρAS

(4)

ρDS,1 ρBE,1

(5)

Introducing adsorption ηA and elution ηD efficiencies, we can compute the amount of protein that can be adsorbed during the second adsorption cycle (n = 2) using eq 6:

(2)

R R ρAS,1 = ρAS − ρBE,0 + ρDS,0 = ρAS [1 − ηA (1 − ηD)]

(6)

After the second adsorption, the amount of protein attached to ligands is computed by the change in concentration of the protein solution during the adsorption process, taking into account proteins that are not linked specifically to the matrix. During the second adsorption cycle, the amount adsorbed is again affected by the adsorption efficiency, taking into account that not all sites available after the first adsorption/elution can be occupied by the molecules of protein, so this amount of protein per unit of surface can be calculated according to eq 7: R ρBE,2 = ηA ρAS [1 − ηA (1 − ηD)] = (C0 − C E|2 )ϕm

(7)

In turn, equilibrium protein concentration, CE, of the second adsorption cycle will be related to the adsorption efficiency ηA and elution process according to eq 8: C E|2 = C0 −

R ηA ρAS

ϕm

[1 − ηA (1 − ηD)]

(8)

The elution efficiency, ηD, is used to estimate the amount of protein eluted per unit of surface in the second elution process (n = 2) as shown is eq 9:

R ρAS T ρAS

R ρAS

The stochastic nature of the adsorption process and modifications to the adsorption sites due to the adsorption/ elution processes, measured by the cycle adsorption efficiency, ηA, and common to all adsorption cycles, must be taken into account separately from structural effects represented by the structural efficiency, ηA0. After adsorption and an intermediate rinse with phosphate buffer at pH 4.5, adsorbed protein was eluted using a 0.2 M solution of imidazole in a PBS buffer at pH 7.4. The amount of protein removed by elution is somewhat smaller than the amount of protein adsorbed. When proteins remain attached to their adsorption sites, not all original sites will be available for the next adsorption cycle, as shown schematically in Figure 5. Due to residual imidazole, this cannot be removed after the elution process; as well, the protein molecules cannot be eluted and can block adsorption sites for the next cycle. Thus the combination of incomplete elution and site modifications diminishes the monolith adsorption capacity for the next cycle. To take into account these complex processes, we introduce an elution efficiency to relate the number of moles of protein that are released on the adsorbent surface in the elution process ρDS,1 for n = 1 to the surface sites available for protein adsorption after the first adsorption process ρBE,1.

The correction term in eq 2, ϕ ≈ 1, is a fraction less than but close to one and was introduced to take into account the proteins trapped inside the column, but not linked to the sites, considered as a correction to the real variation in the concentration of protein C0 − CE|0. The internal volumes of the agarose are negligible, because its porosity is negligible therefore the adsorption process is carried out exclusively on the adsorbent surface. Equation 1 allows conversion of protein solution concentration into surface concentration on the monolith, while eq 2 is used to compute the theoretical maximum amount of protein that can be adsorbed on the surface. We make use of these definitions to track changes in adsorption behavior of the monolith columns based on two types of adsorption efficiencies. The first efficiency, ηA0, is the relationship between real amounts of protein that can be adsorbed on the surface of monolith (ρRAS (mmol/cm2) which cannot be measured directly and must be estimated by extrapolation of adsorption equilibrium profile) and the theoretical amount of protein that can be adsorbed, ρTAS. This last variable depends on the coating structure and can only be measured by extrapolation during the first adsorption cycle (n = 1). ηA0 =

ρBE,1

(3)

The second adsorption efficiency, ηA, is due to the natural transformation that monoliths undergo with successive reuses and relates the amount of protein adsorbed during the first adsorption step per unit area of surface and the real amounts of protein that can be adsorbed on the surface of monolith.

R ρDS,2 = ηDηA ρAS [1 − ηA (1 − ηD)]

(9)

The analysis performed for the first two cycles can be easily extended to an arbitrary number of n cycles, where the equilibrium concentration CE is given by eq 10: 15462



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Figure 8. Production rates per unit area of column per unit time as a function of the number of adsorption/elution cycles before regeneration. Adsorption process are run for tA = 5 (equilibrium), 3, and 2 h. Regeneration time is equal to 18 h.

C E | n = C0 −

R ηA ρAS

ϕm

[1 − ηA (1 − ηD)]n − 1

The solid trendlines in Figure 7 are fitted using an exponential function, with values of R = 0.999 for both monoliths. The intersection of the trendline with the line for n = 0 allows us to compute an experimental value of ρRAS. Values of ρRAS for the five monoliths are collected in Table 3. With this value, it is possible to calculate the adsorption structural efficiency and adsorption efficiency, ηA, according to eqs 4 and 5, respectively. Experimental values of ηA0 and ηA for the five monoliths are shown in Table 3.

(10)

In logarithm form, the surface concentration of protein adsorbed during the nth cycle is given by eq 11: R ln(ρBE, n ) = ln(ηA ρAS ) + (n − 1)ln[1 − ηA (1 − ηD)]

(11)

Equation 11 represents a straight line in a semilogarithmic plot with slope = ln[1 − ηA(1 − ηD)] and intersection at n = 1 equal to ln(ηAρRAS). In Figure 6 are semilogarithmic plots of ln(ρBE,n) vs n − 1 for the flow rates studied. From the intersection of the straight line with the origin, we find the first surface adsorption amount, ρBE,1 = ηAρRAS, and from its slope, we find the value of [1 − ηA(1 − ηD)]. The slope can be calculated from the values of elution efficiency, ηD, if the values of adsorption efficiency, ηA, are known. Values of ηD for the five monoliths are shown in Table 3. To estimate the real amounts of protein that can be adsorbed on the surface of monolith ρRAS, we introduce a graph relating the surface concentration of protein adsorbed, ρBE,n, to the number of cycles, n. The real available surface concentrations are computed assuming that extrapolation of protein adsorbed surface concentration toward values of n → 0 provide a fair estimate of the value of available surface concentration:

lim[ρBE, n ] ≅

n→0

R ρAS

5. THROUGHPUT PRODUCTION BY MONOLITH REUSE Assuming that there is no loss of protein during the recovery process, we can estimate protein production as the amount of protein desorbed during the elution step. At the end of the nth cycle, the amount of protein desorbed is computed by the recursive relationship: R ρDS, n = ηDηA ρAS [1 − ηA (1 − ηD)]n − 1

(13)

Adding the protein produced in the successive cycles of reuse, the amount of protein produced per unit area of monolithic column is given by

(12) 15463



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Figure 9. Production rates per unit area of column per unit time as a function of the number of adsorption/elution cycles before regeneration. Adsorption process are run for tA = 5 (equilibrium), 3, and 2 h. Columns are exchanged assuming a downtime of 5 h.

number of cycles, the downtime would be based on the time needed to flush the system and place a new/regenerated column in place. To take into account the adsorption time, we assume that adsorption steps are extended for the same amount of time for all cycles. Thus, if the adsorption steps are performed for 300 min each, we can also assume adsorption has reached equilibrium. For shorter adsorption times, we ran the column during 180 min where roughly 85−90% of equilibrium is achieved or for 120 min where roughly 70−80% of equilibrium is achieved. Figure 8 represents the production rate of protein in millimolar per unit area (cm2) per unit time (h) taking into account a regeneration time of 18 h. If the only operating criteria was the amount of protein produced per unit time and unit volume of column, and on the basis of a single monolith column that must be regenerated after a number of adsorption/ elution cycles, production rates indicate that, if equilibrium is achieved in each cycle, a maximum production rate with the same column will be obtained if we use the column during four cycles before regeneration. However, if we decrease the adsorption steps to 180 min, after five cycles the column is still slowly increasing production after five cycles. Interestingly, reducing the duration of the adsorption step to 120 min is not an attractive proposition since the duration of the intermediate washing and elution steps become determinant. Since production rates are shown per unit time, if we assume a continuous operation of the column, the annual production rates will show similar characteristics as a function of the number of reuse cycles and adsorption times. If the process can be operated with columns that are exchanged and regenerated offline, the amount of downtime needed to change the columns becomes one of the optimization criteria. Certainly, decreasing downtime makes

ρprod | n (mmol/cm 2·cycle) n R = ηDηA ρAS ∑ [1 − ηA (1 − ηD)]j − 1 j=2 n

= α ∑ β j−1 (14)

j=2

ηDηAρRAS

The constants in eq 14 are defined as, α = and β = [1 − ηA(1 − ηD)]. The time needed to complete one cycle was already defined at the end of section 3 as tcycle = tads + 5 . If the monolith is used for n cycles, the total duration of the reuse process of production includes also the downtime needed to regenerate or change the column, tdownt. Finally, production per unit time is computed by dividing eq 15 by the total duration of the reuse process: n

2

ρprod | n (mmol/cm ·h) =

α ∑j=2 β j−1 n(tads + 5) + tdownt

(15)

Assuming that washing and elution steps take always the same amount of time, eq 15 represents the production of protein per unit area of monolith column and per unit time and has three operating variables: (1) the amount of time allowed for adsorption, tads, (2) the number of cycles the column is used between regeneration, and (3) the downtime needed to regenerate the column or to change the column if regeneration is done as an independent step. A rough estimate of the regeneration time can be found by following the production steps of a monolith column described in section 2. A conservative estimate of regeneration time from our experiments is tdownt = treg = 18 h. If on the other hand, we operate the process by changing the monolith column after a certain 15464



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reuse of columns less attractive if annual throughput is the only criterion. To give an indication of how throughput is affected by column downtime, Figure 9 shows annual production per unit volume of column per hour for a downtime of 5 h. As before, we are able to compare production when columns are allowed to reach equilibrium during adsorption with production rates when adsorption is interrupted after 180 and 120 min, respectively. If the adsorption process is allowed to reach equilibrium, a maximum production rate is reached by allowing the monolith to be reused twice (three cycles). If adsorption is terminated after 3 h, production rates per unit area of column are slightly larger than the equilibrium case and also have a maximum for a 3 cycle operation. Finally, if the adsorption process is terminated after only 2 h, for every cycle, the production rate per unit area of column is now smaller than the equilibrium (5 h) and the 3 h adsorption runs. It is evident that reusing monolith columns will increase the throughput of protein for a given process operation. However, since the additional cost of regenerating the monolith column after a few uses is not included in the analysis, we do not have a full economical evaluation. The cost of regenerating the column will certainly be larger for smaller adsorption times since the columns will have to be regenerated more frequently. Also, we have not taken into account wear and tear that takes place during the process and the whole column replacement that will take place after a number of several-cycle processes.

Article

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by funds from the Ministerio de Economia y Competitividad, Project reference CTQ201124486, “Desarrollo de una nueva tecnologiá de separación de ́ proteinas basada en cromatografía IMAC y monolitos cerámicos como soporte”. The authors gratefully acknowledge the financial support.

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REFERENCES

(1) Porath, J.; Carlsson, J.; Olsson, I.; Belfrague, G. Metal chelate affinity chromatography, a new approach to protein fractionation. Nature 1975, 258, 598. (2) Porath, J. Immobilized metal ion affinity Review. Protein Expr. Purif. 1992, 3, 263. (3) Gutierrez, R.; Martín del Valle, E. M.; Gálan, M. Immobilized metal-ion affinity Chromatograhy: Status and Trends. Sep. Purif. Technol. 2007, 36:1, 71. (4) Oyrton, A. C.; Monteiro, Jr.; Airoldi, C. Some thermodynamic data on copper−chitin and copper−chitosan biopolymer interactions. J. Colloid Interface Sci. 1999, 212, 212. (5) Arnold, F. H. Metal-affinity separations: a new dimension in protein processing. Bio-Technology 1991, 9, 150. (6) Hemdan, E. S. Surface topography of histidina residues: a facile probe by immobilized metal ion affinity chromatography. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 1811. (7) Yip, T. T. Evaluation of the interaction of peptides with Cu(II), Ni(II) and Zn(II) by high performance immobilized metal ion affinity chromatography. Anal. Biochem. 1989, 83, 159. (8) Gutiérrez, R.; Martín Del Valle, E.; Galán, M. A. Adsorption and mass transfer studies of Catalase in IMAC chromatography by dynamics methods. Process Biochem. 2006, 4, 142. (9) Yi-Miao, Ke Preparation of the immobilized metal affinity membrane with high amount of metal ions and protein adsorption efficiencies. Process Biochem. 2010, 45, 500. (10) Zou, H.; Luo, Q.; Zhou, D. Affinity membrane chromatography for the analysis and purification of proteins. J. Biochem. Biophys. Methods 2011, 49, 199. (11) Martin Del Valle, E. M.; Galan, M. A.; Cerro, R. L. Use of ceramic monoliths as stationary phase in affinity chromatography. Biotechnol. Prog. 2003, 19, 921. (12) Muvdi Nova, C. J.; Paolucci-Jean, M. P.; Barboui, M.; Rios Rivallin, G. Elaboration, characterization and study of a new hybrid chitosan/ceramic membrane for affinity membrane chromatography. J. Membr. Sci. 2008, 321, 81. (13) Vijayalakshmi, M. A. Pseudobiospecific ligand affinity chromatography. TIBTECH 1989, 7, 71. (14) Ç etinus, S. A.; Sahin, E.; Saraydin, D. Preparation of Cu(II) adsorbed chitosan beads for catalase immobilization. Food Chem. 2009, 114, 962. (15) Sulkowski, E. Purification of proteins by IMAC. Trends Biotechnol. 1985, 3, 1. (16) McPhil, D. Tris buffer- a case for caution in its use in coppercontaining systems. BJ Lett. 1984, 559. (17) Tamagawa, R. E.; Azzoni, A. R.; Miranda, E. A.; Vijayalakshmi, M. A. Aprotinin recovery: Comparison beteween biospecific and pseudobiospecific affinityadsorption. Braz. J. Chem. Eng. 1999, 16, 2. (18) Vançan, S.; Everson, M.; Alves, S. IMAC of human IgG: studies with IDA-immobilized cooper, nickel, zinc and cobalt ions and different buffer systems. Process Biochem. 2002, 37, 573.

6. CONCLUSIONS In this work a theoretical model for successive monolith reuses has been developed on the basis of maximum production rate per volume of chromatographic columns From the experimental work it can be found that despite a smaller specific interfacial area than bead columns, monolith columns are well-suited for high throughput protein production for their low pressure drop to liquid flow, fast mass transfer rates, and mechanical robustness. Due to their mechanical robustness and high flow rates, monoliths can be easily used in a sequence of cycles of adsorption/elution. As in all similar chromatographic columns, some of the original adsorption activity is lost upon reuse. Our experimental program involving several monolith columns was devised to characterize the evolution of adsorption activity where ceramic monolith coated with agarose were used as a support to separate BSA from solution. In addition, the simple, accurate mathematical model developed to simulate the dynamics and equilibrium characteristics of our model system can be developed in a similar way for any other chromatographic system. There are three main parameters that must be experimentally evaluated for any system: (1) fraction of adsorption completion, referred to equilibrium saturation of the column and measured as time of adsorption, (2) number of adsorption/ elution cycles before regeneration, and (3) downtime between multiple cycle processes due to regeneration or change of the monolith column. Indeed, to extend the scope of this work to the analysis of industrial operating policies, one must introduce not just operating costs but the overall context of the separation process within an associated bioprocess system. In general, although the model used to evaluate throughput does not take into account costs, only production rates, it is evident that column reuse is a very attractive policy for industrial bioseparation processes. 15465



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(19) Daqiq, L.; Fellows, C.; Bekes, F.; Lees, E. Methodologies for symmetrical-flow field-flow fractionation analysis of polymeric glutenin. J. Texture Stud. 2006, 38, 273. (20) Foose, L.; Blanch, H.; Radke, C. J. Kinetics of Adsorption and Proteolytic Cleavage of a MultilayerOvalbumin Film by Subtilisin Carlsberg. Langmuir. 2008, 24, 7388.

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Productivity Model for Separation of Proteins Using Ceramic Monoliths

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