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Dynamic liquid chromatography column models in separation processes

1

protein

E. Vasquez-Alvarez1 and J. M. Pinto1,2

University of So Paulo, Department of Chemical Engineering, So Paulo SP, Brazil, elsa@lscp.pqi.ep.usp.br 2 Polytechnic University, Othmer Department of Chemical and Biological Sciences and Engineering, Brooklyn NY, USA, jpinto@poly.edu,

Abstract. Chromatographic methods that use solid stationary and liquid mobile phases are increasingly important processes for the isolation and purification of pharmaceutical products, bio-molecules and fine chemicals. Peptides and proteins are most frequently separated using ion-exchange chromatography but issues related to the mass transfer in the chromatographic separations of bio-molecules are still unsolved. Mathematical modeling plays an essential role for the prediction of chromatographic peaks under different operation modes. Dynamic models can thus provide a basis for the optimization of a chromatographic process operated either in preparative scale or in high performance liquid chromatography mode, since the chromatographic peaks are predictable with a given change in the conditions that affect process operation. The objective of this work is to propose a dynamic mathematical model for the chromatography-based purification of multiple component mixtures, as well as its parameter estimation. The equilibrium-dispersive dynamic model representation is used that considers differential mass balances in the mobile phase, the concentrations in the stationary phase are related to the mobile phase concentration by adsorption isotherms, and mass transfer resistance is described by the dispersion apparent coefficient. An example for the separation of a mixture with multiple components (Thaumatin, BSA, Ovalbumin and SBTI) is presented and compared to experimental data. Keywords: Chromatographic separation, Protein purification, Parameter estimation, Equilibriumdispersive model.

1. Introduction
The pharmaceutical industry started to show great interest for the preparative chromatography of high performance since the early 80s (Guiochon et al., 1994). Preparative chromatography aims at isolating a certain amount of a purified component and using it for a further goal. Furthermore, the exact amount desired has a secondary effect which makes its perspective different from that of the analytical (Guiochon, 2002). Mathematical modeling plays an essential role for the prediction of chromatographic peaks under different operation modes. Dynamic models can thus provide a basis for the optimization of a chromatographic process operated either in preparative scale or in high performance liquid chromatography mode, since the chromatographic peaks are predictable with a given change in the parameters that affect chromatographic processes. Diverse dynamic models of preparative chromatography have been developed. The simplest model is the ideal one that assumes no axial dispersion and infinite mass transfer (Guiochon et al. (1994); Guiochon (2002)). Rate models have been developed by Gu et al. (1993), Guiochon et al. (1994), Gu (1995) and Garrido (1997). This model involves the interaction of complex hydrodynamics, thermodynamics and kinetics phenomena. Guiochon (2002) indicates that this model attempts to simultaneously account for all the possible contributions to the mass transfer that arises in chromatography.

To

whom all correspondence should be addressed. E-mail: jpinto@poly.edu,

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Guiochon et al. (1994), Teoh et al. (2001) and Guiochon (2002) studied the equilibrium-dispersive model. In this model, mass transfer in the chromatographic column is controlled by the molecular diffusion of the mobile phase that percolates through a porous particle bed and by the flow exchange between the stationary and mobile phases. The concentrations in the mobile and stationary phases are also considered to be in equilibrium throughout the whole column. The contributions of the axial dispersion and possible additional mass resistance can cause band broadening, which can be described through an apparent dispersion coefficient. Moreover, Teoh et al. (2001) used this model as a basis for the optimization of a chromatographic separation process. The objective of this work is to propose dynamic mathematical models for the chromatography-based purification of multiple component mixtures, as well as their parameter estimation for protein standard separation. The approach relies on the equilibrium-dispersive model. An example for the separation of a mixture with multiple components is presented and compared to experimental data.

2. Formulation of the equilibrium-dispersive model

The equilibrium-dispersive model used in this work was originally proposed by Teoh et al. (2001) for the separation of an aromatic mixture that consists of differential mass balances, equilibrium isotherms as well as initial and boundary conditions. The model formulation considers the following assumptions: (a1) (a2) (a3) (a4) (a5) the column is radially homogeneous, i.e. unidimensional. there is no temperature gradient in the column. the compressibility in the mobile phase is negligible. the contributions of the axial dispersion and possible additional resistance can cause band broadening that can be described through the apparent dispersion coefficient (Dap). the concentrations in the mobile and stationary phases are considered to be in equilibrium throughout the whole column. This assumption is valid as long as the number of theoretical plates in highresolution chromatography is larger than 102. The mass balance for component p in a differential control volume in the column is given in Eq. (1). The Langmuir competitive adsorption isotherm is used in the model (Teoh et al., 2001) that describes the ratio of component phases, as shown in Eq. (2).
2 Cm p x 2

Cm p t Cs p =

+F

Cs p t

+v

Cm p x

= Da p

p p

(1) (2)

a p Cm p 1 + b p Cm p

where a p, b p
Cm p

Langmuir isotherm coefficients of component p concentration of component p in the mobile phase 2

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Cs p

concentration of component p in the stationary phase axial dispersion coefficient of component p geometric constant, defined as the volume ratio of the stationary and mobile phases ( F = time mobile phase velocity column axial position porosity of the stationary phase
1 )

Da p F t v x

Note from Eq. 2 that the concentration of component p is independent of the concentrations of other components. The axial dispersion coefficient of component p (Dap) can be estimated from the number of theoretical plates of the column (Npp) as shown in Eq. (3).

Da p =

v Lcol 2 Np p

(3)

Npp is calculated from the Knox dimensionless equation shown in Eq. (4) that provides acceptable estimation according to Morgenstern (1998).
2 up + u p 10

3 h p = u1/ p +

(4a)

where
up = v.dp Dm p

p p

(4b) (4c)

hp =

Lcol Np p dp

where
dp Dmp hp Lcol Npp up

particle average diameter molecular diffusion coefficient of component p in the mobile phase reduced plate height column length number of theoretical plates reduced linear velocity

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There are several methods to obtain an approximate value of the diffusion coefficient (Dmp). Correlations have been presented by Young et al. (1980), Guiochon et al. (1994), and Teoh et al. (2001) among others for the determination of this parameter as a function of the solute molar volume. According to Young et al. (1980), specific volumes of proteins lie in the 0.69 - 0.78 cm3/g interval and can be represented by a Gaussian distribution with 0.73 cm3/g average. Equation (5) is used to predict the diffusion coefficient of more than 300 proteins (including the BSA) based on their molecular mass and specific volume of 0.73cm3/g. According to Guiochon et al. (1994) and Miyabe and Guiochon (2000), Eq. (5) generates very accurate results.

Dm p = 8.34.105 (

1/ 3 solv MW p

(5)

where MWp is the molecular mass of protein p (Da), solv is the viscosity of the solvent (Pa.s) and T is the absolute temperature (K). The velocity is expressed in terms of the length and retention time of an unretained component, or dead time
t0 that corresponds to the time required for the elution of the first component.
Lcol t0

v=

(6)

The initial and boundary conditions are given in Eqs. (7) to (9). Equation (7) describes the state of the column when the experiment begins, i.e. at this time the column has no contaminants. The boundary condition described by Eq. (8) is given in three time intervals. During the feed injection period the solute concentration of component p at the column inlet is equal to that of the feed sample, where CE is the injection profile that is usually rectangular in shape (CE = Cinjtinj). Finally, during product collection or conventional elution the pure solvent of the mobile phases is pumped into the column. The second boundary condition in Eq. (9) at the column outlet is the Danckwerts condition (Rice and Do, 1994).
Cm p ( 0, x ) = 0

(7)

0 Cm p (t , 0) = CE 0

0 t < tinj tinj t < te t te

initial injection elution

(8)

Cm p x
x = Lcol

=0

(9)

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3. Model implementation and case study

Chromatography models contain nonlinear partial differential equations whose solution requires numerical approaches. There are several alternatives to obtain numerical solutions, and two of them are mostly used in the solution of the chromatography models. These are the methods of finite differences and orthogonal collocation on finite elements. According to Guiochon et al. (1994), the method of finite differences could underestimate or overestimate the extent to which axial dispersion affects the profile of single component bands. A more accurate method is necessary to account for the elution profiles of multicomponent mixture, especially when the column efficiency is moderate. The finite element method presents this advantage, but the computational time is larger. There are many methods of finite elements, but the method of orthogonal collocation is most used to obtain of the elution profile (Guiochon et al., 1994). The orthogonal collocation method can solve problems with partial differential equations, particularly elliptical and parabolic. This method with domain [0, 1] (any [a, b] subdomain can be transformed in [0, 1]) is called global orthogonal collocation (Rice and Do, 1994). A variation of this method that is useful to handle problems with acute profiles is called orthogonal collocation on finite elements. This method divides the domain in several subdomains (elements) and orthogonal collocation is applied to each element. These elements are connected by imposing physical continuity conditions on the state variables and flows (Rice and Do, 1994). The equilibrium-dispersive model was implemented in the gPROMS platform (v. 2.3.0) - general PROcess Modeling System - software of modeling simulation and optimization processes from Process System Enterprise Ltd. (2004). Teoh et al. (2001) used a third-degree polynomial with 80 elements to predict the separation of an aromatic mixture. In the current work, the space is discretized with the method of orthogonal collocation on finite elements that makes use of a polynomial of 3rd degree with 200 elements. A protein mixture is used to validate the model using one of the chromatographic techniques applied, which were obtained by numerical experiments, to a previously study on the synthesis of protein purification processes (Vasquez-Alvarez and Pinto, 2004). The protein to be separated is Bovine Serum Albumin (BSA) from a mixture of four proteins (BSA - p1, Ovalbumin - p2, Thaumatin - p3 and Soybean Tripsin Inhibitor - p4) using an anion exchange column with Q-Sepharose FF matrix and buffer Bis-Tris propane at pH 7.0. Table 1 shows the parameters and conditions used in the case study. The model requires parameters that in the case of these proteins are not available in the literature. Hence, it becomes necessary to predict the protein separation based on physical property information (see Table 2) such as isoeletric points (pI), protein maximum capacity in the ion exchange and the dissociation constant protein-ion are required for the target protein and for the contaminants. In this case, the pI values shown in Table 2 were taken from Schmidt and Asenjo (1994). The non-competitive Langmuir isotherm coefficients for p1 were obtained from Garrido (1997) and those for the other mixture components (p2, p3 and p4) were estimated as functions of the isoelectric points (pI).

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Table 1. Initial conditions for protein mixture separation.

Symbol Cinjp dp Lcol F T tinj t0 solv

Parameter Sample concentration Particle diameter Column length Phase ratio Temperature Injection time Column dead time Viscosity of solvent

Units kg/m3 m m K s s mPa.s

Value 2.0 9.310-6 0.25 0.25 293 7.0 9.0 1.0

Reference Lienqueo et al. (1998) Bosma and Wesselingh (1998) Proposed Golshan-Shirazi et al. (1989) Guiochon et al. (1994) Proposed Proposed Guiochon et al. (1994)

Table 2. Parameter of the Langmuir non-competitive isotherm.

Protein p1 p2 p3 p4

pI 4.7 4.9 12.5 4.6

qmp (kg/m3) 130.0 124.7 48.9 132.8

kip (kg/m3) 0.117 0.112 0.044 0.119

Reference Garrido (1997) Estimate Estimate Estimate

In Table 2, the coefficients relate to those in (2) as follows: a p = qm p ki p and b p = 1 ki p . Parameters Dmp,
Npp and Dap are shown in Table 3 for each protein as well as the equations that were used for their calculation.

The molecular mass of the proteins is required to calculate the diffusion coefficient.
Table 3. Addional parameters for the case study.

Protein p1 p2 p3 p4 Reference

MWp (Da) 67000 43800 22200 24500 Lienqueo et al. (1998)

Dmp (m2/s) 6.0010-11 6.9110-11 8.6610-11 8.3810-11 Eq. (5)

Npp (-) 60 69 86 83 Eq. (4a 4c)

Dap (m2/s) 0.578710-4 0.503210-4 0.403710-4 0.418310-4 Eq. (3)

4. Computational results of the base case

The elution concentration profiles are presented in Figure 1. It is important to note that the dynamic behavior of the concentration generated from the formulated model is similar to the experimental results presented by Lienqueo et al. (1998) for anion exchange at pH 7.0 in the separation of the same mixture. The process eliminates almost all thaumatin (p3) and elutes the mixture (BSA, ovalbumin and SBTI). The process synthesis model (Vasquez-Alvarez and Pinto, 2003; Vasquez-Alvarez and Pinto, 2004) using the same technique also eliminated almost all thaumatin (initial concentration 2.0 mg/mL to 0.05 mg/mL); SBTI (p4) is also eliminated, which does not happen in the concentration profile shown in Figure 1.

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0.8 Cm (mg/mL) 0.6 0.4 0.2 p2

p1

0.15 0.1 p4 0.05 p3 0

0 0 10 20 t (min) 30 40 50

Fig. 1. Concentration profiles generated from the model.

In the concentration profiles shown in Figure 1 it can be observed that the elution of proteins p1, p2 and p3 takes place at almost the same time and reach similar heights (axis on the right hand side). Figure 2 shows a detailed view of Figure 1.

0.2 Cm (mg/mL) 0.15 0.1 0.05 0 6 7 8 t (min) 9 10

p1

p2

p4

Fig. 2. Detailed elution profiles for the example.

Note that the chromatographic curves, although having a similar behavior to the experimental ones, are very wide. With the purpose to analyze the quality of the simulated results in terms of the retention time, these must be expressed in dimensionless values (Kdp) for each component, such as in Lienqueo et al. (1998), Lienqueo (1999) and Vasquez-Alvareaz and Pinto (2004). The calculation of the dimensionless retention time for each component is denoted by Eq. (10):

Kd p =

Trp min Tin p '

p'

max Tf p min Tin p '

p'

{ }

p'

} }

(10)

where Trp is the time at which the concentration profile of protein p reaches its maximum height, Tinp indicates the initial time of the chromatographic curve of protein p with non-null concentration (Cmp = 10-6 mg/mL) and
Tfp is the final time in which the concentration profile in the elution of protein p has a significant concentration

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(Cmp = 10-3 mg/mL). The dimensionless time (Kdp) is determined considering the value of Trp of each protein p, the smallest Tinp and the largest Tfp values. The dimensionless retention times calculated from the equilibrium dispersive model for the example are shown in Table 4 for each one of the proteins. These indicate that Kdp1 is very similar to the experimental value (Lienqueo, 1999), i.e. that the isotherm coefficients (kip1) and (qmp1) represent a good approach. The values for the other proteins are similar, except for p4 that presents a considerable difference. These experimental values were obtained from a mixture of the same proteins with an anion exchange column of Q-20 (pores) at pH 7.0. Table 4 also shows results obtained from mathematical correlations of physicochemical properties by Lienqueo et al. (1998) that were used in a synthesis process model developed by Vsquez-Alvarez and Pinto (2004).
Table 4. Values of dimensionless retention times. Protein p1 p2 p3 p4 Reference Equilibrium dispersive 0.165 0.154 0.001 0.167 Calculated Kdp (-) models Experimental 0.17 0.18 0.00 0.28 Lienqueo (1999) Mathematical correlations 0.13 0.21 0.00 0.27 Lienqueo et al. (1998)

5. Parameter estimation
The objective of the analysis is to determine the effect of parameters on the elution profiles of the protein separation for the equilibrium dispersive model, which generates a variation of the base case. The detailed steps of the estimation process are omitted in this paper because of space restrictions. First, consider a more precise value for the phase ratio (F=0.385) obtained from the literature (Teoh et al., 2001). The larger value of the phase ratio corresponds to a smaller overal porosity of the column. Hence the time period it takes for the elution of the first component (dead time) is larger than that of the base case. The values for parameters Dmp, Npp and Dap estimated from correlations are shown in Table 5 for each protein that constitutes the mixture. Note that the number of theoretical plates (Npp) is larger than that of the base case and therefore the axial dispersion coefficient is smaller. The values of isotherm coefficients qmp and kip for each protein were adjusted to the experimental results of the dimensionless times and are presented in Table 5. Additionally, the simulation required the same initial conditions presented in Table 1, except for the injection time (tinj = 9.0 s) and the dead time (t0 = 11.0 s), whose values were adjusted. The orthogonal collocation on finite element methodwith the same parameters is used for the solution of this particular case. The simulated elution profile for the new set of parameters and initial conditions is shown in Figure 3. Note that protein p3 is eliminated and that there is a clear separation of p4; it can be also observed that curves p1 and
p2 elute later than on the base case.

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Table 5. Estimated parameters.

Protein p1 p2 p3 p4

MWp (Da)

67000 43800 22200 24500

Dmp (m2/s) 6.0010-11 6.9110-11 8.6610-11 8.3810-11

Npp (-) 73 84 104 101

Dap (m2/s)

0.38910-4 0.33810-4 0.27310-4 0.28110-4

qmp (kg/m3) 143.0 149.6 44.0 172.7

kip (kg/m3) 0.1053 0.0896 0.0484 0.0833

0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0

Cm (mg/mL)

p3

p1

p2

p4

10 20 30 40 50 60 70 80 90 100 110 120 t (min)

Fig 3. Concentration profiles for the variation of the base case.

The results in terms of the dimensionless retention times for this case are presented in Table 6. It is important to note that these values are very close to the ones obtained experimentally (Lienqueo, 1999) that are presented in Table 4. The purity levels obtained for the proteins are shown in Table 6 by calculating the intersection of the chromatographic curves for each protein. Table 6 clearly indicates the large sensitivity for the purity value of p1 and that this operation is sutitable to separate completely protein p3 and partially protein p1.
Table 6. Results for the variation of the base case. Protein p1 p2 p3 p4 Equilibrium dispersive Kdp (-) 0.17 0.18 0.0003 0.26 Purity (%) Base Case Variation 30.4 38.5 99.9 99.9 -

6. Conclusions
This paper presented a detailed phenomenological model, which is based on the equlibrium dispersive assumptions, for dynamic liquid chromatography column models in protein separation processes. Results indicate that the prediction of several parameters from the literature, in particular those for isotherms from isoelectric points, provide an initial estimate for the performance of chromatographic separation. Nevertheless, precision can only be attained with a detailed estimation of the main model parameters. Finally, such models can be applied to synthesize, design and operate optimally chromatographic bioprocesses.

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References
Bosma, J. C., Wesselingh, J. A. (1998). pH dependence of ion-exchange equilibrium of proteins. AIChE J. 44, 2399. Garrido, C. V. S. (1997). Modelacin y simulacin de procesos cromatogrficos de separacin de protenas. Undergraduate Thesis, University of Chile, Santiago, Chile (In Spanish). Golshan-Shirazi, S., Lin, B. C, Guiochon, G. (1989). Effect of mass-transfer kinetics on the elution of a binary mixture in nonlinear liquid chromatography. J. Phys. Chem. 93, 6871. Gu, T. (1995). Mathematical modelling and scale-up of liquid chromatography. Springer: Berlin. Gu, T., Tsai, G-J., Tsao, G. T. (1993). Modeling of Nonlinear Multicomponent Chromatography. Adv. Bioch. Eng. Biotech. 49, 45. Guiochon, G. (2002). Preparative Liquid Chromatography, Review. J. Chromatog. A. 965, 129. Guiochon, G., Golshan-Shirazi, S., Katti, A. M. (1994). Fundamentals of Preparaive and Nonlinear Chromatography. Academic Press: Boston, MA. Lienqueo, M. E. (1999). Desarrollo de un sistema experto para la seleccin racional de procesos de purificacin de protenas: optimizacin de criterios de seleccin de secuencias. PhD Thesis, University of Chile, Santiago, Chile, (In Spanish). Lienqueo, M. E., Salgado, J. C., Asenjo, J. A. (1998). Design of an expert system for selection of protein purification processes: comparison between different selection criteria. Comp. Appl. Biotech. CAB7, Osaka, Japan, 321. Miyabe, K., Guiochon, G. (2000). Morgenstern, A. S. (1998). Magazine 26, 46. Process System Enterprise Ltd. (2004). gPROMS Introductory User Guide v. 2.3.0, Rainbow Technologies, Inc: London. Rice, R. G., Do, D. D. (1994). Applied Mathematics and Modeling for Chemical Engineers. John Wiley & Sons, Inc: New York, NY. Schmidt, A. S., Asenjo, J. A. (1994). Modelling the partition behaviour of proteins in aqueous two-phase systems. In: Separations for Biotechnology III. London, 463. Teoh, H. K., Turner, M., Titchener-Hooker, N. E Sorensen, E. (2001). Experimental verification and optimization of a detailed dynamic high performance liquid chromatography column model. Comp. Chem. Eng. 25, 893. Vasquez-Alvarez, E., Pinto, J. M. (2003). A mixed integer linear programming model for the optimal synthesis of protein purification processes with product loss. Chem. Biochem. Eng. Q. 17, 27. Vasquez-Alvarez, E., Pinto, J. M. (2004). Efficient MILP formulations for the optimal synthesis of chromatographic protein purification processes. J. Biotech. 110, 295. Young, M. E., Carroad, P. A., Bell, R. L. (1980). Estimation of diffusion coefficients of proteins. Biotech. Bioeng. 22, 947. Kinetic study of the mass transfer of bovine serum albumin in anion-exchange Anal. chromatography. J. Chromatog. A. 866, 147. Optimization and comparison of different modes of preparative chromatography.

Acknowledgment
The authors acknowledge financial support from PADCT /CNPq under grant 62.0239/97 QEQ, and from VITAE under grant B-11487/10B006.

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