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Ind. Eng. Chem. Res. 1998, 37, 4090-4098
Synthesis and Characterization of High-Affinity, Low Molecular Weight Displacers for Cation-Exchange Chromatography Abhinav A. Shukla,† Sung Su Bae,‡ J. A. Moore,‡ Kristopher A. Barnthouse,† and Steven M. Cramer*,† Department of Chemical Engineering and Department of Chemistry, Rensselaer Polytechnic Institute, Troy, New York 12180
This paper describes the synthesis and characterization of novel low molecular weight displacers for protein purification in cation-exchange systems. A series of displacers based on the pentaerythritol geometry are examined to identify high-affinity, low molecular weight displacers. The steric mass action (SMA) model is employed to evaluate the relative affinities of these displacers. A linear gradient method is described for the measurement of SMA model parameters of molecules. This work demonstrates that, in addition to the number of charges on a displacer molecule, the presence of aromatic moieties can have a profound effect on affinity. In particular, the placement of aromatic groups near the surface of a molecule is shown to result in significant increases in displacer efficacy. Finally, the efficacy of these displacers is examined in displacement experiments using a model protein. These results indicate that nonspecific interactions can play a major role in determining the affinity of a molecule for ion-exchange systems. Introduction Displacement chromatography has attracted significant attention over the past decade for its potential as a high-resolution/high-throughput purification process.1-5 Recent reports have demonstrated the efficacy of displacement chromatography for the purification of proteins from industrial process streams.6,7 Conventionally, large polyelectrolytes have been employed as displacers for ion-exchange systems.8-11 Jen and Pinto12 have used a 2 kD molecular weight poly(vinyl sulfonic) acid as a displacer. An important advance has been the discovery that still lower molecular weight displacers can also be employed as effective displacers.13 A variety of low molecular weight displacers have been identified, including protected amino acids,13 dendrimers,14 and antibiotics.15 Furthermore, low molecular weight displacers have been successfully employed for high-resolution separations.16 Low molecular weight displacers have significant operational advantages compared with large polyelectrolyte displacers and have generated significant interest from the industry. First and foremost, if there is any overlap between the displacer and the protein of interest, these low molecular weight materials can be readily separated from the purified protein during subsequent downstream processing involving size-based purification methods. The relatively low cost of synthesizing low molecular weight displacers can be expected to significantly improve the economics of displacement chromatography. Furthermore, the saltdependent adsorption behavior of these low molecular weight displacers greatly facilitates column regeneration. The use of low molecular weight displacers also opens up the possibility of performing selective displacement chromatography.6,17 * Author to whom all correspondence should be sent. Email:
[email protected]. Telephone: 518-276-6198. † Department of Chemical Engineering. ‡ Department of Chemistry.
Although low molecular weight displacers have been successfully employed for protein purification in ionexchange systems, to date these displacers have possessed moderate affinities and have thus been unable to displace highly retained biomolecules. Clearly, to broaden the scope of this technique it is necessary to develop high-affinity, low molecular weight displacers. Previous work with dendritic molecules based on the pentaerythritol geometry14 with molecular weights ranging from 500 to 5000, indicated that increasing the number of charges from 4 to 36 had minimal effect on the affinity of these displacers. In this paper, several novel displacers containing aromatic moieties are synthesized and evaluated for their efficacy as protein displacers. Theory The Steric Mass Action (SMA) model18 has been shown to successfully predict complex behavior in ionexchange chromatography19-23 and has been adopted for methods development for displacement chromatography.6,9,13-17 The SMA model involves three parameters: the characteristic charge (ν), which is the average number of sites that a molecule interacts with on a surface; the equilibrium constant (K); and the steric factor (σ), which is the average number of sites on the surface that are sterically shielded by the molecule. The equation for the SMA isotherm is:
C)
( )(
)
Cs Q K Λ - (ν + σ)Q
ν
(1)
where C and Q are the solute concentrations in the mobile and stationary phases respectively, Cs is the background salt concentration, Λ is the total ionic bed capacity, and ν, σ, and K are the SMA parameters. To date, isocratic retention data have been employed to evaluate the SMA equilibrium parameters for modeling retention on ion-exchange chromatography.24 To
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Ind. Eng. Chem. Res., Vol. 37, No. 10, 1998 4091
obtain the linear SMA parameters (ν and K) directly from gradient experiments, one can employ the technique of Parente and Wetlaufer25 with minor modification. Equation 2 relates a solute retention volume (Vg) to initial and final carrier salt concentrations (xi, xf), the gradient volume (VG), the column dead volume (Vm), and the SMA linear parameters (ν and K). In this equation, β is the column porosity and Λ the total ionic capacity of the stationary phase.
Vg )
{[
xν+1 + i
]
VmKβΛν(ν + 1)(xf - xi) VG
1/(ν+1)
}
- xi × VG
(xf - xi)
(2)
This expression can be employed with at least two gradient experiments to solve for ν and K (using a leastsquares fit). This gradient technique enables a rapid evaluation of the linear SMA parameters for both displacers and proteins. In addition, for displacers, it is often desirable to obtain an independent measure of the induced salt gradient produced during a frontal experiment. Once the parameters are obtained, they can be employed to determine the dynamic affinity of a given molecule using the relation:
λ)
1/ν
(K∆)
(3)
where λ is the dynamic affinity and ∆ ) Q/C. Taking logarithms of both sides of eq 3 yields an expression for the dynamic affinity line:
log K ) log ∆ + ν log λ
(4)
Thus a plot of log K versus ν (dynamic affinity plot)26 defines two regions: a region below the dynamic affinity line in which all solutes have a lower dynamic affinity than the given molecule (and thus cannot displace it) and a region above the affinity line in which all solutes can displace the given molecule. In this research, a dynamic affinity plot is employed to compare the efficacies of the synthesized displacers. Experimental Section Materials. A strong cation-exchange column (SCX, 8-µm particle size, 100 × 5 mm i.d.) was obtained from Waters Corp. (Milford, MA). A C3 reversed-phase column (Zorbax, 4.6 × 250 mm) was obtained from BTR Separations (Wilmington, DE). Lysozyme, R-chymotrypsinogen A, sodium monobasic phosphate, and sodium dibasic phosphate were purchased from Sigma (St. Louis, MO). Pentaerythrityl tetrabromide, p-toluenesulfonyl chloride, potassium hydride, N,N-dimethylethanolamine, benzyl chloride, sodium bromide, iodomethane, triethyl orthoacetate, pyridinium p-toluene sulfonate, potassium hydroxide, benzene sulfonyl chloride, sodium azide, and lithium aluminum hydride were from Aldrich (Milwaukee, WI). Dipentaerythritol was from Perstorp (Toledo, OH). PETMA4 [pentaerythrityl trimethylammonium (4)], DPE-TMA6 [dipentaerythrityl trimethylammonium (6)], Ph-TMA6 [phenyl dipentaerythrityl trimethylammonium (6)], and PE-DMABzCl4 [pentaerythrityl benzyl dimethylammonium (4)] were synthesized as described in the Procedures Section.
Apparatus. All gradient and isocratic experiments were carried out using a Pharmacia FPLC system (Pharmacia Biotech, Uppsala, Sweden) consisting of two P-500 pumps controlled by a LCC-500 Plus controller and connected to a Spectroflow 757 absorbance detector (Kratos, Ramsey, NJ). Data acquisition and processing were carried out using a 820 Maxima chromatography workstation (Waters, Milford, MA) and a Millenium 2010 chromatography workstation (Waters). Fractions of the column effluent were collected using an LKB 2212 Helirac fraction collector (LKB Bromma, Sweden). 1H and 13C nuclear magnetic resonance (NMR) spectra were recorded on a 500-MHz Varian Unity-500 spectrometer. Infrared (IR) spectra were obtained with a Perkin-Elmer Paragon 1000 FT-IR. Melting points were determined on a Thomas-Hoover Meltemp apparatus and are uncorrected. Elemental analyses were performed by Galbraith Laboratories, Inc. (Knoxville, TN). Procedures. A. Displacer Analysis. Online UV detection was employed to detect PhTMA6 and PEDMABzCl4 in the column effluent during gradient elution. For displacers with no significant UV absorbance above 237 nm (i.e., PETMA4 and DPE-TMA6), fractions of the column effluent were collected and analyzed using a reversed-phase chromatographic assay. A linear gradient from 0 to 10% (v/v) acetonitrile in 20 min was run on the C3 column with UV detection at 215 nm. B. Determination of Displacer SMA Parameters. The characteristic charge (ν) was determined as an average value from the induced salt gradients produced during nonlinear frontal experiments24 (using displacer concentration of 5 and 10 mM and a pH 6, 20 mM phosphate buffer). The same experiments also furnished the breakthrough volumes of the displacer that were used to calculate the steric factor (σ). The equilibrium constant (K) was fit to the retention volumes of the displacers (corrected for the instrument dwell volume) during linear gradient elution by eq 2. Linear gradients from 0 to 2 M NaCl (in 20 mM, pH 6 phosphate buffer) using 20 to 60 column volumes were employed. A minimum of three gradient lengths were employed to estimate each equilibrium constant. C. Determination of Protein SMA Parameters. The characteristic charge (ν) and the equilibrium constant (K) were fitted to the retention volumes of the proteins during linear gradient elution by eq 2. A minimum of three gradient lengths were employed to estimate these parameters. Gradient slopes were selected such that the elution volume of the proteins varied between 4 and 20 column volumes. D. Synthesis of PETMA4. PETMA4 was prepared by the method presented in Figure 1. The first step involved the synthesis of PE-DMA4. First, 5.2 g (0.13 mol) of potassium hydride was washed twice with hexane under an argon atmosphere and 100 mL of dimethyl formamide (DMF) was added. The mixture was then cooled to 0 °C, and a solution of 10.7 g (0.12 mol) of N,N-dimethylethanolamine in 100 mL of DMF was added in a dropwise manner and stirred at room temperature for 3 h. Subsequently, a solution of 7.8 g (0.02 mol) of pentaerythrityl tetrabromide in 100 mL of DMF was added in a dropwise manner and the mixture was heated at 80 °C for 8 h. The temperature was raised to reflux for 24 h, and the resulting mixture cooled to 50 °C and poured into 300 mL of ice water. The solvents were removed on a rotary evaporator, the
4092 Ind. Eng. Chem. Res., Vol. 37, No. 10, 1998
Figure 1. Synthesis schemes for PETMA4 and PE-DMABzCl4.
residue was extracted with 800 mL of ethyl ether in several portions, and the combined extracts were dried over MgSO4. The drying agent was removed by filtration, the ether was evaporated, and the crude product was distilled under reduced pressure (
