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Synthesis and Characterization of High-Affinity, Low-Molecular-Mass Displacers for Anion-Exchange Chromatography Nihal Tugcu,†,‡ Sun K. Park,§ J. A. Moore,§ and Steven M. Cramer*,† Departments of Chemical Engineering and Chemistry, Rensselaer Polytechnic Institute, Troy, New York 12180
In this paper, the synthesis and characterization of various low-molecular-mass anion-exchange displacers is described. A homologous series of displacers based on either triazine or phloroglucinol were synthesized. The displacer molecules were then characterized using the steric mass action (SMA) isotherm model and dynamic affinity lines resulting from this model. In addition to the dynamic affinity analysis, log P descriptors were calculated as well as several other molecular descriptors to characterize these molecules. The displacers were used to screen for their behavior on agarose-based Q Sepharose HP, hydrophilized poly(styrene-divinylbenzene)(PS-DVB-) based Source 15Q, and poly(methyl methacrylate)-based ToyoPearl Super Q-650S stationary-phase materials. This work demonstrates that aromaticity/hydrophobicity is very important in increasing the affinity of displacers for anion-exchange resins regardless of their backbone chemistry. The results also indicate that a benzene ring is superior to a triazine ring in increasing the affinity of these anionic displacers. In addition, the data indicate that the location of an aromatic ring in the core enables the molecule to approach the stationary phase in a flat geometry, thereby increasing the number of charges interacting with the stationary phase. Finally, the results of a dynamic affinity analysis and displacement experiment confirm that this class of displacers can be readily used for protein purification by anion-exchange displacement chromatography. 1. Introduction Ion-exchange displacement chromatography has been shown to be a promising technique for preparative protein separations.1-3 In addition, displacement chromatography of proteins has been successfully carried out on hydroxyapatite4-6 and hydrophobic-interaction and reversed-phase chromatographic systems.7,8 Various classes of displacers such as polyelectrolytes,9-13 polysaccharides,14,15 low-molecular-mass dendrimers,16 amino acids,17 and antibiotics18 have been identified for ion-exchange systems. The ability to use low-molecularmass displacers ( HPB-SO3Na over the entire range of delta values. A similar ranking was observed on the ToyoPearl material, whereas the order on the Sepharose material for the first group was HPBSO3Na > TA-PhSO3Na at the higher delta values, which correspond to low displacer concentrations. In addition, PG(EO1)-PhSO3Na, the results for which are not included in these figures because it did not elute from the
columns, can be assumed to have the highest affinity of the molecules in group 1 on all three resins. PG(EO1)-PhSO3Na exhibits a higher affinity than both of the other displacers in group 1. As seen in Table 2, PG(EO1)-PhSO3Na has a higher log P (i.e., is more hydrophobic) than TA-PhSO3Na. Furthermore, it has four benzene rings as compared to three benzene rings and one triazine ring. Thus, it is not surprising that it exhibits more affinity than TA-PhSO3Na. On the other hand, HPB-SO3Na has both higher hydrophobicity and more aromatic rings than PG(EO1)-PhSO3Na. One possible explanation for the higher affinity of PG(EO1)PhSO3Na is that the aromatic rings in PG(EO1)-PhSO3Na have a better chance of interacting with the stationary phase because of the increased flexibility.
Ind. Eng. Chem. Res., Vol. 41, No. 25, 2002 6489 Scheme 3. Synthesis Scheme for HPB-SO3Na
Table 2. Properties of the Displacers molecule
MW
HPB-SO3Na 1146.9 PG(E01)Ph-SO3Na 792.7 TA-PhSO3Na 663.5 PG(E01)-PSNa3 690.6 PG-PSNa3 558.5 TA-PSNa3 561.4 IC-PSNa3 561.4
Fchargea b_arb -6 -3 -3 -3 -3 -3 -3
42 24 24 6 6 6 0
log P number (o/w)c of ringsd a_acce 6.736 2.397 0.946 -1.350 -0.362 -2.804 -2.855
7B 4B 3B + 1T 1B 1B 1T 1
0 6 6 6 3 6 0
a Fcharge ) formal charge on the molecule. b b_ar ) number of aromatic bonds. c log P(o/w) ) octanol/water partition coefficient. d B ) benzene, T ) triazine. e a_acc ) number of hydrogen-bond acceptor atoms.
There are several possible explanations for the elevated affinity of the group 1 displacers. Cation-π interactions38 could play a role in all three resins. For the Source material, π-π interactions could also be playing a role because of the aromatic content of the resin (PS/DVB). This could also be the reason the equilibrium constants obtained on the Source 15Q resin were higher than those obtained on the other two resins evaluated in this study. As described above, the second group of displacers contained a single or no aromatic ring in their structure. The affinity order for the group 2 displacers on the Source 15Q material was PG-PSNa3 > IC-PSNa3 ) PG(EO1)-PSNa3 > TA-PSNa3 (Figure 1). On the Sepharose material, the order was PG-PSNa3> TA-PSNa3 > PG(EO1)-PSNa3 ) IC-PSNA3. On the ToyoPearl material, the affinity order of the group 2 displacers was PGPSNa3 > TA-PSNa3 ) PG(EO1)-PSNa3 > IC-PSNa3. These results indicate that the ToyoPearl and Sepharose
materials exhibited similar behaviors with respect to this set of displacers. In contrast, for the Source 15Q material, the order was different, with IC-PSNa3 exhibiting intermediate affinity in the group 2 displacers. For this group, PG-PSNa3 exhibited the highest affinity on all three resins. log P values indicate that PG-PSNa3 (log P ) -0.362) is more hydrophobic than the other molecules in this group, indicating that hydrophobicity plays a role in this system. As mentioned earlier, the only difference between PG-PSNa3 and TAPSNa3 is the type of core ring structure. These results indicate that benzene rings contribute significantly more affinity to anionic displacers than triazine rings. It also indicates that, even though TA-PSNa3 has more hydrogen-bonding capability than PG-PSNa3, the higher aromaticity of PGPSNa3 is the more important property. Further, the only difference between PG-PSNa3 and PG(EO1)-PSNa3 is the linker between the core benzene ring and the charged moieties at the termini. For PG(EO1)-PSNa3, there is an additional ethylene oxide on each linker that makes the molecule more flexible and more hydrophilic. Because PG(EO1)-PSNa3 has a lower affinity than PG-PSNa3, this indicates that hydrophobicity is more important than flexibility for this particular case. However, the results discussed above for the group 1 molecules PG(EO1)PhSO3Na3 and HPBSO3Na indicate that flexibility is also important. However, in that case, both molecules were sufficiently hydrophobic and contained a relatively large number of aromatic rings. These results taken together indicate that, when displacers have sufficient hydrophobicity/ aromaticity, flexibility can further enhance the affinity.
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Figure 1. Ranking of dynamic affinities of displacers and proteins on Source 15Q.
Figure 2. Displacement separation of 18 mg of β-lactoglobulin mixture using PG-PSNa3 as a displacer. Column, 50 × 5 mm i.d.; Source 15Q; carrier, 20 mM Tris-HCl + 55 mM NaCl; displacer, 5 mM PG-PSNa3; flow rate, 0.2 mL/min. Table 3. Linear SMA Parameters of the Displacers on Different Stationary Phases stationary phase
Q Sepharose HP
Source 15Q
ToyoPearl SuperQ-650S
displacer
ν
K
ν
K
ν
K
HPB-SO3Na TA-PhSO3Na PG-PSNa3 TA-PSNa3 PG(EO1)-PSNa3 IC-PSNa3
4.11 ( 0.39 2.22 ( 0.45 2.18 ( 0.17 2.81 ( 0.22 2.55 ( 0.22 2.54 ( 0.19
102.0 ( 13.43 29.00 ( 7.50 1.59 ( 0.17 0.416 ( 0.05 0.374 ( 0.07 0.352 ( 0.06
4.36 ( 0.49 1.9 ( 0.19 3.04 ( 0.08 3.02 ( 0.11 2.56 ( 0.15 3.07 ( 0.10
478.0 ( 52.5 169.0 ( 17.47 9.93 ( 1.28 1.18 ( 0.18 3.12 ( 0.50 3.21 ( 0.32
3.76 ( 0.18 1.67 ( 0.09 2.54 ( 0.12 2.69 ( 0.08 2.61 ( 0.13 2.7 ( 0.20
54.8 ( 8.70 83.7 ( 12.29 2.61 ( 0.80 0.39 ( 0.08 0.454 ( 0.11 0.040 ( 0.02
In general, the results indicate that the affinity of this class of displacers is relatively independent of the stationary-phase backbone chemistry. For all of the resins examined in this study, group 1 displacers had significantly higher affinities than group 2 displacers.
Clearly, aromaticity/hydrophobicity is playing an important role in increasing the efficacy of these anionic displacers. Interestingly, the agarose material exhibited a strong dependence on the aromatic content of the displacers. These results are in contrast to previously
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published results with cation-exchange displacers24 where agarose-based materials were relatively insensitive to the aromatic and hydrophobic contents of the displacers. Again, we believe that this is due to cation-π interactions that would not be present in cationexchange systems. In addition to obtaining SMA parameters for the various displacers, we also obtained SMA parameters for a set of model proteins on the Source 15Q stationary phase. As can be seen in Figure 1, where the bottom four dynamic affinity lines correspond to the model proteins used in this work, all of the displacers synthesized in this study had dynamic affinities greater than those of the model proteins. This result indicates that, according to the theory,22 these molecules should be able to act as effective displacers of these proteins over a wide range of conditions. As seen in Figure 2, displacement separation of β-lactoglobulin A and B was indeed successfully carried out using one of the synthesized molecules (PG-PSNa3) as a displacer. Conclusions In this paper, the synthesis and characterization of high-affinity low-molecular-mass displacers is reported for anion-exchange systems. Several conclusions can be drawn from the present work about the design of highaffinity anionic displacers. The common trend for all of the resins employed in this study was that the displacers with a high degree of aromaticity (increasing number of aromatic bonds) had relatively high affinities. This indicates that displacer candidates should not only contain several charged moieties to facilitate electrostatic interactions, but should also include aromatic rings. Specifically, these aromatic rings should be benzene rather than triazine rings. In addition, the characteristic charges obtained for these displacers indicate that most of the charges on these molecules actually interact with the stationary phase. This supports our hypothesis that locating the aromatic ring in the core enables the molecule to approach the stationary phase in a flat geometry, which would increase the number of charges interacting with the stationary phase. The results also indicate that locating additional aromatic rings at the periphery of the molecule close to the charged groups further increases the affinity. Thus, the combination of a high number of charges and appropriately located aromatic rings will generate highaffinity anionic displacers. Finally, as analyzed for the Source 15Q stationary phase, all of the displacers synthesized in this work can be readily employed for the purification of protein mixtures. The availability of very high-affinity anionic displacers now opens up the possibility of using these displacers at very low concentrations, which has the potential of resulting in extremely high-resolution separations of complex biological mixtures. This will be the subject of a future report. Acknowledgment The authors acknowledge NIH Grant GM 47372-04A2 in support of this research. The authors thank Ashley Thomas and Yelena Shapshaikhes for their help in performing the experiments. Literature Cited (1) Barnthouse, K. A.; Trompeter, W.; Jones, R.; Inampudi, P.; Rupp, R.; Cramer, S. M. Cation-Exchange Displacement Chromatography for the Purification of Recombinant Protein Therapeutics from Variants. J. Biotechnol. 1998, 66, 125.
(2) Shukla, A. A.; Hopfer, R. L.; Chakravarti, D. N.; Bortell, E.; Cramer, S. M. Purification of an Antigenic Vaccine Protein by Selective Displacement Chromatography. Biotechol. Prog. 1998, 14, 92. (3) Gerstner, J. A.; Morris, J.; Hunt, T.; Hamilton, R.; Afeyan, N. B. Rapid Ion-Exchange Displacement Chromatography of Proteins on Perfusive Chromatographic Supports. J. Chromatogr. A 1995, 695, 195. (4) Freitag, R.; Breier, J.; Displacement Chromatography in Biotechnological Downstream Processing. J. Chromatogr. A 1995, 691, 101. (5) Vogt, S.; Freitag, R. Comparison of Anion-Exchange and Hydroxyapatite Displacement Chromatography for the Isolation of Whey Proteins. J. Chromatogr. A 1997, 760, 125. (6) Kasper, C.; Breier, J.; Vogt, S.; Freitag, R. Protein displacement chromatography on hydroxy- and fluoroapatite columns. Bioseparations 1996, 6, 247. (7) Horvath, C.; Nahum, A.; Frenz, J. High-Performance Displacement Chromatography. J. Chromatogr. 1981, 218, 365. (8) Shukla, A. A.; Sunasara, K. M.; Rupp, R. G.; Cramer, S. M. Hydrophobic Displacement Chromatography of Proteins. Biotechnol. Bioeng. 2000, 68, 672. (9) Gerstner, J. A.; Cramer, S. M. Heparin as a Nontoxic Displacer for Anion-Exchange Displacement Chromatography of Proteins. BioPharm 1992, 5, 42. (10) Jayaraman, G.; Gadam, S. D.; Cramer, S. M. Ion-Exchange Displacement Chromatography of ProteinssDextran-Based Polyelectrolytes as High-Affinity Displacers. J. Chromatogr. 1993, 630, 53. (11) Torres, A. R.; Peterson, E. A. Purification of Monoclonal Antibodies by Complex-Displacement Chromatography on CM Cellulose. J. Chromatogr. 1990, 499, 47. (12) Jen, S.-C. D.; Pinto, N. G. Use of the Sodium Salt of Poly(vinylsulfonic acid) as a Low Molecular Weight Displacer for Protein Separations Ion-Exchange Displacement Chromatography. J. Chromatogr. 1990, 519, 87. (13) Jen, S.-C. D.; Pinto, N. G. Dextran Sulfate as a Displacer for the Displacement Chromatography of Pharmaceutical Proteins. J. Chromatogr. Sci. 1991, 29, 478. (14) Lee, A. L.; Liao, A. W.; Horvath, C. Tandem Separation Schemes for Preparative High-Performance Liquid Chromatography of Proteins. J. Chromatogr. 1988, 443, 31. (15) Liao, A. W.; Horvath, C. Purification of Beta-Galactosidase by Combined Frontal and Displacement Chromatography. Ann. N. Y. Acad. Sci. 1990, 589, 182. (16) Jayaraman, G.; Li, Y.-F.; Moore, J. A.; Cramer, S. M. IonExchange Displacement Chromatography of Proteins Dendritic Polymers as Novel Displacers. J. Chromatogr. A 1995, 702, 143. (17) Kundu, A.; Vunnum, S.; Jayaraman, G.; Cramer, S. M. Protected Amino Acids as Novel Low-Molecular-Weight Displacers in Cation-Exchange Displacement Chromatography. Biotechnol. Bioeng. 1995, 48, 452. (18) Kundu, A.; Vunnum, S.; Cramer, S. M. Antibiotics as LowMolecular-Mass Displacers in Ion-Exchange Displacement Chromatography. J. Chromatogr. A 1995, 707, 57. (19) Kundu, A.; Cramer, S. M. Low-Molecular-Weight Displacers for High-Resolution Protein Separations. Anal. Biochem. 1997, 248, 111. (20) Kundu, A.; Barnthouse, K. A.; Cramer, S. M. Selective Displacement Chromatography of Proteins. Biotechnol. Bioeng. 1997, 56, 119. (21) Shukla, A. A.; Barnthouse, K. A.; Bae, S. S.; Moore, J. A.; Cramer, S. M. Synthesis and Characterization of High-Affinity, Low Molecular Weight Displacers for Cation-Exchange Chromatography. Ind. Eng. Chem. Res., 1998, 37, 4090. (22) Shukla, A. A.; Barnthouse K. A.; Bae, S. S.; Moore, J. A.; Cramer, S. M. Structural Characteristics of Low-Molecular-Mass Displacers for Cation-Exchange Chromatography. J. Chromatogr. A 1998, 814, 83. (23) Tugcu, N.; Bae, S. S.; Moore, J. A.; Cramer, S. M. Stationary Phase Effects on the Dynamic Affinity of Low-Molecular-Mass Displacers. J. Chromatogr. A 2002, 954, 127. (24) Shukla, A. A.; Bae, S. S.; Moore, J. A.; Cramer, S. M. Structural Characteristics of Low-Molecular-Mass Displacers for Cation-Exchange Chromatography II. Role of the Stationary Phase. J. Chromatogr. A 1998, 827, 295.
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(25) Stahlberg, J.; Jonsson, B.; Horvath, C. Combined Effect of Coulombic and Vanderwaals Interactions in the Chromatography of Proteins. Anal. Chem. 1992, 64, 3118. (26) Roth, C. M.; Lenhoff, A. M. Electrostatic and Vanderwaals Contributions to Protein AdsorptionsComputation of Equilibrium Constants. Langmuir 1993, 9, 962. (27) Roth, C. M.; Unger, K. K.; Lenhoff, A. M. Mechanistic Model of Retention in Protein Ion-Exchange Chromatography. J. Chromatogr. A 1996, 726, 45. (28) Barron, R. E.; Fritz, J. S. Effect of Functional-Group Structure on the Selectivity of Low-Capacity Anion-Exchangers for Monovalent Anions. J. Chromatogr. 1984, 284, 13. (29) Masuda, T.; Nishimura, Y.; Tonegawa, M.; Kitahara, K.; Arai, S.; Yamashita, J.; Takai, N. High-Performance Liquid Chromatographic Separation of Carbohydrates on Stationary Phases Prepared from Polystyrene-Based Resin and Tertiary AminessEffect of Chemical Structure of Anion-Exchange Sorbents. J. Chromatogr. A 1999, 845, 401. (30) Staby, A.; Jensen, I. H. Comparison of Chromatographic Ion-Exchange Resins II. More Strong Anion-Exchange Resins. J. Chromatogr. A 2001, 908, 149. (31) Tugcu, N.; Mazza, C. B.; Breneman, C. B.; Sanhgvi, Y. S.; Cramer, S. M. High Throughput Screening and Quantitative Structure Efficacy Relationship Models for Designing Displacers for Antisense Oligonucleotide Purification in Anion-Exchange Systems. Sep. Sci. Technol. 2002, 37, 1667.
(32) Shukla, A. A.; Deshmukh, R. R.; Moore, J. A.; Cramer, S. M. Biotechnol. Prog. 2000, 16, 1064. (33) Tugcu, N.; Deshmukh, R. R.; Sanhgvi, Y. S.; Moore, J. A.; Cramer, S. M. Purification of an Oligonucleotide at High Column Loading by High-Affinity, Low-Molecular-Mass Displacers. J. Chromatogr. A 2001, 923, 65. (34) Kril, M. B.; Fung, H. L. Influence of Hydrophobicity on the Ion-Exchange Selectivity Coefficients for AromaticsAmines. J. Pharm. Sci. 1990, 79, 440. (35) Leach, A. R. Molecular Modelling Principles and Applications; Longman: London. 1998. (36) Brooks, C. A.; Cramer, S. M. Steric Mass-Action IonExchangesDisplacement Profiles and Induced Salt Gradients. AIChE J. 1992, 38, 1969. (37) Boschetti, E. Advanced Sorbents for Preparative Protein Separation Purposes. J. Chromatogr. A 1994, 658, 207. (38) Dougherty, D. Cation-π Interactions in Chemistry and Biology: A New View of Benzene, Phe, Tyr, and Trp. Science 1996, 271, 163.
Received for review April 5, 2002 Revised manuscript received September 9, 2002 Accepted September 10, 2002 IE020255G
