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High-performance ion-exchange chromatography of proteins: The current status
ANALYTICAL
BIOCHEMISTRY
126, 1-7 (1982)
REVIEW High-Performance
Ion-Exchange Chromatography The Current Status’
of Proteins:
FRED E. REGNIER Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907 Received March 5, 1982 Ion-exchange chromatography of proteins and peptides has been most successfully achieved historically on hydrophilic gel matrices, The poor mechanical strength of these organic gels has necessitated the development of new supports for high-performance separations. Highperformance supports are of three types: totally inorganic, totally organic, and composite inorganic-organic materials. Several ionic species such as diethylaminoethyl ethanol and polyethylene imine have been used as stationary phases with similar results. Pore-diameter selection has been shown to be important in both resolution and loading capacity. Capacity is maximum for proteins of 50 to 100 kilodaltons on 300~A-pore-diameter supports. Maximum resolution of high-molecular-weight species also requires macroporous supports. Interestingly, column length is of minor importance in the resolution of proteins. Columns of 5-cm length have approximately the same resolution as those of 30-cm length. Application of high-performance ion-exchange chromatography to a variety of protein mixtures has now been reported. These supports generally give recoveries of enzyme activity equivalent to the classical supports.
A major limitation of conventional geltype ion-exchange media is their lack of mechanical strength. It is obvious that the high-mobile-phase velocities used in highperformance liquid chromatography (HPLC) require packing materials that are rigid. Interestingly, the first rigid ion-exchange supports for proteins were prepared before diethylaminoethyl (DEAE)- and carboxymethyl (CM)‘-cellulose. In 1954, Boardman (1,2) prepared a cation exchanger in a two-step process consisting of styrene polymerization on the surface of diatomaceous earth followed by sulfonation of the polystyrene matrix. This strong cation exchanger was used in the chromatography of chymotrypsinogen
and cytochrome c. Unfortunately, the very hydrophobic nature of polystyrene causes an irreversible adsorption of many proteins and limits the utility of this packing in protein separations. More recently, Eltekov (3) prepared rigid anion-exchange packings by derivatizing the surface of controlled porosity glass with aminopropyltrimethoxy silane (APS). The resulting weak anion exchanger separated proteins but showed a high degree of nonspecific adsorption. This undesirable property is probably the result of residual silanols on the support surface. When an inorganic surface is exposed to this silylating agent, two types of bonding may occur simultaneously: (i) siloxane bond formation at the surface and (ii) ion pairing of the APS amino groups with surface silanols. Siloxane bond formation and the concomitant sequestering of surface silanol is inhibited by ion pairing. The undesirable properties of packings with a high level of surface silanols have been noted repeatedly (4-7). The propensity of APS monomer to form
’ This is Journal Paper No. 8930 from the Purdue University Agricultural Experiment Station. 2 Abbreviations used: CM, carboxymethyl; APS, aminopropyltrimethoxy silane; HPIEC, high-performance ion-exchange chromatography; PEI, polyethylenimine; SEC, size-exclusion chromatography; BSA, bovine serum albumin.
1
0003-2697/82/15000
1-07$02.00/O
Copyright Q 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
2
FRED E. REGNIER
siloxane polymers in the presence of traces of water is another property of triethoxysilanes that has bearing on the quality of ionexchange coatings (8). Unfortunately, these siloxane polymers are not completely stable when eluted with thousands of volumes of aqueous buffer. Aminopropyl silane monomer gradually leaks from the surface of inorganic supports. The erosion of ion-exchange stationary phase causes both the ionexchange capacity and separation characteristics of columns to change with aging. Chang (9,10) found that thin surface layers of organic polymers produce far more stable ion exchangers than organosilane monomer bonded phases. Both epoxy and polyamine polymers produced packings that behaved like the conventional gel-type ion-exchange packings used in the chromatography of proteins but achieved separations in less than 30 min. Based on these studies it was concluded (10) that microparticulate inorganic supports coated with a thin film of ion-exchanging organic polymer had a substantial advantage in protein separations over both gel-type and organosilane monomer ion-exchange packings. PACKING MATERIALS
The extensive literature on both conventional gel-type ion-exchange resins for proteins and HPLC packings indicate that the ideal HPIEC packing for proteins should be (i) mechanically stable to mobile phase velocities of 1 mm/s, (ii) completely hydrophilic, (iii) of high ion-exchange capacity, (iv) chemically stable over a broad pH range, (v) available in 5 to IO-pm particle size, (vi) available in pore diameters from 300 to 1000 A, (vii) spherical, (viii) of a pore volume between 0.5 and 1.O ml/g, (ix) easy to pack, and (x) inexpensive. No packing material available at the present time has all of these prop erties. Available HPIEC materials for proteins are of three types: (i) organic resins, (ii) composite inorganic-organic matrices, and (iii) inorganic supports with a thin surface layer
of organic bonded phase. More experience with HPIEC is necessary to judge which of these materials is superior. Organic Resins The only known organic ion-exchange resin that can be classed as an HPLC packing for proteins is the polymethacrylate-based Spheron (11) material of Lachema. Separation of proteins on both the CM and DEAE versions of Spheron have been reported by Mikes et al. (12,13). Unfortunately, these ion exchangers are not commercially available in less than 20-pm particle size. The weak cation-exchange resin Bio-Rex 70 is also useful in protein separations but is only available in greater than 40-pm particle size ( 1,14- 16). Van der Wal (17) has examined the utility of several nonrigid ion exchangers in the fractionation of cytochrome c and its derivatives by HPIEC. Cation exchangers with a polystyrene-divinylbenzene or cellulose matrix were generally found to be unsatisfactory. Polymethacrylate matrices had much greater utility. In general, retention increased as either pH or ionic strength was decreased. It was reported that temperature had minimal effect on retention of derivatized cytochrome c. Composite Organic-Inorganic
Packings
Vanecek and Regnier (18) have recently reported the preparation of weak anion-exchange packings with polymer layers 100 A thick in lOOO- and 4000-A pores. Since the whole polymer layer exchanged ions and was permeable to small molecules, the ion-exchange capacity of the packings was much higher than that of a silica support with a surface monolayer of ion-exchange groups. After heavy polymer layers in the silica-support pores were bonded, the functional porosity to macromolecules was still greater than 600 A with an ion-exchange capacity of 300 meq/g. The function of the inorganic support was to provide a rigid matrix within which gel-type ion-exchangers could survive the pressure of a high-performance system.
CHROMATOGRAPHY
The principal advantage of packings with such large pores is said to be in the analysis of proteins exceeding 10’ daltons. Determination of the ultimate utility of these composite materials will require much more testing Surface-Modified
Inorganic
Packings
The most widely used technique for the preparation of HPIEC packings for proteins is through the application of a 20- to 30-A polymer layer to the surface of inorganic sup ports. Ion-exchange groups are either coupled to the surface (i) during the polymerization reaction, (ii) after the polymer layer has been deposited, or (iii) as preformed ionic polymers. An example of the first technique may be found in Chang’s preparation of an inorganic DEAE ion exchanger ( 19). Deposition of diethylaminoethanol and a multifunctional oxirane on the surface of a glycidoxypropylsilane-bonded phase support followed by polymerization caused both DEAE and glyceryl residues to become incorporated into the epoxy polymer formed. The DEAE groups served the function of partitioning proteins electrostatically while the glyceryl residues anchored the coating to the surface at multiple sites. Gupta (20) has reported a second synthetic route to polymeric ion-exchange coatings. The first step in this process is the bonding of a simple organosilane monomer with a reactive organic functional group to the surface of a silica support. This silylation reaction is followed by the covalent attachment of polyethylenimine (PEI) at multiple sites to the silica support through functional groups on the organosilanes. This PEI packing may either be used directly as a weak anion exchanger, further derivatized with oxiranes to form a tertiary amine ion exchanger, or reacted with acid anhydrides (succinic or diglycolic) to produce weak cation exchangers. A single synthetic route is used in the preparation of both anion- and cation-exchange packings. A third type of HPIEC packing has been
3
OF PROTEINS
described by Alpert (2 1). In this process, PEI is first adsorbed onto the surface of inorganic supports as a monolayer and then crosslinked into a thin polyamine skin with multifunctional oxiranes. This crosslinked coating is adsorbed to the surface at many sites and held so tightly that it cannot be desorbed even under severe conditions. The high surface density of amino groups completely titrates all surface silanols and prevents them from interacting with proteins. In 1978, SynChrom introduced the first commercial HPIEC supports for proteins. These weak anion exchangers had a polyamine-bonded phase that produced separations similar to DEAE-cellulose. Although the materials were available in lOO-, 300-, 500-, and 1OOO-A-pore-diameter sizes, the 300-A-pore-diameter packing was the most widely used because of its greater loading capacity. This weak anion exchanger was followed by a weak cation-exchanging CM support in 1982. Pharmacia and Toya Soda Corporation also introduced complete lines of HPIEC columns for proteins in 1982. Although these columns are so new that few people have examined them, they are reported to give separations equivalent to the conventional CM and DEAE columns. It should also be noted that the strong cationexchange material from Whatman, Partisil SCX, has been used effectively in the separation of low-molecular-weight proteins. PORE-DIAMETER
EFFECTS
The ratio of support pore diameter to molecular size can contribute to both the resolution and loading capacity of a column. Resolution Retention in ion-exchange chromatography is controlled by two independent phenomena: (i) the inherent size-exclusion contribution from differential penetration by solutes of macroporous matrices and (ii) electrostatic partitioning at the surface of the ion exchanger. It has been observed in sizeexclusion chromatography (SEC) that as sol-
4
FRED E. REGNIER
ute dimensions approach those of the sup port pores, severe limitations of molecular diffusion occur within the pores (22). The net effect of this phenomenon is resistance to mass transfer between the stationary and mobile phases and a concomitant loss of resolution. The inherent SEC properties of HPIEC packings cause this same bandspreading phenomenon to occur as solute size approaches pore diameter. These porediameter limitations of stagnant mobile-phase mass transfer are easily overcome in HPIEC by using larger pore-diameter packings (18). The optimum relationship between pore diameter and solute size has not been determined. Preliminary studies would indicate that 300-A-pore-diameter packings give less than optimum resolution with proteins greater than lo5 daltons. Loading Capacity Because ion-exchange chromatography is a surface-directed process, it would seem that ion-exchange capacity should be proportional to the surface area of a packing. Actually, not all of the surface of a packing is available to macromolecules. Greater than 95% of the surface area of a porous support is inside the pore network, and a molecule must be able to penetrate the pore matrix to reach the surface. The surface area (AJ available for ion-exchange partitioning is expressed by the equation A, = A() + K&i where A0 is the surface area on the outer surface of particles in a bed, Ai is the internal surface area of the pores of particles in the bed, and K, is a surface accessability coefficient. The term K, indicates the fraction of the internal surface available for ion-exchange adsorption and thus ranges from 0 to 1. Obviously, K, will be different for each molecular species and approach zero as molecular dimensions approach the dimensions of pores. For molecules such as hemoglobin and bovine serum albumin (BSA), 250- to 300-A-pore-diameter packings have the
greater value of KJi and therefore give the highest ion-exchange capacity. A BSA loading capacity of 100 mg/g of packing is common (23). Smaller proteins such as carbonic anhydrase exhibit highest loading capacity on 100-A-pore-diameter packings as opposed to proteins of several hundred thousand kilodaltons that require 500- to 1000-A pores for maximum loading. For both loading capacity and resolution, 250- to 300-A-pore-diameter ion-exchange packings are probably of broadest utility because they bracket the 50- to 100~kilodalton range of proteins. A 0.41 X 25-cm column will show no signs of overloading with up to 10 mg of ovalbumin (23). Under overloading conditions, the column will still separate most impurities in an ovalbumin sample even at a 50-mg injection. COLUMN LENGTH
It has been determined that column length plays a minimal role in resolution of proteins (23). Resolution on 5-cm columns is almost equivalent to that of 25-cm columns. These small columns have some definite advantages in analytical work such as (i) the elution of proteins in a smaller elution volume and therefore a more concentrated form, (ii) up to 6 times greater sensitivity because eluants are more concentrated, (iii) fewer problems with underloading columns, (iv) lower operating pressures, (v) columns last longer, and (vi) columns are cheaper. The disadvantages of short (~5 cm) columns are that maximum loading capacity is a few milligrams of protein and the small elution volumes promote extracolumn band spreading in some equipment. Both low dead-volume connecting tubing and flow cells are essential. MOBILE-PHASE
VELOCITY
The influence of mobile-phase velocity on band spreading and resolution has been treated in depth by Giddings (24) and is discussed in a number of books (25-27). The Giddings treatment indicates that as the diffusion coefficient of a molecule decreases, its
CHROMATOGRAPHY
ability to difke into and out of support pores decreases accordingly. Thus, as molecular weight increases, mass transfer problems are expected to escalate because of the decreasing diffusion coefficients. Obviously increasing mobile-phase velocity in a column TABLE
OF PROTEINS
5
aggravates the mass transfer problem. It has been calculated that to obtain equivalent column efficiency, mobile-phase velocity must be 10 times slower with a molecule of 70 kilodaltons than one of several hundred daltons (28). Practically speaking, this means 1
PROTEINSPURIFIEDBYHIGH-PERFORMANCEION-EXCHANGECHROMATOGRAPHY
Protein Lactate dehydrogenase isoenzymes Creature kinase isoenzymes Alkaline phosphatases Hexokinase &enzymes Arylsulfatase isoenzymes Hemoglobins
Cytochrome c Mozyme Myoglobin Soybean trypsin inhibitor Interferon Lipoxygenase Trypsin Chymotrypsinogen Immunoglobulin Ovalbumin
G
Albumin Apolipoproteins Adenylsuccinate synthetase Insulin &Lactoglobulin Carbonic anhydrase Monoamine oxidase
Column
Type of ion exchanger
DEAE-glycopha& SynChropak AX300e DEAE-glcyophase SynChropak AX300 DEAE-glycophase SynChropak AX300 DEAE-glycophase SynChropak AX300 DEAE-glycophase IEX 545 DEAE’ IEX 535 CM’ Bio-Rex 70g CM-polyamide CM-polyamide CM-polyamide IEX 535 CM CM-glycophased Partisil SCX* SynChropak AX300 DEAE-glycophase SP-glycophase IEX 535 CM SynChropak AX300 SynChropak AX300 IEX 545 DEAE SynChropak AX300 DEAE-Glycophasc IEX 545 DEAE SynChropak AX300 SynChropak AX300 Partisil SCX IEX 535 CM Partisil SCX Partisil SCX SynChropak AX300
WAX” WAX WAX WAX WAX WAX WAX WAX WAX WAX wcx* wcx wcx wcx wcx wcx wcx SCX’ WAX WAX sex wcx WAX WAX WAX WAX WAX WAX WAX WAX sex wcx sex sex WAX
References (10,19,30,31,33) (30) (31-34) (3Q35) (36) (37) (38) (39,41,43,45) (l&19) (44) (44) (42)
(20) (20) (20) (46) (19) (48)
(18) (19) (19) (46) (54) (23) (47) (23) (19) (46) (5655) (W (49) (46) (49) (49) (51)
a WAX designates weak anion exchanger. * WCX designates weak cation exchanger. ’ SCX indicates strong cation exchanger. d DEAE and CM-glycophase are products of Pierce Chemical Company, Rockford, Illinois. e SynChropak AX300 is produced by SynChrom, Linden, Indiana. /IEX 535 CM and 545 DEAE are the products of Toya Soda Corporation, Yamaguchi, Japan. s Bio-Rex 70 is supplied by Bio-Rad, San Francisco, California. h Partisil SCX is manufactured by Whatman, Clifton, New Jersey.
6
FRED E. REGNIER
that good resolution will only be obtained at a linear velocity of 0.1 mm/s or less. Columns of 0.41 X 25 cm will give best resolution at 0.25 ml/min when developed in a gradient of several hours duration (23). Although molecules of 200 kilodaltons may be eluted from columns in less than 10 min, a very large price is paid in resolution. In most cases, the demands on the column for resolution are sufficiently low that 0.5- to l-ml/ min flow rates and 30-min total analysis times are tolerable. MOBILE PHASES
Relatively little work has been done on mobile-phase selection in HPIEC. Because most commercial columns are silica based, there is an upper limit of pH 8.0 to prevent destruction of the silica support matrix. Other than this limitation, column packing materials will withstand a broad range of organic and aqueous mobile phases. Ion-exchange chromatography of proteins is based on the use of mobile-phase pH to manipulate the charge on a protein. At any pH above its pZ a protein will have a net negative charge as opposed to a positive charge below the pZ. Depending on the pZ of a protein, one selects either an anion-exchange or cation-exchange column. Proteins with a high pZ are best separated on cation exchangers, and those with a low pZ separate best on anion exchangers. Both cation- and anion-exchange columns may be eluted with an ionic strength or pH gradient. A variety of mobile phases have been used in the elution of conventional ion-exchange columns that function well in HPIEC columns. The elution profiles obtained with both the conventional and HPIEC columns are usually very similar. Differences will be in ionic strength required for elution, resolution, and separation time. Because the ionic strength required for protein desorption is a function of a packing’s ligand density and it is unlikely that different manufacturers materials will be the same, only relative elution position in chromatograms can be compared.
As a general rule, recoveries of enzyme activity from HPIEC columns are equivalent to those obtained with conventional ion-exchange columns (2 1,30). The short residence time on HPIEC columns will also be useful in recovery of enzyme activity from very labile proteins. Addition of stabilizing agents to mobile phases will be an additional technique for recovering labile proteins from HPIEC columns. APPLICATIONS
HPIEC has now been applied to the resolution of a variety of proteins. As noted above, there is considerable similarity among the mobile phases and elution protocols used to develop all ion-exchange columns. Rather than discuss each case of HPIEC individually, see Table 1 for a list of the essential features of the chromatography of a spectrum of proteins. ACKNOWLEDGMENT The author gratefully acknowledges the support of U. S. Public Health Grant GM 25431.
REFERENCES 1. Boardman, N. K. (1955) Biochim. Biophys. Acfa 18, 290. 2. Boardman, N. K., and Partridge, S. M. (1955) Biochem. J. 59, 543. 3. Eltekov, Y. A., Kiselev, A. V., Kbokblova, T. D., and Nikitin, Y. S. (1973) Chromatographia 6, 187-189. 4. Mizutani, T., and Mizutani, A. (1975) J. Chromatog.
111,214-215.
5. Regnier, F. E., and Noel, R. (1976) J. Chromatogr. Sci. 14, 316-320. 6. Engelhardt, H., and Mathers, D. (1977) J. Chromatogr. 142, 3 1 I-320. 7. Schmidt, D. E., Jr., Giese, R. W., Conron, D., and Karger, B. L. (1980) Anal. Chem. 52, 177-182. 8. Pluddeman, E., Dow Coming Corporation, Midland, Mich., personal communication. 9. Chang, S. H. (1976) PhD Thesis, Purdue University. 10. Chang, S. H., Gooding, K. M., and Regnier, F. E. (1976) J. Chromatogr. 125, 103-l 14. 11. Mikes, O., Strop, P., Zbrozek, J., and Coupek, J. J. (1976) J. Chromatogr. 119, 339-354. 12. Mikes, O., Strop, P., and Sedlackova, J. ( 1978) J. Chromatogr. 148, 237-245. 13. Mikes, O., Sedlackova, J., Rexova-Benkova, L., and Omelkova, J. (1981) J. Chromatogr. 207, 99.
CHROMATOGRAPHY 14. Davis, J. E., McDonald, J. M., and Jarett, L. ( 1978) Diabetes 21, 289. 15. Cole, R. A., Soeldener, J. S., Dunn, T. J., and Bunn, H. F. (1978) Metabolism 27, 289. 16. Wajcman, H., Dastugue, B., and Iabie, D. (1979) Clin. Chem. Acta 92, 33. 17. Van Der Wal, S., and Huber, J. F. K. (1980) Anal. B&hem. 105,219-229. 18. Vanecek, G., and Regnier, F. E. (1982) Anal. Biothem., in press. 19. Chang, S. H., Noel, R. N., and Regnier, F. E. (1976) Anal. Chem. 48, 1839-1845. 20. Gupta, S., Pfannkoch, E., and Regnier, F. E. (1982) Anal. Biochem., in press. 2 1. Alpert, A. J., and Regnier, F. E. (1979) J. Chromatogr. 185, 375-392. 22. Pfannkoch, E., Lu, K. C., Regnier, F. E., and Barth, H. ( 1980) J. Chromatogr. Sci. 18,430-441. 23. Vanecek, G., and Regnier, F. E. (1980) Anal. Biothem. 109,345-353. 24. Giddings, J. C. (1965) in Dynamics of Chromatography, pp. 13-94, Dekker, New York. 25. Synder, L. R., and Kirkland, J. J. (1979) in lntroduction to Modem Liquid Chromatography, p. 27, Wiley-Interscience, New York. 26. Karger, B. L., Synder, L. R., and Horvath, C. (1973) in An Introduction to Separation Science, p. 135, Wiley-Interscience, New York. 27. Morris, C. J. 0. R., and Morris, P. (1976) in Separation Methods in Biochemistry, 2nd ed., p. 17, Halsted Press, New York. 28. Chang, S. H., Gooding, K. M., and Regnier, F. E. (1977) Contemp. Top. Clin. Anal. Chem. I, l-27. 29. Schroeder, R. R., Kudirka, P. J., and Toren, E. C., Jr. (1977) J. Chromatogr. 134,83-90. 30. Schlabach, T. D., Alpert, A. J., and Regnier, F. E. (1978) Clin. Chem. 24, 1351-1360. 31. Schlabach, T. D., Fulton, J. A., Mockridge, P. B., and Toren, E. C., Jr. (1979) Clin. Chem. 25, 1600-1607. 32. Fulton, J. A., Schlabach, T. D., Kerl, J. E., Toren, E. C., Jr., and Miller, A. R. ( 1979) J. Chromatogr. 175,269-281. 33. Schlabach, T. D., Fulton, J. A., Mockridge, P. B., and Toren, E. C., Jr. (1980) Anal. Chem. 52,729-
733. 34. Denton, M. S., Bostick, W. D., Dinsmore, S. R., 35. 36. 37. 38.
and Mrochek, J. E. (1978) Clin. Chem. 24,14081413. Bostick, W. D., Denton, M. S., and Dinsmore, S. R. (1980) C/in. Chem. 26, 712. Schlabach, T. D., Chang, S. H., Gooding, K. M., and Regnier, F. E. (1977) J. Chromatogr. 134, 91-106. Alpert, A. J. (1979) PhD Thesis, Purdue University. Bostick, W. D., Dinsmore, S. R., Mrochek, J. R.,
OF PROTEINS
39. 40. 41. 42.
7
and Waalkes, T. P. (1978) Clin. Chem. 24, 13051316. Goading, K. M., Lu, K C., and Regnier, F. E. (1979) J. Chromatogr. 164,506-509. Rudolph, F. B., and Clark, S. W. ( 1981) Paper No. 202, International Symposium on HPLC of Proteins and Peptides, Washington, D. C. Hanash, S. M., and Shapiro, D. N. ( 1980) Hernoglobin 5, 165. Abraham, E. C., Cope, N. D., Braziel, N. N., and Huisman, T. H. J. (1979) Biochim. Biophys. Acta
577, 159-169. 43. Hanash, S. M., Kavadella, M., Amanulla,
44.
45.
46.
47.
48.
A., Scheller, K., and Bunnell, K. (198 1) in Advances in Hemoglobin Analysis (Hanash, S. M., and Brewer, G. J., eds.), pp. 53-67. Umino, M., Watanbe, H., Komiya, K., and Mori, N. (198 1) Paper No. 208, International Symposium on HPLC of Proteins and Peptides, Washington, D. C. Gardiner, M. B., Wilson, J. B., Carver, J., Abraham, B. L., and Huisman, T. H. J. (1981) Paper No. 203, International Symposium on HPLC of Proteins and Peptides, Washington, D. C. Umino, M., Watanabe, H., and Komiya, K. (198 1) Paper No. 204, International Symposium on HPLC of Proteins and Peptides, Washington, D. C. Kato, Y., Komiya, K., and Hashimoto, T. (1981) Paper No. 214, International Symposium on HPLC of Proteins and Peptides, Washington, D. C. Radhakrishnan, A. N., Stein, S., Licht, A., Gruber, K. A., and Udenfiiend, S. (1977) J. Chromatogr.
132,552-555. 49. Frolick, C. A., Dart, L. L., and Spom, M. B. (1981)
50. 5 1.
52. 53. 54. 55.
Paper No. 205, International Symposium on HPLC of Proteins and Peptides, Washington, D. C. Alpert, A. J., and Beaudet, A. L. (1981) Paper No. 2 10, International Symposium on HPLC of Proteins and Peptides. Washington, D. C. Ansari, G. A. S., Patel, N. T., Fritz, R. R., and Abell, C. W. ( 198 1) Paper No. 2 11, International Symposium on HPLC of Proteins and Peptides, Washington, D. C. Barford, R. A., Sliwinski, B. J., and Rothbart, H. L. (1979) J. Chromatogr. 185,393-402. Jones, B. N., Lewis, R. V., Paabo, S., Kojima, K., Kimura, S., and Stein, S. (1980) .I. Liq. Chromatogr. 3, 1373-1383. Lu, K.-C., Gooding, K. M., and Regnier, F. E. (1979)Clin. Chem. 25, 1608-1612. Gtt, G. S., and Shore, V. G. (1981) Paper No. 201, International Symposium on HPLC of Proteins and Peptides, Washington, D. C.
BIOCHEMISTRY
126, 1-7 (1982)
REVIEW High-Performance
Ion-Exchange Chromatography The Current Status’
of Proteins:
FRED E. REGNIER Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907 Received March 5, 1982 Ion-exchange chromatography of proteins and peptides has been most successfully achieved historically on hydrophilic gel matrices, The poor mechanical strength of these organic gels has necessitated the development of new supports for high-performance separations. Highperformance supports are of three types: totally inorganic, totally organic, and composite inorganic-organic materials. Several ionic species such as diethylaminoethyl ethanol and polyethylene imine have been used as stationary phases with similar results. Pore-diameter selection has been shown to be important in both resolution and loading capacity. Capacity is maximum for proteins of 50 to 100 kilodaltons on 300~A-pore-diameter supports. Maximum resolution of high-molecular-weight species also requires macroporous supports. Interestingly, column length is of minor importance in the resolution of proteins. Columns of 5-cm length have approximately the same resolution as those of 30-cm length. Application of high-performance ion-exchange chromatography to a variety of protein mixtures has now been reported. These supports generally give recoveries of enzyme activity equivalent to the classical supports.
A major limitation of conventional geltype ion-exchange media is their lack of mechanical strength. It is obvious that the high-mobile-phase velocities used in highperformance liquid chromatography (HPLC) require packing materials that are rigid. Interestingly, the first rigid ion-exchange supports for proteins were prepared before diethylaminoethyl (DEAE)- and carboxymethyl (CM)‘-cellulose. In 1954, Boardman (1,2) prepared a cation exchanger in a two-step process consisting of styrene polymerization on the surface of diatomaceous earth followed by sulfonation of the polystyrene matrix. This strong cation exchanger was used in the chromatography of chymotrypsinogen
and cytochrome c. Unfortunately, the very hydrophobic nature of polystyrene causes an irreversible adsorption of many proteins and limits the utility of this packing in protein separations. More recently, Eltekov (3) prepared rigid anion-exchange packings by derivatizing the surface of controlled porosity glass with aminopropyltrimethoxy silane (APS). The resulting weak anion exchanger separated proteins but showed a high degree of nonspecific adsorption. This undesirable property is probably the result of residual silanols on the support surface. When an inorganic surface is exposed to this silylating agent, two types of bonding may occur simultaneously: (i) siloxane bond formation at the surface and (ii) ion pairing of the APS amino groups with surface silanols. Siloxane bond formation and the concomitant sequestering of surface silanol is inhibited by ion pairing. The undesirable properties of packings with a high level of surface silanols have been noted repeatedly (4-7). The propensity of APS monomer to form
’ This is Journal Paper No. 8930 from the Purdue University Agricultural Experiment Station. 2 Abbreviations used: CM, carboxymethyl; APS, aminopropyltrimethoxy silane; HPIEC, high-performance ion-exchange chromatography; PEI, polyethylenimine; SEC, size-exclusion chromatography; BSA, bovine serum albumin.
1
0003-2697/82/15000
1-07$02.00/O
Copyright Q 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
2
FRED E. REGNIER
siloxane polymers in the presence of traces of water is another property of triethoxysilanes that has bearing on the quality of ionexchange coatings (8). Unfortunately, these siloxane polymers are not completely stable when eluted with thousands of volumes of aqueous buffer. Aminopropyl silane monomer gradually leaks from the surface of inorganic supports. The erosion of ion-exchange stationary phase causes both the ionexchange capacity and separation characteristics of columns to change with aging. Chang (9,10) found that thin surface layers of organic polymers produce far more stable ion exchangers than organosilane monomer bonded phases. Both epoxy and polyamine polymers produced packings that behaved like the conventional gel-type ion-exchange packings used in the chromatography of proteins but achieved separations in less than 30 min. Based on these studies it was concluded (10) that microparticulate inorganic supports coated with a thin film of ion-exchanging organic polymer had a substantial advantage in protein separations over both gel-type and organosilane monomer ion-exchange packings. PACKING MATERIALS
The extensive literature on both conventional gel-type ion-exchange resins for proteins and HPLC packings indicate that the ideal HPIEC packing for proteins should be (i) mechanically stable to mobile phase velocities of 1 mm/s, (ii) completely hydrophilic, (iii) of high ion-exchange capacity, (iv) chemically stable over a broad pH range, (v) available in 5 to IO-pm particle size, (vi) available in pore diameters from 300 to 1000 A, (vii) spherical, (viii) of a pore volume between 0.5 and 1.O ml/g, (ix) easy to pack, and (x) inexpensive. No packing material available at the present time has all of these prop erties. Available HPIEC materials for proteins are of three types: (i) organic resins, (ii) composite inorganic-organic matrices, and (iii) inorganic supports with a thin surface layer
of organic bonded phase. More experience with HPIEC is necessary to judge which of these materials is superior. Organic Resins The only known organic ion-exchange resin that can be classed as an HPLC packing for proteins is the polymethacrylate-based Spheron (11) material of Lachema. Separation of proteins on both the CM and DEAE versions of Spheron have been reported by Mikes et al. (12,13). Unfortunately, these ion exchangers are not commercially available in less than 20-pm particle size. The weak cation-exchange resin Bio-Rex 70 is also useful in protein separations but is only available in greater than 40-pm particle size ( 1,14- 16). Van der Wal (17) has examined the utility of several nonrigid ion exchangers in the fractionation of cytochrome c and its derivatives by HPIEC. Cation exchangers with a polystyrene-divinylbenzene or cellulose matrix were generally found to be unsatisfactory. Polymethacrylate matrices had much greater utility. In general, retention increased as either pH or ionic strength was decreased. It was reported that temperature had minimal effect on retention of derivatized cytochrome c. Composite Organic-Inorganic
Packings
Vanecek and Regnier (18) have recently reported the preparation of weak anion-exchange packings with polymer layers 100 A thick in lOOO- and 4000-A pores. Since the whole polymer layer exchanged ions and was permeable to small molecules, the ion-exchange capacity of the packings was much higher than that of a silica support with a surface monolayer of ion-exchange groups. After heavy polymer layers in the silica-support pores were bonded, the functional porosity to macromolecules was still greater than 600 A with an ion-exchange capacity of 300 meq/g. The function of the inorganic support was to provide a rigid matrix within which gel-type ion-exchangers could survive the pressure of a high-performance system.
CHROMATOGRAPHY
The principal advantage of packings with such large pores is said to be in the analysis of proteins exceeding 10’ daltons. Determination of the ultimate utility of these composite materials will require much more testing Surface-Modified
Inorganic
Packings
The most widely used technique for the preparation of HPIEC packings for proteins is through the application of a 20- to 30-A polymer layer to the surface of inorganic sup ports. Ion-exchange groups are either coupled to the surface (i) during the polymerization reaction, (ii) after the polymer layer has been deposited, or (iii) as preformed ionic polymers. An example of the first technique may be found in Chang’s preparation of an inorganic DEAE ion exchanger ( 19). Deposition of diethylaminoethanol and a multifunctional oxirane on the surface of a glycidoxypropylsilane-bonded phase support followed by polymerization caused both DEAE and glyceryl residues to become incorporated into the epoxy polymer formed. The DEAE groups served the function of partitioning proteins electrostatically while the glyceryl residues anchored the coating to the surface at multiple sites. Gupta (20) has reported a second synthetic route to polymeric ion-exchange coatings. The first step in this process is the bonding of a simple organosilane monomer with a reactive organic functional group to the surface of a silica support. This silylation reaction is followed by the covalent attachment of polyethylenimine (PEI) at multiple sites to the silica support through functional groups on the organosilanes. This PEI packing may either be used directly as a weak anion exchanger, further derivatized with oxiranes to form a tertiary amine ion exchanger, or reacted with acid anhydrides (succinic or diglycolic) to produce weak cation exchangers. A single synthetic route is used in the preparation of both anion- and cation-exchange packings. A third type of HPIEC packing has been
3
OF PROTEINS
described by Alpert (2 1). In this process, PEI is first adsorbed onto the surface of inorganic supports as a monolayer and then crosslinked into a thin polyamine skin with multifunctional oxiranes. This crosslinked coating is adsorbed to the surface at many sites and held so tightly that it cannot be desorbed even under severe conditions. The high surface density of amino groups completely titrates all surface silanols and prevents them from interacting with proteins. In 1978, SynChrom introduced the first commercial HPIEC supports for proteins. These weak anion exchangers had a polyamine-bonded phase that produced separations similar to DEAE-cellulose. Although the materials were available in lOO-, 300-, 500-, and 1OOO-A-pore-diameter sizes, the 300-A-pore-diameter packing was the most widely used because of its greater loading capacity. This weak anion exchanger was followed by a weak cation-exchanging CM support in 1982. Pharmacia and Toya Soda Corporation also introduced complete lines of HPIEC columns for proteins in 1982. Although these columns are so new that few people have examined them, they are reported to give separations equivalent to the conventional CM and DEAE columns. It should also be noted that the strong cationexchange material from Whatman, Partisil SCX, has been used effectively in the separation of low-molecular-weight proteins. PORE-DIAMETER
EFFECTS
The ratio of support pore diameter to molecular size can contribute to both the resolution and loading capacity of a column. Resolution Retention in ion-exchange chromatography is controlled by two independent phenomena: (i) the inherent size-exclusion contribution from differential penetration by solutes of macroporous matrices and (ii) electrostatic partitioning at the surface of the ion exchanger. It has been observed in sizeexclusion chromatography (SEC) that as sol-
4
FRED E. REGNIER
ute dimensions approach those of the sup port pores, severe limitations of molecular diffusion occur within the pores (22). The net effect of this phenomenon is resistance to mass transfer between the stationary and mobile phases and a concomitant loss of resolution. The inherent SEC properties of HPIEC packings cause this same bandspreading phenomenon to occur as solute size approaches pore diameter. These porediameter limitations of stagnant mobile-phase mass transfer are easily overcome in HPIEC by using larger pore-diameter packings (18). The optimum relationship between pore diameter and solute size has not been determined. Preliminary studies would indicate that 300-A-pore-diameter packings give less than optimum resolution with proteins greater than lo5 daltons. Loading Capacity Because ion-exchange chromatography is a surface-directed process, it would seem that ion-exchange capacity should be proportional to the surface area of a packing. Actually, not all of the surface of a packing is available to macromolecules. Greater than 95% of the surface area of a porous support is inside the pore network, and a molecule must be able to penetrate the pore matrix to reach the surface. The surface area (AJ available for ion-exchange partitioning is expressed by the equation A, = A() + K&i where A0 is the surface area on the outer surface of particles in a bed, Ai is the internal surface area of the pores of particles in the bed, and K, is a surface accessability coefficient. The term K, indicates the fraction of the internal surface available for ion-exchange adsorption and thus ranges from 0 to 1. Obviously, K, will be different for each molecular species and approach zero as molecular dimensions approach the dimensions of pores. For molecules such as hemoglobin and bovine serum albumin (BSA), 250- to 300-A-pore-diameter packings have the
greater value of KJi and therefore give the highest ion-exchange capacity. A BSA loading capacity of 100 mg/g of packing is common (23). Smaller proteins such as carbonic anhydrase exhibit highest loading capacity on 100-A-pore-diameter packings as opposed to proteins of several hundred thousand kilodaltons that require 500- to 1000-A pores for maximum loading. For both loading capacity and resolution, 250- to 300-A-pore-diameter ion-exchange packings are probably of broadest utility because they bracket the 50- to 100~kilodalton range of proteins. A 0.41 X 25-cm column will show no signs of overloading with up to 10 mg of ovalbumin (23). Under overloading conditions, the column will still separate most impurities in an ovalbumin sample even at a 50-mg injection. COLUMN LENGTH
It has been determined that column length plays a minimal role in resolution of proteins (23). Resolution on 5-cm columns is almost equivalent to that of 25-cm columns. These small columns have some definite advantages in analytical work such as (i) the elution of proteins in a smaller elution volume and therefore a more concentrated form, (ii) up to 6 times greater sensitivity because eluants are more concentrated, (iii) fewer problems with underloading columns, (iv) lower operating pressures, (v) columns last longer, and (vi) columns are cheaper. The disadvantages of short (~5 cm) columns are that maximum loading capacity is a few milligrams of protein and the small elution volumes promote extracolumn band spreading in some equipment. Both low dead-volume connecting tubing and flow cells are essential. MOBILE-PHASE
VELOCITY
The influence of mobile-phase velocity on band spreading and resolution has been treated in depth by Giddings (24) and is discussed in a number of books (25-27). The Giddings treatment indicates that as the diffusion coefficient of a molecule decreases, its
CHROMATOGRAPHY
ability to difke into and out of support pores decreases accordingly. Thus, as molecular weight increases, mass transfer problems are expected to escalate because of the decreasing diffusion coefficients. Obviously increasing mobile-phase velocity in a column TABLE
OF PROTEINS
5
aggravates the mass transfer problem. It has been calculated that to obtain equivalent column efficiency, mobile-phase velocity must be 10 times slower with a molecule of 70 kilodaltons than one of several hundred daltons (28). Practically speaking, this means 1
PROTEINSPURIFIEDBYHIGH-PERFORMANCEION-EXCHANGECHROMATOGRAPHY
Protein Lactate dehydrogenase isoenzymes Creature kinase isoenzymes Alkaline phosphatases Hexokinase &enzymes Arylsulfatase isoenzymes Hemoglobins
Cytochrome c Mozyme Myoglobin Soybean trypsin inhibitor Interferon Lipoxygenase Trypsin Chymotrypsinogen Immunoglobulin Ovalbumin
G
Albumin Apolipoproteins Adenylsuccinate synthetase Insulin &Lactoglobulin Carbonic anhydrase Monoamine oxidase
Column
Type of ion exchanger
DEAE-glycopha& SynChropak AX300e DEAE-glcyophase SynChropak AX300 DEAE-glycophase SynChropak AX300 DEAE-glycophase SynChropak AX300 DEAE-glycophase IEX 545 DEAE’ IEX 535 CM’ Bio-Rex 70g CM-polyamide CM-polyamide CM-polyamide IEX 535 CM CM-glycophased Partisil SCX* SynChropak AX300 DEAE-glycophase SP-glycophase IEX 535 CM SynChropak AX300 SynChropak AX300 IEX 545 DEAE SynChropak AX300 DEAE-Glycophasc IEX 545 DEAE SynChropak AX300 SynChropak AX300 Partisil SCX IEX 535 CM Partisil SCX Partisil SCX SynChropak AX300
WAX” WAX WAX WAX WAX WAX WAX WAX WAX WAX wcx* wcx wcx wcx wcx wcx wcx SCX’ WAX WAX sex wcx WAX WAX WAX WAX WAX WAX WAX WAX sex wcx sex sex WAX
References (10,19,30,31,33) (30) (31-34) (3Q35) (36) (37) (38) (39,41,43,45) (l&19) (44) (44) (42)
(20) (20) (20) (46) (19) (48)
(18) (19) (19) (46) (54) (23) (47) (23) (19) (46) (5655) (W (49) (46) (49) (49) (51)
a WAX designates weak anion exchanger. * WCX designates weak cation exchanger. ’ SCX indicates strong cation exchanger. d DEAE and CM-glycophase are products of Pierce Chemical Company, Rockford, Illinois. e SynChropak AX300 is produced by SynChrom, Linden, Indiana. /IEX 535 CM and 545 DEAE are the products of Toya Soda Corporation, Yamaguchi, Japan. s Bio-Rex 70 is supplied by Bio-Rad, San Francisco, California. h Partisil SCX is manufactured by Whatman, Clifton, New Jersey.
6
FRED E. REGNIER
that good resolution will only be obtained at a linear velocity of 0.1 mm/s or less. Columns of 0.41 X 25 cm will give best resolution at 0.25 ml/min when developed in a gradient of several hours duration (23). Although molecules of 200 kilodaltons may be eluted from columns in less than 10 min, a very large price is paid in resolution. In most cases, the demands on the column for resolution are sufficiently low that 0.5- to l-ml/ min flow rates and 30-min total analysis times are tolerable. MOBILE PHASES
Relatively little work has been done on mobile-phase selection in HPIEC. Because most commercial columns are silica based, there is an upper limit of pH 8.0 to prevent destruction of the silica support matrix. Other than this limitation, column packing materials will withstand a broad range of organic and aqueous mobile phases. Ion-exchange chromatography of proteins is based on the use of mobile-phase pH to manipulate the charge on a protein. At any pH above its pZ a protein will have a net negative charge as opposed to a positive charge below the pZ. Depending on the pZ of a protein, one selects either an anion-exchange or cation-exchange column. Proteins with a high pZ are best separated on cation exchangers, and those with a low pZ separate best on anion exchangers. Both cation- and anion-exchange columns may be eluted with an ionic strength or pH gradient. A variety of mobile phases have been used in the elution of conventional ion-exchange columns that function well in HPIEC columns. The elution profiles obtained with both the conventional and HPIEC columns are usually very similar. Differences will be in ionic strength required for elution, resolution, and separation time. Because the ionic strength required for protein desorption is a function of a packing’s ligand density and it is unlikely that different manufacturers materials will be the same, only relative elution position in chromatograms can be compared.
As a general rule, recoveries of enzyme activity from HPIEC columns are equivalent to those obtained with conventional ion-exchange columns (2 1,30). The short residence time on HPIEC columns will also be useful in recovery of enzyme activity from very labile proteins. Addition of stabilizing agents to mobile phases will be an additional technique for recovering labile proteins from HPIEC columns. APPLICATIONS
HPIEC has now been applied to the resolution of a variety of proteins. As noted above, there is considerable similarity among the mobile phases and elution protocols used to develop all ion-exchange columns. Rather than discuss each case of HPIEC individually, see Table 1 for a list of the essential features of the chromatography of a spectrum of proteins. ACKNOWLEDGMENT The author gratefully acknowledges the support of U. S. Public Health Grant GM 25431.
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