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High performance capillary electrophoresis John Frenz and William S. Hancock The application of recombinant-DNA methods for the production of therapeutic proteins has, over the past decade, driven the development of new technology for the analysis and characterization of biological molecules. High performance capillary electrophoresis (HPCE) has generated enormous interest among biochemists, analytical chemists and chromatographers, and is emerging as an extremely high-resolution separation technique, that may rival high performance liquid chromatography (HPLC) in its efficiency and breadth of application.

High performance capillary electrophoresis (HPCE), is expected to become an important tool of the biotechnology industry, with analytical applications particularly in the area of highprecision characterization of the products of recombinant DNA (rDNA) technology. Until now, the 'workhorse' techniques in this area have been HPLC (for the separation and purification of proteins), and gel electrophorefis (for the analysis of proteins and nucleic acids). HPCE was developed as a refinement of earlier electrophoretic separation methods 1.2, with the diameter of the capillary in which separation is carried out being made smaller in order to improve heat dissipation and facilitate on-column detection3.4. HPCE complements existing analytical techniques by providing, in certain cases, features that are unavailable in the older methods. For example, capillary zone electrophoresis separates species according to net charge, and thus provides an alternative basis for selectivity to that 0¢ reversed-phase HPLC, which separates accordhag to solute hydrophobicity. HPCE is a rapid instrumep~ technique that, like HPLC, allows convenient quantitative analysis of the components of a mixture, and thereby complements the qualitative analysis afforded by gd dectrophoresis. Capillary electrophoresis further complements chromatographic techniques because, in principle, the analyte interacts only with the buffer and the dectric fidd, and not with charged or hydrophobic surfaces. Thus, differences among closely related components of the analyte mixture can be the basis for separation even if these differences do not lie on the face of the protein presented to the adsorbing surface. These

and other potential advantages account for the promise of HPCE, and the breadth of applications for the technique that have been reported. Instrmnental electrophoresis Capillaries

The capillary, like the column in HPLC, is the heart of the HPCE system. Typically composed of polyimide-clad silica, the inner diameter usually measures 25-200 pm, while the outer diameter is 300-500pLm. The narrow dimensions provide relatively efficient dissipation of the heat generated by electric current flow through the capillary, thus minimizing convective mixing and changes in buffer properties that are associated with elevated temperature. Open tube - coated and uncoated

Open-tube free-zone or free-solution capillary electrophoresis is the simplest and most widely practiced mode of HPCE. The high rates of heat loss from the capillary obviate the need for an anticonvective medium such as that employed in gel electrophoresis. Hence, electrophoresis can be carried out efficiendy in an open capillary tube, without the presence of a gel or packing material. Both coatedS and uncoated silica and polymeric3 capillaries have been used in applications involving biological macromolecules. Coating the inner wall of the capillary may render the surface more inert with respect to the analyte, and improve the effidency and selectivity of the separation process s. An important feature ofcoated capillaries is that the direction and magnitude of buffer flow occurring during electrophoresis can be manipulated by modifying the charge at the inner wall of the capillary by an appropriate coating treatment. The wall of an uncoated silica capillary, for example, .]. Frenz and W. $. Hancock are at the Depamnent of Medicinal and Analytical Chemistry, Genentech, Inc., 460 Point San Bruno bears acidic silanol groups that are negatively charged when in contact with the buffers that are Boulevard, South San Francisco, CA 94080, USA. ~) 1991, ElsevierScience PublishersLtd (UK) 0167- 9430/91/$2:00

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reviews commonly employed in dectrophoresis. These negative charges are immobile while their cationic counterions can be mobilized by the electric field applied during electrophoresis. This mobility yields a bulk flow of the solvent (termed dectroendoosmotic flow), towards the cathode buffer reservoir. The flow rate by this pumping mechanism can be manipulated by adjusting buffer pH, or by the addition to the buffer of components that interact strongly with, and mask, the surihce charge. The overall result of electroendoosmotic flow is that all components of a mixture (acidic, basic and neutral species), in an uncoated silica capillary, flow in the same direction as the solvent and can be detected in free-zone electrophoresis. Basic components of the analyte mixture migrate more quickly than neutral species, while acidic species migrate more slowly. A neutral coating that shidds the acidic silanol groups can be applied to the surface to eliminate electroendoosmotic flow s. In free-zone electrophoresis in these capillaries, neutral species and the buffer are immobile, while acidic species migrate to the anode and basic species to the cathode. Since detection normally takes place at only one end of the capillary, only one class of species at a time can be detected in an analysis using a coated capillary.

Gel filled

ployed, or the sample must be at high concentration to be detected. For biological molecules, the most widely used detection method in HPCE is absorbance of ultraviolet (UV) light, and detection by this technique, in practice, requires concentrated samples, compared with those detectable by HPLC s. Among methods in development to address this problem are fluorescence techniques, including laser-induced fluorescence9, which can increase by several orders of magnitude the detectability, compared with UV absorbance, of compounds with the appropriate excitation and emission characteristics. The other (related) challenge for detection methods in HPCE is the difficulty of identifying compounds electrophoresed through the capillary. This problem also stems from the small sample capacity of the technique, which hampers the collection of a sufficient quantity of all but the largest peaks for characterization by the conventional tools of protein chemistry or molecular biology. The sensitivity of photodiode-array technology 1°, which can provide a portion of the UV absorbance spectrum and facilitate identification, is hindered by the limited quantity of light that can be passed through the capillary. Mass spectrometry (MS), with either a fast atom bombardment (FAB) 11, or an electrospray 12 interface between the capillary and the mass spectrometer has shown great promise for the identification of peaks electrophoresed from the capillary. Figure 1 shows the reconstructed ion current profiles (i.e. 'electropherograms', by analogy to the 'chromatograms' produced in chromatography) of a four-component mixture of proteins separated by HPCE and detected by MS. The four proteins in this mixture elute with little separation from each other, but selective ion monitoring permits identification of the mixture components as distinct peaks. In certain respects, HPCE is well matched to MS, as the latter technique, carried out at high vacuum, is better suited to the low sample volumes characteristic of HPCE than, for instance, the volumes typical of bands eluted from an HPLC column 12.

Methods for casting and anchoring polyacrylamidev gels within capillaries have been developed to mimic the selectivity and Mr-based separations obtained in slab-gel electrophoresis of biopolymers. The ability to obtain Mr information on a protein, for example, can aid in identification of peaks in the electropherogram 7. Capillary SDSPAGE of proteins and PAGE of oligonucleotides yield rapid, high-efficiency separations (exceeding one million theoretical plates per meter), and allow more accurate quantification of components of the mixture than can be obtained by conventional staining protocols employed for slab gels. Gelfilled capillaries have high separation efficiencies (i.e. narrow peak widths), in part because the analyte mixture is focused at the inlet of the capillary, in a manner analogous to the stacking that occurs at the top of the separating gel in slab Operating modes electrophoresis. Free.zone electrophoresis The operating modes of free-zone and gel-filled Detection capillary electrophoresis have been noted above. In Detection in capillary electrophoresis poses these modes, the sample is loaded into one end of among the most daunting challenges of the tech- the capillary either gravimetrically, pneumatically nique, due to the extremely low sample volumes or electrophoreticallyTM. The first two of these and masses associated with HPCE relative to, for methods ensure that the aliquot that enters the instance, HPLC. The small sample size is dictated capillary has a composition representative of the by the narrow dimensions of the capillary that also sample, while dectrophoretic sample-loading limits the path length available for on-column biases the load to the more electrophoretically optical detection. In on-column detection, light is mobile components of the mixture. Following passed across the capillary, such that a portion of injection, the buffer is replaced at the inlet end of the the capillary acts, effectively, as the flow cell. Since capillary, the power turned on, and electrophoresis the path length is relatively short, either an ex- proceeds with control of the electric current, tremely efficient detection scheme must be em- voltage or power input across the capillary. lIBl'ECHJULY 1991 NOL 9)

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Micellar electrokinetic chromatography A technique closely related to free-zone HPCE is micellar electrokinetic capillary chromatography (MECC) in which a micellar solution is used as the running buffer is. Sample components partition into the micelles, and separation can be achieved according to the partition coefficients of the sample components between the micellar and bulk phases. Neutral molecules or mixtures of similarly charged species may require this approach to achieve HPCE separations. This technique has been applied mostly to analyses of small organic molecules, but may have applications to peptides and proteins as well.

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lsoelectricfocusing Isoelectric focusing (IEF) in HPCE is a two-step process involving the focusing of the sample in an ampholyte mixture, followed by mobiliTation of the resulting bands past the detector to produce the electropherogram. During the focusing step, the carrier ampholytes mixed with the sample create a pH gradient along the length of the capillary and the sample components focus at points in the gradient corresponding to their pL Electroendoosmotic flow must be eliminated in the capillary, by, for exan~ple, coating the inner walls, so that the static pH gradient can form. After focusing, the bands are mobilized past the detector either pneumatically, or electrophoretically by changing the buffer ionic strength16.

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lsotachophoresis lsotachophoresis is also known as 'displacement electrophoresis '17 because of its similarity to displacement chromatography TM. This technique involves mobilization of the sample by a moving front of an dectrolyte at relatively high concentration behind the sample zone. The sample bands all migrate with the velocity of this front, which is higher than their individual velocities in free-zone dectrophoresis. The advantage of this technique is that larger sample masses and volumes can be loaded into the capillary, while still retaining high resolution owing to the focusing effect achieved by use of the displacer electrolyte. This operating mode has been employed to overcome the sample size limitation of HPCE and to permit efficient detection of peptides and proteins by electrospray mass spectrometry ~3.

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the complementarity of the technique to reversed phase HPLC 19. The mobility of peptides in freezone HPCE can be correlated with mass and charge In a variety of applications, free-zone capillary (Fig. 2). Since retention times in HPLC correlate electrophoresis has been shown to be, like reversed- less well with these parameters, mixtures that are phase HPLC, a convenient means for the rapid difficult to separate by HPLC can often be resolved analysis of peptide mixtures. In this respect, it readily by HPCE. Figure 3 shows the separation of extends the range of applications of electrophoretic a pair oftryptic peptides that co-elute in HPLC, but techniques, which are, in general, better suited to are well resolved in capillary electrophoresis. Thus, separations of larger biopolymers. An important HPCE provides a convenient means of assessing advantage of HPCE for the separation ofpeptides is the purity of fractions collected from HPLC. An

.Applications Peptides

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added advantage is that the concentration of peptides collected from reversed-phase separations is often high enough to allow direct analysis of an aliquot of the column effluent by HPCE. An important application of peptide separations is the mapping of fragments obtained by enzymatic digestion of a protein, as is frequently performed by reversed-phase HPLC. The high resolution and speed of HPCE make it a promising means of enzymatic mapping that, because of altemative selectivity compared with HPLC, can yield additional information on the digested protein. Figure 4 shows tryptic maps of recombinant methionyl human growth hormone obtained by both reversed-phase HPLC and free-zone HPCE under acidic conditions. Comparison of the two maps confirms the expected differences in selectivity of the two methods, and the high resolution that is obtained by both procedures. Owing to the suppression of charge at the capillary wall, low pH buffers frequently yield more reproducible results for HPCE of peptides, but under these conditions most tryptic peptides have a net charge of +2. In order to achieve greater differentiation in the charge state of the peptides and thereby improve selectivity, the use of other enzymes, which cleave at other than basic residues, can be investigated21, but, in general, these enzymes are less selective than trypsin and yield ill-defined fragmentation patterns.

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Time (rain) Rgure 3 Analysis of a fraction collected from a Wptic map of hGH by (e) HPLC and (b) HPCE at low pH. Peptides that are not resolved by HPLC may be well separated by HPCE, since the criteria for separation for the two techniques are largely independent. Reprinted, with permission, fron, Ref. 19. TIBTECHJULY1991 (VOL9)

Slab electrophoresis has become the method of choice for separating oligonucleotide mixtures, including those obtained by Sanger DNA sequencing protocols 22. The wide use of this important application has prompted considerable interest in developing higher-resolution methods for separation of these mixtures by capillary electrophoresis~-26. Among the attractions of HPCE h~ sequencing applications are the speed of the separation relative to slab electrophoresis, and the ability to use a variety of detection methods, including fluorescent26 or isotopic27 labelling of individual nucleotides, to facilitate sequence determination. Labelled dideoxynucleotides are used such that oligonucleotides terminating at a particular residue have a characteristic emission spectrum, or an isotopic marker. The former label can be monitored by fluorescence detection, and the latter by mass spectrometry. An application of HPCE not yet fully explored is the restriction fragment mapping of polynucleotides28, which has numerous uses for confirmation ofplasmid integrity and identification of DNA or RNA samples. Since these mapping procedures are commonly carried out by gel electrophoresis, HPCE provides a simple means of obtaining fingerprint patterns rapidly and on an automated instrument. To achieve the high resolution demanded by these applications, both gel-filled and open-tube capillary electrophoresis approaches have been

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developed. As noted above, gel-filled capillaries provide high-resolution, size-based separations that have shown their greatest potential in separations of oligonucleotides. Figure 5 shows the separation of a mixture of polythymidilic acid fragments consisting of 20-160 base pairs (bp).

Close examination of Fig. 5 reveals the unit bp resolution is obtained, suggesting that the technique would be suitable in DNA-sequencing applications. Open-tube HPCE with viscous, hydrophihc-polymer-containing running buffers can also resolve relatively closely-related oligoTIBTECHJULY1991(VOL9)

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Separation of proteins forms the other major area of analytical application of electrophoresis, and is an area of rapid development of HPCE for both charge- and size-based separation procedures. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) systems have been reported for Mr-based separations of denatured model proteins (Fig. 6). Broad acceptance of this technique awaits commercialization of gelfilled capillaries produced by proprietary methods. Charge-based separations of proteins have been

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Rgure 6 Capillary SDS-PAGE of intact myoglobin and fragments. The inset shows a plot of log Mr ofthe sample components vs. their mobility [measured in (cmZ/V.s) x 10s] in the gel-filled capillary. The linearity of the plot validates estimates of protein size by this approach in a manner analogous to slab gel SDS-PAGE of proteins. Peak No. 5 is myoglobin, peaks ] - 4 are smaller fragments. Reprinted, with permission, from Ref. 7.

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Figure 7 Capillary electrophoresis of (a) intact and (b) neuraminidasedigested recombinant soluble CD4 at pH 4.5. Digestion with neuraminidase removes sialic acid from the carbohydrate groups on the glycoprotein, thereby limiting its charge heterogeneity and collapsing the pattern obtained by free-zone electrophoresis, a technique that separates according to charge.

demonstrated in both free-zone capillary electrophoresis and isoelectric focusing. Figure 7a shows the resolution ofglycosylation variants of recombinant soluble CD4, a glycoprotein with potential for use in AIDS therapy. The charge heterogeneity of the protein can be reduced by removal of sialic acid residues with neuraminidase digestion, yielding the electropherogram shown in Fig. 7b. HPCE offers a rapid means of assessing carbohydrate heterogeneity, thereby providing insight to cell-culture conditions or the extent ofdeglycosylation achieved with glycosidic enzymes. Capillary IEF is another means of achieving charge-based separations in HPCE. After the protein is focused to a particular position accord•ng to its isoelectric point (pI), the entire contents of the capillary are mobilized past the detector, yielding an IEF electropherogram in which elution time can be related to pI. IEF provides a powerful means of focusing and detecting minor charge variants of a mixture, but quantification is hampered compared with free-zone HPCE by the precipitation of the more abundant proteins 19.

Outlook for HPCE The research potential for HPCE for highefficiency separation of macromolecules has already been demonstrated by examples similar to those described in this review. Further rapid growth of research and development (R&D) in this area will, however, be contingent on the commercial availability of capillaries with a variety of surface coatings, as well as continued advances in detectors. The future of HPCE is indeed exciting with advances in microelectronics permitting applications of HPCE to such areas as single-cell analyses, on-line fermentation monitoring as well a~ rapid quality control of biosynthetic proteins and the investigation of transcription/translation processes. References | Vesterburg, O. (1989)J. Chromatogr. 480, 3-|9 2 Kuhr, W. G. (1990) Anal. Chem. 62, 403R-414R 3 Mikkers, F. E. P., Everaerts, F. M. and Verheggen, Th. P. E. M. (1979)J. Chromawgr. 169, 11-20 4 Jorgensen, J. W. and DeArman-Lukacs, K. (1981)J. Chromato,~r. 218, 209-216 5 Hjertdn, S. (1985)J. Chromawgr. 347, 191-198

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reviews 20 Nidsen, R. G., Riggin, R. M. and Rickard, E, C. (1989) 6 McCormack, R. M. (1988) Anal. Chem. 60, 2322-2328 J. Chromatogr. 480, 393-401 7 Cohen, A. S. and Karger, B. L. (1987)J. Chromatogr. 397, 40921 Frenz, J., Battersby, J. and Hancock, W. S. (1990) in Peptides: 417 Chemistry, Structure attd Biology: Proceedingsof tire l lth American 8 Ewing, A. G., Wallingford, R. A, and Olefirowicz, T. M. (1989) PeptideSymposium,July 9-14, 1989, LaJolla, CA (River, J. E. and Anal. Chem. 61,292A-303A Marshall, G. R., eds), pp. 430-432, ESCOM 9 Gozel, P., Gassmann, E., Michdsen, H. and Zare, R. N. (1987) 22 Sanger, F., Nicklen, S. and Coulson, A. R. (1977) Doc. Natl Acad. Anal. Chem. 59, 44-49 Sci. USA 74, 5463-5467 10 Kobayashi, S., Ueda, T. and Kikumoto, M. (1989)J. Chromatogr. 23 Drossman, H., Luckey, J. A., Kostichka, A.J., D'Cunha, J. end 480, 179-184 Smith, L. M. (1990) Anal. Chem. 62, 900-903 11 Minard, R. D., Chin-Fatt, D., Curry, P. and Ewing, A. G, (1988) Proceedingsof the 36th ASMS Conferenceon Mass Spectrometry 24 Swerdlow, H. and Gesteland, R. (1990) "Nucleic Acids Res, ii~, 1415-1419 and AIlied Topics, San Francisco,CA,June 5-10, 1988, p. 950, Am. 25 Cohen, A. S., Najarian, D. R. and Karger, B. L. (1990)~ Soc. Mass Spectrometry J. Chromatogr. 516, 49-60 12 Olivares, J. A., Nguyen, N. T., Yonker, C. R. and Smith, R. D, 26 Smith, L. M., Sanders, J. Z., Kaiser, R. J., Hughes, P., Dodd, (1987) Anal. Chem. 59, 1230-1232 C., Connell, C. R., Heiner, C., Kent, S. B. H. and Hood, L. E. 13 Smith, R. D., Loo, J. A., Barinaga, C. J., Edmonds, C. G. and (1986) Nature 321, 674-679 Udseth, H. R. (1989)J. Chromatogr.480, 211-232 14 WaUingford, R. A. and Ewing, A. G. (1987) Anal. Chem. 59, 27 Brennan, T., Chakd, J., Verity, p. and Fidd, M. (1990) in Biological Mass Spectrometry: Doceedings of the 2rid Intemaeional 678--681 Symposh#n on Mass Spectrometry in tire Health and Life Scimces, 15 Terabe, S., Otsuka, K., lchikama, K., Tsuchiya, A. and Ando, September 28, 1989, San Francisco, CA (Burlingame, A. L. and T. (1984) Anal. Chem. 56, !11-113 McCloskey, J. A., eds), pp. 159--177, Elsevier 16 Hjertdn, S. and Zhu, M. (1985)J. Chromatogr.346, 265-270 17 Mardn, A.J.P. and Everaerts, F. M. (1967) Anal. Chim. Acta 38, 28 Heiger, D. N., Cohen, A. S. and Karger, B. L. (1990) J. Chromatogr. 516, 33-48 233-237 18 Frenz, J. and Horv~th, Cs. (1989) in HPLC - Advances and 29 Paulus, A., Gassmann, E. and Field, M. (1990) ElearophoresisI1, 702-708 Perspectives, Vol. 5 (Horv~th, Cs., ed.), pp, 212-314, Academic 30 Zhu, M., Hansen, D. L., Burd, S. and Gannon, F. (1989) Press J. Chromatogr. 480, 311-319 19 Frenz,J., Wu, S-L and Hancock, W. S. (1989)J. Chromatogr.480, 379-391

Analysing lymphokine-receptor interactions of IL.1 and IL-2 by recombinant-DNAtechnology Gerard Zurawski The development of lymphokines as pharmaceutical agents is at the forefront of current biotechnological research. Lymphokinesexert their regulatory action on cellular function through interaction with cell-surface receptors. An understanding of the biochemical nature of these molecular interactions should facilitate the design of small peptide analog pharmaceuticals which mimic lymphokines or their receptors. Lymphokines are immunoregulatory protein hormones made by lymphoid cells, and they mediate their effects on cell activation through interaction with specific cell-surface receptors. Lymphokines include y-interferon, colony stimulating factors (CSFs), tumor necrosis factors (TNFs), and some of the interleukins1. Their

receptors are related to receptors of other protein hormones such as growth hormone and prolactin2. Some lymphokines are useful in therapy for cancer3 and viral infections 4, and in immunosuppressed individuals s. There is little doubt that lymphokines have enormous potential as therapeutics in the areas of cancer, allergy inflammation, and infectious diseases. Mutagenic analysis by recombinantG. Zurawski is at tile D N A X Research hzstitute of Molecular and DNA (rDNA) technology can be used to study Celhdar Biology Inc., 901 California Avome, Palo Alto, C A lymphokine--receptor interactions. Such studies 94304.1104, USA. should facilitate the discovery of small-molecule

TIBI"ECHJULY 1991 (VOL9]

~) 1991, ElsevierScience PublishersLtd (UK) 0167 - 9430/91/$2.00