Anal. Chem. 1994,66, 2148-2154
Incorporation of Hydrophobic Selectivity in Capillary Electrophoresis: Analysis of Recombinant Insulin-like Growth Factor I Variants Wassim Nashabeh, Kimberly F. Greve, Dan Kirby, Frantisek Foret, and Barry L. Karger' Barnett Institute, Northeastern University, Boston, Massachusetts 02 1 15 David H. Relfsnyder and Stuart E. Builder Genentech, Inc., South San Francisco, Californh 94080
A highly selective electrophoreticsystem employingdifferential hydrophobic interaction was evaluated for the quantitative determination of recombinant insulin-like growth factor I (ICF-I) variants. The system consisted of mixed aqueousorganic buffers containing suitable amounts of a zwitterionic detergent. In addition, a neutral hydrophilic coating was attached to the wall of the capillary to minimize analyte adsorptionand electroosmotic flow. The zwitterionic detergent acted as a hydrophobic selector, allowing independent optimization of the electrophoretic and hydrophobic selectivities in the separation system. The extent of hydrophobicinteraction was conveniently adjusted by varying the type and amount of organic modifier. Complete resolution of a mixture of IGF-I variantswith closely related mass-to-chargeratioswas achieved. Quantitative analysis of ICF-I process samples agreed well with HPLC results. Finally, the approach was found to be compatible with on-line capillary electrophoresis-mass spectrometry.
Human insulin-like growth factor I (IGF-I) is a basic polypeptide composed of 70 amino acid residues with three disulfide bonds (see Figure 1). First isolated in 1976 from human serum,' IGF-I has been shown to mediate many of the anabolic effects of human growth hormone.2 IGF-I occurs in blood plasma at concentrations ranging from 20 to 80 nmol/L and at even lower concentrations in most other tissues.3 Thus, to obtain large quantities of the molecule, it is necessary to synthesize IGF-I by recombinant DNA technology. However, during the cellular production of the protein, several variants are obtained. One of these variants, designated as improperly folded IGF-I, has the disulfide bonds between cysteines 6 and 47 and cysteines 48 and 52 instead of between residues 6 and 48 and residues 47 and 52, as seen in native IGF-I.4 In addition, another form in which the single methionine residue at position 59 is oxidized to methionine sulfoxide (Met-0) (see Figure l)s and other IGF-I variants, in which N-terminal amino acids are cleaved, are alsoobtained. (1) Rinderknecht, E.; Humbel, R. Proc. Nafl.Acad. Sci. W.S.A.1976,73,23652369. ( 2 ) Spencer, E. M.; Skover, G.; Hunt, T. K. In GrowfhFacforsand Orher Aspecfs of Wound Healing. Biological and Clinical Implications; Alan, R., Ed.;Liss, Inc.: New York, 1988; pp 103-116. (3) Hejnaes, K.;Bayne, S.; Nsrskov, L.; Ssrensen, H. H.; Thomsen, J.; SchPffer, L.; Wollmer, A.; Shriver, L. Protein Eng. 1992, 5, 797-806. (4) Raschdorf, F.; Dahinden, R.; Maerki, W.; Richter, W. J.; Merryweather, J. P. Biomed. Environ. Mass. Spectrom. 1988, 16, 3-8.
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Flgwe 1. Aminoacidsequenceof 1-4. Disulfklebondsfor correctly folded IW-1 r"wr are indicated (S-S). Improperly folded IW-1 occurs when bonds are formed between Cyssand Cys" and Cysa and cyss*. ~ S - I O F - Ispecies refer to removal of amino acids trom N-terminus. MethioninesQmay be oxidized to the sulfoxide.
High-resolution and rapid methods are needed for the identification and quantitation of IGF-I and its variants in order to monitor the quality of process samples. Currently, reversed-phase HPLC is the method of choice; however, other methods arevery desirable in order to provide a morecomplete assessment of product purity. Capillary zone electrophoresis (CZE) is increasingly being used for the separation of a wide variety of biological and pharmaceutical compounds. The rapid growth of the technique is due to many beneficial features such as speed, high separation efficiency,quantitation, and automation. In CZE, the separation of ionic species is mainly a result of differential migration under the influence of an applied electric field. Thus, CZE separates on the basis of differences in mass-to-charge ratios of the analytes. Separation in terms of hydrophobic differences, as required in part for the separation of IGF-I and its variants, necessitates some modification of the buffer system. An important development in this regard was the introduction of micellar electrokinetic chromatography (MEKC),6in which a surfactant, e.g., sodium dodecyl sulfate (SDS), is added to the background electrolyte at a concentration above the critical micelle concentration (CMC). This approach permits the separation of small molecular weight compounds by their differential partitioning between the ( 5 ) Frosberg, G.; Palm, G.; Ekebacke, A.; Josephson, S.; Hartmanis, M. Biochem. J . 1990, 271, 375-376. (6) Terabe, S.; Otsuka, K.; Ichikama, K.; Tsuchiya, A.; Ando, T. Anal. Chem. 1984, 56, 1 11-1 13.
0003-2700/94/03862 148$04.50/0
0 1994 American Chemlcal Society
micellar pseudostationary phase and the aqueous buffer phase. Neutral solutes migrate in order of increasing hydrophobic character, whereas ionic species separate on the basis of a combination of mass-to-charge ratio, hydrophobic character, and, in some cases, ion pairing with the charged detergent. Using uncoated fused silica capillaries, MEKC has proven to be useful for the analysis of a wide variety of species, e.g., nucleic acid constituents, chiral compounds, aromatics, and amino a c i d ~ . ~ -However, I~ the technique has two limitations: (1) a finite separation time window which can limit peak capacity and resolution of the system and (2) poor selectivity for the separation of highly hydrophobic low-molecularweight compounds, as well as larger species, such as oligopeptides and proteins. This loss in separation may be a result of strong interactions of the hydrophobic parts of the analytes with the micellar phase. To overcome this problem, detergents of less hydrophobic character such as surfactants with shorter alkyl chains13or large polar moieties,14 micelles having adjustable surface charge density,'s and a monomolecular pseudostationary phase16 have been used. Alternatively, various buffer additives shift the partition equilibrium toward the aqueous phase. These additives include urea,17 cyclodextrins,18J9bile salts,8and various organic solvents.2g22Very recently, for example, it has been shown that the addition of acetonitrile to charged micelles [i.e., SDS or CTAB (cetyltrimethylammonium bromide)] improves the separation of closely related peptides and small proteins,23 as well as neutral Triton-X series oligomers.24 It should be noted that the poor selectivityfor larger species in MEKC may also be a result of the inability of the solute to penetrate into the micelle. Moreover, interaction of free detergent molecules with such species may be significant in its own right. This paper investigates the potential of capillary electrophoresis in the separation and routine analysis of recombinant insulin-like growth factor I variants, differing mainly in hydrophobic character, using mixed aqueous4rganic electrolytes and suitable amounts of a zwitterionic detergent. The capillary column utilizes a neutral hydrophilic coating to prevent adsorption of themoleculesto thesurface. In addition,
the minimizationof electroosmoticflow in such a system leads to ease of optimizing electrophoretic behavior and high reproducibility. It is shown that the method results in the rapid and quantitative determination of several IGF-I variants having closely related mass-to-charge ratios but subtle differences in hydrophobicity. In addition, the approach is shown to be compatiblewith on-line capillary electrophoresismass spectrometry (CE-MS), enabling the identification of a mixture of five IGF-I variants.
EXPERIMENTAL SECTION CE Instrumentation and Methods. A Beckman P/ACE instrument, version 2100 (Beckman, Palo Alto, CA), with System Gold version 7.1 1 was used for all CE measurements. The electropherograms were monitored at 214 nm with a data collection rate of 10 Hz. A 75-pm i.d., 375-pm o.d., fused silica capillary (Polymicro Technologies, Phoenix, AZ) was coated with a cross-linkedpoly(vinylmethylsi1oxane) sublayer and then a layer of poly(a~rylamide).~~ The capillary, maintained at 25 OC, had an effective length of either 40 or 50 cm and a total length of 47 or 57 cm. Samples were lowpressure injected for 8 s. A chromatographic software system, Chrom Perfect, was employed to analyze electropherograms (Justice Innovations, Inc., Palo Alto, CA). To ensure reproducible separations, the capillary column was purged with fresh buffer for 3 min before each injection. The pH of the buffer was not adjusted if the organic solvent content was less than 10% (slight changes in the pH will occur with the addition of organic solvent). The injection volume was estimated from measuring the time required for a 10% (v/v) aqueous-acetone solution to traverse the effectivelength of the capillary under low-pressureinjection. An 8-s injection would then correspond to approximately 36 nL of injected volume. Reversed-Phase HPLC Instrumentation and Methods. Separations were conducted using a Hewlet-Packard 1090M liquid chromatographic system (Palo Alto, CA). The instrument was equipped with a Vydac CISreversed-phase column (4.6 mm X 25 cm 5-pm particle size, 300-A pore size) (Hesperia, CA). Analytical separationswere performed using a constant flow rate of 2 mL/min and a shallow linear gradient of 29-30% acetonitrile in 0.1% trifluoroacetic acid (TFA) (7) Cohen, A. S.; Terabe, S.;Smith, J. A.; Karger, B. L. Anal. Chem. 1987,59, 1021-1027. over 11 min. The column was regenerated with 60% (8) Dobashi, A.; Ono, T.; Hara, S.; Yamaguchi, J. J . Chromatogr. 1989, 480, acetonitrile and equilibrated with 20% acetonitrile in 0.1% 413420. (9) Nishi, H.; Fukuyama, T.; Matsuo, M.; Terabe, S.1.MicmolumnSep. 1989, TFA prior to each injection. The total time between injections 1 , 234-241. was 20 min. (10) Cole,R.O.;Sepaniak,M.J.;Hinze, W.L.;Gorse, J.;Oldig*l,K.J.Chromarogr. 1991,557, 113-123. CEMS Imtnunentation and Methods. A Fmnigan TSQ700 (11) Liu, J.; Cobb, K. A.; Novotny, M. J . Chromatogr. 1988, 468,55-65. spectrometer (Finnigan MAT, San Jose, CA) was used to (12) Bushey. M. M.; Jorgenson, J. W. Anal. Chem. 1989. 61, 491-493. (13) Balchunas, A. T.; Gpaniak, M. J. Anal. Chem. 1987,59, 1466-1470. obtain mass spectra from 1000 to 2000 amu in 1.5 s in the (14) Swedkrg, S. A. J. Chromatogr. 1990,503,449-452. profile mode. The electrospray interface (ESI) was (15) Cai, J.; El R a d , 2. J . Chromarogr. 1992, 608, 31-45. (16) Palmer, C. P.; Khaled, M. Y.; McNair, H. M. J . High Resolur. Chromatogr. modified with a 22-gauge stainlesssteel capillary as the liquid 1992, 15, 756-762. sheath tube, with the CE capillary inserted through it. The (17) Terabe, S.; Ishihama,Y.;Nishi,H.;Fukuyama,T.;Otsuka, K. J. Chromatogr. 1991, 545, 359-368. ESI interface was operated at -3.9 kV, 0 2 sheath gas at 210 (la) Terabe, S.; Miyashita, Y.; Shibata, 0.;Barnhart, E. R.; Alexander, L. R.; cc/min, and N2 drying gas at 150 OC with a flow rate of -3 Patterson, D. 0.;Karger, B. L.; Hosoya, K.;Tanaka, N . J. Chromatogr. 1990, 516, 23-31. L/min. The liquid sheath was 2275: 1 H20:MeOH:HAc (19) Liu, J.; Cobb. K. A.; Novotny, M. J. Chromatogr. 1990, 519, 189-197. (v/v) at a flow rate of 1.2 pL/min. The separation capillary (20) Gorse, J.; Balchumas, A. T.; Swaile, D. F.; Sepaniak, M. J. J . High Resoluf. Chromatogr. 1988,11,554-559. was 50 cm long, 50 pm i.d., and coated as described above. (21) Bushey, M. M.; Jorgenson, J. W. J. Microcolumn Sep. 1989, I , 125-130. A CZE lOOOR power supply (Spellman HVCE, Plainview, (22) Vindevogcl, J.; Sandra, P. Anal. Chem. 1991,63. 1530-1536.
(23) Yashima, T.; Tsuchiya, A.; Morita, 0.;Terak, S. Anal. Chem. 1992, 64, 2981-2984. (24) Bullock, J. J. Chromatogr. 1993, 645, 169-177.
(25) Schmalzing, D. K.; Piggce, C. A.; Foret, F.; Carrilho, E.; Karger, B. L. J . Chromatogr. 1993, 652. 149-159.
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(mln.)
Flgure 2. Llquld chromatography performed on a Vydac CIS column. Samples were eluted wlth a shallow 0.1 % TFA/acetonMle gradient, as described in the Experlmental Section. Approximately 10 pg each of improperly folded IGF-I (A), Met-0-IGF-I (e) or correctly folded monomer (C) was Injected.
NY) operated at 25 kV was used. The background electrolyte consisted of 20 mM 8-alaninelcitric acid, pH 3.8, 2.5% 1-butanol, and 3 mM zwitterionic detergent. Samples were loaded hydrodynamically. Reagents and Materials. Recombinant human ICF-I and its variants were purified from Escherichia coli and characterized by mass spectrometry at Genentech, Inc. (San Francisco, CA). The zwitterionic detergent, N-dodecyl-N,Ndimethyl-3-amino-1-propanesulfonate (DAPS), was purchased from Calbiochem (San Diego, CA). Analytical grade buffer materials [P-alanine,citric acid, e-amino-n-caproic acid (EACA)] and acetic acid, as well as bromophenol blue, were purchased from Sigma (St. Louis, MO). HPLC grade acetonitrile was obtained from J. T. Baker (Phillipsburg, NJ), and 1-butanol was purchased from Fisher Scientific (Fairlawn, NJ). MilliQ water (18.2 MO water; Millipore, Bedford, MA) was used to prepare all buffer and sample solutions. Buffers were filtered with a 0.2-pm filter (Schleicher and Schuell, Keene, NH). cmc Determination. cmc determinations were made from changes in absorption of a dye, bromophenol blue, that interacts with the micelle as a function of the concentration of the detergent.26J7 The spectra of the solutions were taken with a Beckman DU-60 spectrophotometer (Beckman, Palo Alto, CA) using a scan range of 560-620 nm and a scan speed of 500 nm/min. A spectrum in the visible region was obtained, and maximum absorption wavelength values were plotted against the detergent concentration. The concentration at which a sharp change in the absorbance occurred was taken as the cmc. RESULTS AND DISCUSSION Reversed-Phase HPLC. A typical HPLC separation of IGF-I and several of its variants is shown in Figure 2. Three main peaks were easily separated on a CIS reversed-phase column using a shallow linear gradient of 29-30% (v/v) acetonitrile in 0.1% TFA: improperly folded IGF-I, Met-0-IGF-I, and correctly folded IGF-I. Under the acidic chromatographicconditions,two other variants, des-GlyI- and
des-Gly1Pro2Glu3-IGF-I(N-terminal cleavage), comigrated with thecorrectly folded IGF-I species (data not shown). Since most of these variants are present at early stages of IGF-I purification, an approach which is able to resolve all species would be a valuable tool to assess the degree of purification. Capillary Zone Electrophoresis. The initial attempt at purifying these variants by capillary electrophoresis utilized a conventional linear poly(acry1amide) coating28 and a P-alanine/citric acid (pH 3.80) buffer. As demonstrated in Figure 3, these variants showed virtually no separation by CZE, migrating together as a broad and tailed peak. The poor resolution was attributed to a combination of the solute adsorption at the capillary wall and the similarity of the electrophoreticmobilities of the speciesunder the given acidic buffer conditions. The sensitivityof these solutes to the quality of the coating can be seen in the fact that even with the commonly used coating,28tailed peaks resulted. The use of a recently developed coatingzs allowed CZE separation with little or no solute-wall interactions, as evidenced by the appearance of symmetrical peaks. However, even suppression of adsorption was found to be insufficient for resolution. For example, only partial separation of native and improperly folded IGF-I was observed with either fused silica capillaries with dynamically adsorbed cationic fluorosurfactants at neutral pHZ9or untreated capillaries at elevated pH (1 1.1).3O Since the mixture of Figure 3 was resolved by reversed-phase HPLC (see Figure 2), an electrophoretic system that incorporates hydrophobic selectivity was deemed necessary to achieve the separation of the variants. As a hydrophobic selector, we chose the zwitterionic detergent DAPS, since such a species would allow adjustment of the pH of the running buffer over a wide range without affecting the zwitterionic character of the detergent. Another consideration in using zwitterionic or neutral detergents was the avoidance of strong electrostatic interactions between the hydrophobic selector and the solutes, which can potentially lead to loss in resolution when both detergent and analytes are
~~
(26) Hjerten, S.; Valtchcva, L.;Elenbring, K.; Eaker, D. J . Liq. Chromarogr. 1989, 12, 2471-2499. ( 2 7 ) Mukerjec. P.; Mysels, K. J. Critical Micelle Concentrarionr of Aqueous Surjacranf Sysrems; US. Government Printing Office: Washington, DC, NSRDS-NBS 36, 1971; pp 1-21.
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(28) Hjerten, S. J . Chromrogr. 1985,347, 191-198. (29) Emmer, A.; Jansson, M.;Rocraadc, J. J. High Resolur. Chromatogr. 1991, 14,738-740. (30) Ludi, H.; Gassmann, E.; Grossenbacher, H.;MHrki, W. Anal. Chim. Acra 1988, 213, 215-219.
A 1
C
I
0.004
Timc (d)
Fi~ufe4. Separatbn of native (C), M e t e n ) , and imptaperly fWed IQF-I(A) uslnga sibxanedloHhearpoly(a~mkIe)aated c a ~ l l l a r y. ~ and hydrophobic selecthrity. A# condiU6nrc #e the 8aM as those'tn Figure 3,except 5 mM DAPS and 15 % ACN Were added to the buffer. Migration order is the same as that in Fl$um 2.
of opposite charge. An additional advantage of using zwitterionic or neutral detergents is that they do not contribute to the solution conductivity, allowiQ-the use of high electfic fields. To modulate the hydrophobic interaction be$ween the detergent and the solute, an organic modifier was added. T k organic modifier and its Concentration would affect tht cmc.31132 Organic modifiers could also affect solute properties by either inducing conformational changes or facilitating solubilization (e.g., highly hydrophobic compounds). It is useful to note that with a neutral, hydrophilic coating, the electroosmoticflow was effectively suppressed. In general, zero flow was advantageous, since additives to the buffer will only affect electrophoretic mability and not simultaneously influence electroosmotic flow, as found, for example, with bare s i l i ~ a .With ~ negligible flow, the system was simpler t6 manipulate and should, in principle, l&d to higher reproducibility. Zero electroosmotic flow had theadded advantage of compatibility with electrospray ionization MS when using zwitterionic detergents (see below). A variety of CZE conditionsusing the hydrophobicselectorDAPS were examinedto optimizeseparationof IGF-I variants. Figure 4 shows that with this additive, it was indeed possible to resolve IGF-I from two variants (i.e., improperly folded and Met-0) with high efficiency. The separation was performed with zero electroosmotic flow25 using 20 mM 8-alaninelcitric acid, pH 3.80, oontaining 5 mM DAPS and 15% (v/v) acetonitrile (ACN). Note that the migration order was identical to that obtained in reversed-phase LC under acidic conditions (see Figure 2), quggesting an electrophoretic order related to hydrophobicity. The role of ACN concentration on hydrophobic selectivity was examined with a fixed concentration of DAPS, 5 mM. The results of this study are shown in Figure 5 with a plot of the migration time ratio of IGF-I to improperly folded IGF-I (selectivity 1) or that of IGF-I tia Met-0-IGF-I (selectivity 2) versus the percentage (v/v) Of ACN in the buffer. It can be seen that the selectivity for both solute pairs first increased with added ACN, reaching a maximum around 10% (v/v) ~~~
(31) Almgrem, M.;S w a m p 3 In Surfactants In Solution;Mittal, K. L.. L~ndman, B., Eds.;Plenum R&: New York, 1981; Vol. I, pp 613625. (32) Hinze, W. L. In Ordered Media in 6h'fiuniciilS¶tions;Hinzc. W. L.; Armstrong,D. L., Us.; American Chemical Sociefy: Washington, D(3,1987; PP 6-9.
Percent of Organic Modifier (ACN) Flgwe 5. Effect of organic modifier on relative migration: selecthrity 1 (A), migratiohtlmeratio of IGF-I/lmproperty folded IQF-I; selectMty 2 (El), migration Utne ratio of IW-I/Met-O-IQF 1. All conditions are the same as In Figure 3, except 5 mM DAPS and various percentages of ACN were added to the buffer. Each polnt is an average of four repetitive measurements with an average RSD of 0.48%.
0.01
C
I
I
Figure 6. Separation of native (C), M e t 4 (B), and improperly folded IW-I (A) using 50% (Wv) ACN. All other conditlons are the same as those in Figure 4. Note the Inversion In migration order versus that in Figure 4.
ACN. For selectivity 1, the migration ratio gradually decreased at higher ACN concentrations to a value of approximately 1.025. For selectivity 2, an inversion in the migration order of IGF-I and Met-0-IGF-I at high concentrations of ACN was observed. Figure 6 shows the separation of the three species at 50% (v/v) ACN. The behavior in Figure 5 can be understood, in part, by examining the influence of ACN on micelle formation of the DAPS molecules. This examination was done by measuring the change in absorption maximum of bromophenol blue in the visible region as a function of concentration of ACN for a fixed concentration of DAPS (5 mM) (see Figure 7). In the absence of ACN, the detergent was present in micellar form (cmc in aqueous buffer was 3.6 mM). Thus, at 5 mM DAPS, the low values of selectivities 1 and 2 at 0%ACN (in Figure 5) were obtained under micellar conditions. As the percent of ACN was increased, the total concentration of the micelles in the system decreased (Le., cmc increased) or, in other words, the ratio of micelle concentration to that of monomer DAPS shifted toward that of the monomer. This paralleled an increase in the selectivity of both pairs of solutes in Figure 5, as ACN changed from 0 to 10% (v/v). The
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Am!Ytkl Chemistry, Vd. 66, No. 13, Ju& 1, 1994
2151
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Flgure 7. Maximum absorbance of bromophenol blue versus amount of ACN at 5 mM DAPS to determlne the amount of organic modifier necessary to disrupt micelle. Buffer: 20 mM &alaninelcltric acM, pH 3.8, wlth various amounts of ACN. For additional details, see the Experimental Section.
-
micelle formed in 5.0 mM DAPS appeared to have been destroyed above 11% (v/v) ACN, as can be seen from the intersection of the two straight lines in Figure 7. Separate studies have indeed shown that the cmc of DAPS in the presence of 10% (v/v) ACN was approximately 5 mM.33This concentration of ACN also corresponded closely to the maximum selectivity. It is suggested that the IGF-I interaction with free DAPS molecules may have been more selective than that with the micelles and that this interaction differentially affected the mobility leading to separation. A similar suggestion on the importance of free-detergent interaction with analytes on selectivity has been made in a separate study of large peptides, e.g., g a ~ t r i n s .DAPS ~ ~ can be considered in these cases as a probe of hydrophobic differences of IGF-I and its variants. The decrease in selectivity with increasing ACN above 11% (v/v) was likely due to a decrease in hydrophobic binding of the detergent to the protein. Indeed, selectivity 1 was reduced to a value close to that found in aqueous buffer above 20% (v/v) ACN. For selectivity 2, an inversion of IGF-I and Met-0-IGF-I was found near 50%(v/v) ACN; see Figure 6. Since the changes in migration times of improperly folded IGF-I and IGF-I itself paralleled each other with increasing concentrations of ACN, it is most likely that the migration of the oxidized variant was differentially decelerated at increased concentrations of ACN. A specific structural or conformational change appeared to have occurred in the case of Met-0-IGF-I. Since hydrophobic interactions areexpected to be weak at 50% (v/v) ACN, it is suggested that the conformational change resulted in a lower positive charge on the surface of the molecule, thus differentially decelerating Met-0-IGF-I relative to native IGF-I. As in the case of reversed-phase and hydrophobic interaction chromatogr a p h ~it, ~would ~ appear that organic solvents may permit manipulation of reversible structural changes for selective separations. It is also worth noting that the efficiency of the bands in Figure 6 was 3 times higher than that in Figure 4. It may
-
(33) Greve, K. F.; Nashabeh, W.; Karger, B. L. Manuscript in preparation. (34) Oroszlan, P.; Wicar, S.; Teshima, G.; Wu, S.; Hancock, W. S.; Karger, B. L. Anal. Chem. 1992.64, 1623-1631.
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Fbwo 8. Electropherogram of four IGF-I variants; (D) des-Gty1Pro2oIus-IGF-I, (A) improperly folded IGF-I, (E) des-Glyl-IGF-I, and (C) IGF-I. Electrophoretic conditions: coated 50 cm effective length, 57 cm total length; buffer, 20 mM EACA/acetlc acid, pH 4.4, 5 mM DAPS, and 10% ACN; running voltage, 30 kV; detection at 2 14 nm; 25 'C.
be that the reversibility of the molecular interaction between DAPS and the IGF-I species was sufficiently slow that this kinetic process contributed to a loss in peak efficency. In addition, it is possible that there was a distribution ofcomplexes of IGF-I species with detergent molecules, leading to a distribution of mobilities and thus broadening of the peak. At 50% ACN, with little or no interaction between DAPS and the IGF-I molecules, peak widths were narrower. However, the increased selectivities for low percentages of ACN shown in Figure 5 more than compensate for the somewhat broader bands. We next examined the ability of this system to separate des-Glyl-IGF-I and des-Gly1Pro2-Glu3-IGF-Ifrom the native IGF-I, since both variants may be present during the manufacturing process. The separation of des-Glyl-IGF-I from IGF-I was particularly challenging as the hydrophobic change is expected to be small relative to the native species. In the case of des-Gly1Pro2Glu3-IGF-I,pH could also be used as a selective tool in separation from IGF-I (see Table 1, selectivity3). At pH 3.2, theglutamic acid should be partially ionized in native IGF-I, thus affecting its mobility, whereas for the des-Gly1Pro2Glu3-IGF-I variant, the glutamic acid residue is not present. The highest value of selectivity 3 was obtained at pH 4.4 where Glu should be more ionized. Note further in Table 1 that selectivity for des-Gly' versus that for native IGF-I (selectivity 4) was pH independent from 3.2 to 4.4. Figure 8 shows the separation of IGF-I from three variants: improperly folded, des-GlylPro2Glu3, and desGlyl-IGF-Iat pH 4.4 with 5 mM DAPS and 10% (v/v) ACN. Further improvement can be obtained for the separation of des-Gly' from IGF-I by change of the organic modifier. A variety of n-alcohols were examined, and the best results were obtained with 2.5% 1-butanol and 3 mM DAPS, Figure 9. At room temperature, the hydrophobic interaction of DAPS and the IGF-I species resulted in broadened bands. Improved
Table 1. Effects of pH om SeIectMty of IQF-I Varlanta
a
buffer
PH
20 mM &alanine/citric acid, 10% ACN, 5 mM DAPS 20 mM @-alanine/citricacid, 10% ACN, 5 mM DAPS 20 mM EACA/acetic acid, 10% ACN, 5 mM DAPS
3.2 3.8 4.4
selectivity 3O
selectivity 4 IGF-I/des-Glyl-IGF-I
1.04 1.06 1.14
1.02 1.02 1.02
IGF-I/des-Gly1Pro2GIu~-IGF-I
Selectivity is defined as the ratio of migration times. Table 2. Comparkon of Purf(y 16 IQF-I Process Sampler by CE and HPLC
sample
CEO (76)
HPLCa (a)
A B C D E
57.5 77.6 71.3 92.6 99.9
53.1 75.0 75.6 91.6 99.7
a Values listed are percent of main peak relative to total area units by the various methods.
Tunc (min)
Figure 9. Separation of des-Oly'-IGF-I (E) and IGF-I (C) at (1) 25 O C and (2) 45 OC. CE condltlons are the same as those In Figure 3, except 2.5% (v/v) butanol and 3 mM DAPS were added to the runnlng buffer.
band sharpness with essentially base-line separation could be obtained by elevatingthe temperature from25 to 45 OC.Figure 9 demonstrates that the choice of the organic modifier can provide subtle differences in selectivity that can be used to improve difficult separation^.^^ QuantitativeAnalysis of ICF-I Process Samples. The use of a commercial instrument incorporating column temperature control and a neutral coated capillary enabled high analytical precision to be obtained. With pressure injection as detailed in the Experimental Section, the migration time and timenormalized peak areas for IGF-I under the conditionsof Figure 4 were 0.56% and 1.58% relative standard deviation (n = 6), respectively. In addition, a calibration plot of peak area versus concentration of injected IGF-I showed linear behavior (r2= 0.994) in the concentration range of 8 to at least 5 X lo3 pg/mL, corresponding to an injected mass range of 28 ng-I8 rg* We next compared the purity of processed samples with thoseobtained by reversed-phasechromatography. As shown in Table 2, a close agreement in purity analysis was found for the twomethods. The high efficiency and different selectivities possible, along with automated operation, make CZE a (35) Idei, M.; Mezo, I.; Vadasz, 2.;Horvath, A.; Teplan, I.; Keri,G. J . Chromatogr. 1993, 648, 251-256.
. 40
1171.6
+7
173.3
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1m
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m/z
Figure 10. Reconvolutedmass spectra of Iff-Ivariants showing +4 through 4-7 charge states for each: (D) des-01yiPr&d-IW3F-I, (E) desQlyi-IGF-I, (A) Improperly folded Iff-I,(E) Met-0-IGF-I, and (C) IGF-I. All spectra resulted in correct molecular weight identlficatlon (seeTable 3). CE condltlons are the same as those In Figure 9.
potentially powerful tool to complement HPLC in the assay of process samples. CE-MS. Under the conditions of Figure 8, we could not resolve Met-0-IGF-I from the des-Gly' and IGF-I species. Thus, as in HPLC, a desirable capability of CZE in process analysis is the ability to identify the eluted species by mass spectrometry. The hydrophobically selective system with no electroosmotic flow used in this work was found to be compatible with electrospray MS. At the 100-fmol level, all components were mass resolved. Reconvoluted spectra of the +4 through +7 charge states of each variant were calculated from spectra under each peak and are shown in Figure 10A-E. Single-componentspectra were obtained for three analytes (Figure 10A,C,D). The spectra from the remalning two unresolved (in time) components (Figure
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Table 3. Molecular Welght Determlnatlon ol IGF-I Varlatr calculated expected molecular molecular weight (Da) weight (Da)
des-Gly1ProZGlu3-IGF-I d=-Gly'-IGF-I Met-0-IGF-I improperly folded IGF-I IGF-I
1364.0 f 0.9 1591.2 1.1 1662.9 f 1.3 7648.6 f 0.9 1646.9 f 0.7
*
1365 1591 1664 1648 1648
10B,E) were deconvoluted to give a pair of component masses near 7664 and 7648 amu. The spectra were then reconvoluted, based on each component mass, to yield spectra containing ions and charge states associated with each component (Figure 10B or 10E). All spectra resulted in the correct molecular weights of the species (see Table 3). Note that the spectra were not background subtracted and yet not interfering surfactant ions ( m / z 1007, 1342, and 1677) were observed. This suggests that a CE buffer system containing a zwitterionic detergent can be used successfully in on-line CE-ESI-MS with coated capillaries where no electroosmotic flow is present. A more extensive report on the use of surfactants in CE-MS will be published separately.
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CONCLUSIONS The use of organic solvents with zwitterionic detergents and coated capillaries represents a powerful means of separation and analysis of peptide species which differ only slightly in hydrophobicity. Since CZE is a solution process, the whole surface of the analyte molecule is probed by the detergent for hydrophobic differences. It appears that in the case of IGF-I and its variants, operation just at the cmc of the zwitterionic detergent in the presence of organic modifiers yields the greatest selectivity for hydrophobicity. As in reversed-phase chromatography, the choice of the organic solvent adds a second level of selectivity. In addition, this approach, utilizing coated capillaries with no electroosmotic flow, is compatible with on-line CE-MS. Thus, CZE can be a highly useful tool for purity analysis of process samples. ACKNOWLEDGMENT The authors gratefully acknowledge support of NIH under Grant GM15847. Contribution No. 600 from the Barnett Institute. Recelved for review January 19, 1994. Accepted April 4, 1994.a ~_______
Abstract published in Advance ACS Abstracts, May 15, 1994.
