Anal. Chem. 1992, 64, 1623-1631
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Conformational Effects in the Reversed-Phase Chromatographic Behavior of Recombinant Human Growth Hormone (rhGH) and WMethionyl Recombinant Human Growth Hormone (Met-hGH) Peter Oroszlanlt Stanislav Wicar; Glen Teshima,' Shiaw-Lin WU) William S. Hancock) and Barry L. Karger**t Barnett Institute, Northeastern University, Boston, Massachusetts 02115, and Medicinal and Analytical Chemistry, Cenentech, Inc., South San Francisco, California 94080
Thls paper examines the retention behavior of recomblnant DNA48flvedhunangrowthhormone(rhGH) In reversedpheee chromatography and its separation from the closely related Kmethionyivarlant(MethGH). I t isfirstshown that retentbn for rhGH decreaseswith Increasingcolumntemperature when l-propanol(1-PrOH) bused as organlc modifier. On the other hand, retentionIncreaseswith temperaturewhen acetonitrile (CHSCN) b employed. The differences In behavior for the two organic modifleni could be related to conformational cha~hrtheproteinasdacMnkredbysduHonanda~ intrinsicfluorescence spectroscopy. Specfflcaily, desorption and dutlon of rhGH udng 1-PrOH could be correlated with a soivent-lnduced conformational change, with retention decreadng with increasing temperature due to the increasing ease of structural atteration. On the other hand for CHSCN the increasein retentioncorrelatedwith temperature rbe was related to a partial structural change yieidlng a more hydrophobic specles. I n this case, a surfacedrlven process b suggested. The work then turned to the separatlon of rhGH and Met-hGH where it was found for both organic modifiers optlmum separation occurred at 45 OC and pH 6.5. Separate studler revealed that durlng the conformationalchange MethGH appeared more hydrophobic than rhGH since protelnproteln aggregation was observed at a lower 1-PrOH concentratlon. I t b suggested that thb hydrophobk difference, whlch was optimized under the condltlor#,citedabove, resuited in the separation. The study demonstratesthe importance of conformationalchanges In retentionbehavior and separation of protein samples.
INTRODUCTION The exploration of chromatographicretention mechanisms of proteins is important in the optimization of a separation and extraction of general rules for manipulation of retention. Such studies are particularly important for the analysis of closelyrelated protein varianta or degradationproducts which may have identical structures except for a single residue variation. The understanding of the retention process is confounded by the fact that the 3-dimensional structure of a protein molecule can be altered when the species is in a nonphysiological environment and furthermore can be sensitive to slight changes in this environment. These secondary and tertiary structural changes (as well as quaternary), which depend on a variety of parameters such as temperature, pH, mobile-phase composition, adsorbent surface characteristics,
* To whom reprint request should be sent.
t Northeastern f
University.
Genentech. 0003-2700/92/0364- 1623$03.00/0
and protein concentration, can significantly influence binding to an adsorbent surface and thus retention in all modes of interactive chromatography,'-5 as well as in noninteractive size exclusion.6 For example, in reversed-phase liquid chromatography (RPLC), adsorption coefficients can differ by a factor of 1Olo or more between a folded (native) and unfolded (denatured) species.' Separation of closely related species may often occur within a narrow range of conditions or with the use of specific additives.@ Indeed, in many circumstances, unbeknownst to the researcher, protein structure may have been manipulated to achieve separation. Although, these structural changes may be small and reversible after elution of the sample from the column, multiple peaks1')-12or asymmetrically shaped peake13 can sometimes be chromatographically observed, leading to incorrect conclusions about sample purity. Moreover, the alterations may be kinetically controlled, so that contact time with a surface can influence results.10J1J4J6 Clearly, an understanding of the phenomena involved can be critical to successful optimization of protein separations. Most often, researchers have examined retention processes of proteins from the correlation of chromatographic measurements.Ie While practically useful, such phenomenological approachesare, however, limitedin terms of an understanding of what actually takes place in the system. Moreover, the models developed from such correlations often view the molecule as existing in a single state (folded or unfolded), (I)Shaltiel, 5. Methods Enzymol. 1984,104,69. (2)Cohen, S.A.; Dong, S.; Benedek, K. P.; Karger, B. L. In Affinity Chromatography and Biological Recognition; Chaiken, I. M., Wdchek, M., Parikh, I., Eds.; Academic Press, New York, 1983;p 479. (3)Regnier, F. E. Science 1987,238,319. (4)Hearn, M.T. W.; A g u i i , M. I.; Nguyen, T.; Fridman, M. J. Chromatogr. 1988,436,271. (5)Frenz, J.; Hancock, W. S.; Henzel, W. J. In HPLC of Biological Macromolecules; Gooding, K. M., Regnier, F. E., Eds.; M. Dekker: New York, 1990;p 145. (6) Shalongo, W.; Jagannadham, M. V.; Flynn, C.; Stellwagen, E. Biochemistry 1989,28.4820. (7)Lin,S:; Karger, B. L. J. Chromatogr. 1990,499, 89. (8)Hancock, W.S.;Bishop, C. A.; Prestidge, R. L.; Harding, D. R. K.; Hearn, M. T. W. Science 1978,20,1168. (9)Lindahl, L.;Vogel, H. J. Anal. Biochem. 1984,140, 394. (10)Cohen, S. A.; Benedek, K.; Dong, S.; Tapuhi, Y.; Karger, B. L. Anal. Chem. 1984,56,217. (11)Hearn, M.T. W.; Hodder, A. N.; Anguilar, M. I. J. Chromatogr. 1985,327,47. (12)Ingraham,R. H.; Lau, S. Y. M.; Taneja, A. K.; Hodges, R. S. J. Chromatogr. 1986,327,77. (13)Lu, X.M.;Benedek, K.; Karger, B. L. J. Chromatogr. 1986,369, 19. (14)Benedek, K.; Dong, S.; Karger, B. L. J. Chromatogr. 1984,317, 227. (15)Itoh, H.; Nimura, N.; Kinoshita, T.; Nagae, N.; Nomura, M. AMI. Biochem. 1991,199,7. (16)Snyder, L.R.;Stadalius, M. A. InHighPerformanceLiquid Chromatography. Aduances and Perspectives; Horvath, C., Ed.; Academic Press: New York, 1986;Vol. 4,p 195.
0 1992 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 14, JULY 15, 1992
EXPERIMENTAL SECTION with surface binding solely based on molecular interaction of rigid species. Such models are clearly incomplete and at times Equipment. The apparatus was similar to that reported misleading. elsewhere.2a Fluorescence emission spectra were recorded on an The SPF 500C spectrofluorometer (SLM-Aminco,Urbana, a). Changes in the structure of proteins in the mobile phase, excitation and emission spectral bandwidths were 2 and 4 nm, as a function of the applied chromatographic conditions, can respectively. The samplecompartment,injector,and t u b w were be studied by a variety of solution methods." Other studies thermostated, as previouslydescribed." For the samplerecovery have characterized proteins directly in contact with the surface studies, the outlet of the flow cell was connected to a 1046A of the support using, among other methods, ellipsometry,18 fluorescence LC detector (Hewlett-Packard,Palo Alto, CA). Fourier-transform infrared spectros~opy,'~ and total internal For the surface fluorescence studies, the 35-pL spectroscopic reflectance fluorescencespectroscopy.20 Intrinsic fluorescence flow cell was carefully packed with roughly 25 mg of n-butylbonded Vydac silica gel (Separations Group, Hesperia, CA; has been especially useful for the study of chromatographic particle size 5 pm, pore diameter 30 nm). The column was surface-adsorbed and mobile-phase-dissolvedproteins, where conditioned by injecting 20 times, 10-pL aliquota of 5 mg/mL changes in emissionmaxima and fluorescent yield can indicate protein, followed each time by elution under reversed-phase conformational alteration of the species.21 Recently, we have gradient conditions. A second, empty flow cell (cell 11) in series explored a combined liquid chromatographic-on-column was utilized for studying fluorescencecharacteristicsof the eluted intrinsic fluorescence spectroscopic approach to study in situ species. Because of the very small flow cell volume, the pressure surface- and solution-induced conformational changes of drop was negligible. proteins.22~~3 Chromatographic separation of rhGH and Met-hGH was carried out on a Vydac C4 column (4.6 by 250 mm, 5 pm, 30 nm). As a continuation of these studies, this paper demonstrates, Temperature was controlled using a water jacket connected to using fluorescence spectroscopy, the importance of confora refrigerated circulating bath. The LC system (HP-lWM, mational change in the control of migration and thus Hewlett Packard, Inc., Palo Alto, CA) consisted of a cooled auseparation of recombinant growth hormone (rhGH) and its tosampler,and autoinjector equipped with a 250-pL syringe,and N-methionyl variant (Met-hGH) in reversed-phase chromaa W photodiode array detector (HP-l040A, Hewlett Packard, Inc., Palo Alto, CAI. tography. The effects of a variety of parameters, such as Chemicals. Recombinant DNA-derived human growth hortemperature, pH, and organic modifier are studied. Recommone (rhGH) and N-methionyl growth hormone (Met-hGH) binant hGH (MW = 22 OOO) is a good example for study since samples were supplied in vials containing 5 mg of protein and it is a relatively 3igid'' molecule, highly resistant to irreversible about 50 mg of excipienta (mainly mannitol). For the solution denaturation in a broad range of temperature and solution fluorescence studies, proteins were buffer exchanged by gel condition^.^^ Moreover, it has only one tryptophan (Trp) permeation chroamtography using PD-10 columns packed with fluorophore. In view of the recently solved 3-dimensional Sephadex G-25 M (Pharmacia LKB, Piscataway, NJ). T h e structure of hGHZ5in which Trp (86)in one helix is hydrogen proteins were eluted by the phosphate or TFA buffer solutions used asmobile-phase A in RPLC (see below). HPLC gradewater, bonded to an aspartic acid (169) in a second helix, it would 1-propanol, and acetonitrile were purchased from Burdick & be expected that any changes in Trp fluorescence characJackson (Muskegon, MI) and J. T. Baker (Phillipsburg,NJ). All teristics would reflect an alteration in the environment of buffer solutions were filtered through an 0.45-pm Nylon memthat particular residue. Substantially different retention brane, degassed by vacuum, and sparged with helium before use. patterns using either 1-PrOH or CH3CN as organic modifiers Samples were kept frozen at -10 "C, and sample solutions were for rhGH as a function of column temperature have been stored at 4 "C. The pH values quoted for the different mobile correlated with Trp fluorescence changes and thus conforphases are apparent values measured for the aqueouscomponent mational structure alterations. before addition of the organic modifier. Procedures. Solution fluorescence spectra of the proteins The separation of the two hGH variants (rhGH and Metwere measured in a 1-cmspectroscopicquartzcuvette. For surface hGH) will also be examined. Surprisingly, these variants are fluorescence studies, a 10-pL injected volume of a 1-5 mg/mL separated isocratically or with a shallow gradient near neutral sample of the protein in mobile-phase A (5% organic modifier but not low pH and optimally at elevated t e m p e r a t ~ r e . ~ ~ , ~in~0.1 M phosphate buffer, pH 6.5, or 5% organic modifier in Under these conditions, separation may be a result of subtle 0.1% TFA, pH 2.1) was used. The detection limit (S/N = 2) for differences in hydrophobicity between the proteins that occur the protein at the excitation wavelength of 295 nm, at 45 OC, was 10 pg/mL. The collected spectra were baseline corrected and during a conformational or structural change. The results smoothed by the SLM spectrofluorometer program in order to suggest a potential approach to the chromatographic sepaminimize random noise. ration of closely related variants, an important issue in the The incubation solvent transported a 10-pL sample of the characterization of protein pharmaceuticals produced by protein into the packed flow cell at a flow rate of 0.3 mL/min. recombinant DNA technology. A 10-min linear gradient from 100%solvent A to 100%B (45% 1-propanol or 70% CHsCN in the aqueous buffer of mobilephase A) was used to elute the protein from the flow cell column. (17)Tanford, C. Adu. Protein Chem. 1968,23,121. (18)Morrisey, B. M.; Smith, L. E.; Stromberg, R. R.; Fenstermaker, Both the excitation and the emission monochromatorsof the C. A. J. Colloid Interface Sci. 1976,56, 557. SPF-500spectrofluorometercould be set in 1-nmmultiples; thus (19)McMillin, G. R.; Walton, A. G. J. Colloid Interface Sci. 1974,48, the precision of the direct emission maxima determination could 345. not be better than *1 nm. The precision was made worse by the (20) Darst, S. A.;Robertaon, C. R.; Berzofsky, J. A. J.Colloid Interface noise superimposed on the emission curves. To increase the Sci. 1986,111, 466. (21)Lochmuller, C.H.;Saavedra, S. S. Langmuir 1987,3,433. precision pf the emission maximum determination, the f i t (22) Lu. X.M.: Fimeroa, A.; Karger, B. L. J.Am. Chem. SOC.1988, moment, A, of the emission curve was determined instead of the 110,1978. curve maximum%in the wavelength range 305-395 nm: (23) Oroszlan, P.;Blanco, R.; Lu, X. M.; Yarmush, D.; Karger, B. L. J. Chromtogr. 1990,500,481. (24)Lewis, U. J.; Singh, R. N. P.; Tutwiler, G. F.; Sigel, M. B.; Vanderlann, E. F.; Vanderlann, V. P. Recent Prog. Horm. Res. 1980,36,477. (25) Vos de, A. M.; Ultsch, M.; Kossiakoff, A. A. Science 1992,255, 306. (26)Riggin, R. M.; Dorulla, G. K.; Miner, D. J. Anal. Biochem. 1987, 167,199. (27)Canova-Davis, E.;Teshima, G. M.; Kessler, T. J.; Lee, P. J.; Guzzetta, A. W., Hancock, W. S. In Analytical Biotechnology; Horvath, C.,Nikelly, J. G., Eds.; ACS Symposium Series No. 434, American Chemical Society: Washington, DC, 1990; p 90.
i;=
Cm,%/Cm,
where mi and Xi are the emission intensities in arbitrary unita and the respective wavelengths. The random noise was fist suppressed by summation of 10 successive scans, and then the first moment of the resulting summation curve was calculated. The first moment of the emission curve did not coincidewith the (28) Mulkerrin, M. G.; Wampler, J. E. Anal. Chem. 1982,64, 1778.
ANALYTICAL CHEMISTRY, VOL. 64, NO. 14, JULY 15, 1002
1625
60 1 55
a 45
30 0 0
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25
Flgure 1. Schematicillustration of the structure of rhQH based on that of porclne OH. Helices are represented by cylinders and labeled I-IV. In the case of Metan addltbnal methionyl group would be attached to the N-terminus. Note the Trp residue in helix I1 at position 86. (Adapted from ref 30).
emission maximum due to the emission band asymmetry; the differences between both quantities varied in the range 2-7 nm. Because of band asymmetry, the mean emission wavelength defined by the emission curve first moment is to be preferred over the emission maximum.= The moments were applied to the comparison of the red shifts observed in rhGH and Met-hGH eolutionscontaining different concentrationsof organicmodifiers. The standard deviation of the moment based on three sets of independent data was found to be *0.13 nm. In the study of the behavior of rhGH alone where highest precision was not required, the intensity ratio Z3m/Z360 at short (320-nm) and long (360-nm) emission wavelengths was used instead of the emission first moment to follow the shifts. A linear ratio and the first moment was dependence between the I~ZO/Z~~O found and could be expressed by the equation I320/Im = 23.4 - 0.065i
where xis in nanometers and the error in the shift determination was about A0.5 nm. The linear relationship between X and the Z ratio is a consequence of only one Trp present in the protein.%
RESULTS AND DISCUSSION Before exploring the chromatographic and spectroscopic results, it is useful to examine the structure and some of the properties of rhGH. The 3-dimensionalstructure of rhGH has recently been determined;%it is similar to the 3D structure of porcine growth hormone (pGH) shown in Figure l.30 Human growth hormone (hGH) has 191 amino acids with two disulfide bridges and contains a relatively high proportion of nonpolar residues, including one Trp at position 86 and eight Tyr's. On the basis of the structure, the Trp is buried within the core of the four-helix bundle and hydrogen bonded to Asp 169. Therefore, the Trp is a sensitive reporter group of conformational changes within the molecule, since alterations in the environment of this amino acid should reflect protein structural changes. Under physiological conditions the protein has approximately55 5% a-helical structure, which remains fixed over the pH range 2.5-11.0.31 Human growth hormone is highly resistant to denaturation; the helical regions are stable even when the tertiary structure is destroyed. In the case of Met-hGH an additional methionine group is attached to the N-terminus of the protein. (29) Demchenko, A. P. Eur. Biophys. J. 1988,16,121. (30) Abdel-Megiud, S. S.;Shieh, H. S.; Smith, W. W.; Dayringer, H. E.; Violand, B. N.; Bertle, L. A. Proc. Natl. Acad. Sci. U.S.A. 1987,84, 6434. (31) Wilhelmi, A. E. In Hormone Drugs,Proceedings of FDA-USP Workshop; Guerigrian, J. L., Bransome, E. D., Outshoern, A. S., Eds.; U.S. Pharmacopeial Convention, Inc.: Rockville, MD, 1987; p 369.
0
10
20
30
40
50
60
70
Temperature ['C]
Flgure 2. Effect of temperatureand pH on the RPLC retentlon of rhQH using 1-PrOH and CH&N as organlc modltiers: (0)l-ROH, pH 6.5; (0)l-ROH,pH2.1;(A)CH&N,pH6.5;(V)CH&N,pH2.1. Condltlone: 30-mln linear gradient from 5 % organic modlfh (1-ROHor CHsCN) to 40% 1-ROH or 70% CHaCN, in 0.5 M phosphate buffer (pH 6.5) or 0.1% TFA (pH 2.1).
Growth hormone is known to be very hydrophobic and to form easily associated ~tates.3~ It is interesting to note that under certain conditions a slight turbidity can be observed in aqueous protein solutions at concentrations above a p proximately 0.1 mg/mL, indicating some protein-protein association. In the case of bovine growth hormone (bGH), an intermediate state in the unfolding or refolding process by guanidine hydrochloride has been shown to form associated species, with the main contact area of association along the hydrophobic surface of helix 111.33 Given the a-helical character of this intermediate, the species could be viewed as the molten globule state of the protein, in which the position of the aromatic amino acid side chains are similar to those in the folded state. In contrast to bGH, it was recently shown that the unfolding of hGH in guanidine hydrochloridewas a twostate process with no observed ass~ciation.~~ The apparent absence of an equilibrium intermediate state was attributed to the relative stability of the native state in the specific unfolding pathway of hGH compared to that of bGH. Thus, strong conditions are necessary to alter hGH, and such conditions are presumed to be unfavorable for a stable intermediate in the unfolding pathway. With the above information, we first explored the RPLC behavior of rhGH. Figure 2 shows the effect of temperature on the gradient elution of rhGH using two different organic modifiers (1-PrOHand CH3CN). As can beseen, theretention decreased with temperature in the 1-PrOH buffer system, an is typical for RPLC;36however, the opposite trend was observed in the CH&N system; Le. retention increased with temperature, reaching a maximum at 45 and 56 OC at pH 2.1 and 6.5, respectively. A similar temperature effect was noted in a study on the thermodynamic parameters for the interaction of insulin with a reversed-phase column.% In addition, the pH of the mobile phase was observed to have little, if any, effect on retention in the 1-PrOH buffer system (32) Li, C. H.In Hormonal Proteins and Peptides; Li, C. H., Ed.; Academic Press: New York, 1975; Vol. 3. (33) Brems, D. N.; Plaisted, S. M.; Kauffman, E. W.; Hsvel, H. A. Biochemistry 1986,25,6539. (34) Brems, D.N.; Brown, P. L.; Becker, G. W. J. Biol. Chem. 1990, 265,5504. (35) Melander, W.R.; Nahum, A.; Horvath, C. J. Chromatogr. 1979, 185, 129. (36) Hancock, W. S.;Knighton, D. R.; Napier, J. R.; Harding, D. R. K.J. Chromatogr. 1986,367, 1.
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 14, JULY 15, 1992
(within &2%),whereas for CHsCN, at low pH the protein eluted at significantly higher concentrations of the organic modifier than at pH 6.5 for all temperatures investigated. Changes in pH thus significantly influenced retention in the CH3CN system. The above observations, in agreement with previous protein s t ~ d i e s , ~point ~ q ~to~ potentially *~~ different adsorption-desorption behavior for rhGH when 1-PrOH and CH3CN are used as organic modifiers in RPLC. Given this interesting contrast in retention, we decided to explore the reason for this behavior in more detail by means of intrinsic fluorescence spectroscopy. IntrinsicFluorescenceSpectroscopy. In order to utilize spectroscopic data in the elucidation of chromatographic retention mechanisms,it is necessary to compare the spectra of the protein in solution and on the surface as a function of the applied chromatographic conditions. Changes in fluorescence characteristics of the buried single Trp of rhGH which is hydrogen bonded to an aspartic acid can sensitively reflect changes in structure. Of course, since the whole molecule is not being probed, the lack of fluorescence change does not conclusively prove that some structure alteration far removed from the Trp has not occurred. At excitation wavelengths of 295 nm and above, the absorption and fluorescence emission are due primarily to Trp, minimizing the role of Tyr residues.39 Furthermore, the results presented below did not vary during the time of the experiments (1h);hence, kinetic processes were not considered to be important in this study. In addition the mobile-phase flow rate had no effect on the surface fluorescence measurements in the range 0 . 3 mL/min. 1-PrOHMobile Phase. A. Solution Fluorescence. Parta A and B of Figure 3 show plots of solution fluorescence characteristics (1320/I360) of rhGH at pH 6.5 and 2.1, respectively, as a function of 1-PrOH concentration and temperature (5,25,45, and 65 O C are shown; other temperatures, 15, 35, and 55 "C, were consistent with the results in Figure 3). In a purely aqueous solution of pH 6.5, the 1320/1360 value of approximately 1.0 (corresponding to X = 345 nm) was in approximate agreement with that found for the native state of rhGH.40 It is known that the Trp is buried and an emission maximum of -330 nm would thus be expected; however, the red shift to of 345 nm is a result of the H bond between Trp and Asp at position 169.41 A t temperatures over 25 "C, I320/ 1360 values were found to be slightly lower (i.e. a small red shift) with increasing temperature. As can be further seen, low 1-PrOH concentration (&lo%) did not significantly change the emission characteristics of the protein, relative to that in pure aqueous media. Upon an increase in the amount of 1-PrOH above 10% v/v at pH 6.5, a sharp change in 132o/IW could be observed at all temperatures (Figure 3A). 1320/1360 red-shifted toward 0.55 (X = 351.5 nm) withina relatively narrow concentrationrange of 1-PrOH. Furthermore, the higher the temperature the sharper the transition. The general behavior in Figure 3A is indicative of a cooperativestru~turalchange.~7 This alteration in structure has been separately confirmed by fluorescence lifetime measurements, CD, and UV spectral changes at 305 nm where the Trp-Asp hydrogen bond is measured.42Note
x
~~~
(37)Cohen, K. A.;Schellenberg,K.; Benedek, K.; Karger, B. L.; Greco, B.; Hearn, M. T. A w l . Biochem. 1984,140,223. (38)Hancock, W. S.;Knighton, D. R.; Harding, D. R. K. Peptides 1984;Almqvist and Wikeell International: Stockholm,Sweden, 19&1; p 145. (39)Lakowicz, J. R. In Principles of Fluorescence Spectroscopy; Plenum heas: New York, 1983;p 349. (40)Kauffman, E. W.; Thamann, T. J.; Havel, H. A. J. Am. Chem. SOC. 1989,111,5449. (41)Bewley, T.A.;Li, C. H. Arch. Biochem. Biophys. 1984,233,219. (42)Bathory,G.; Wicar, S.; Mulkerrin, M. G.; Karger, B. L. Manuscript in preparation.
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Flguro 3. Effect of 1-PrOH concentratlon on fluorescence emisslon 1 3 ~ /values 1 ~ of rhGH in solution at various temperatures: (0) 5 OC; (0)25 "C; (A)45 "C; (V)65 OC. Arrows lndlcate moblk-phase elution composltlon of the protein from the C, RP column. Condltlons: sample, 0.5 mg/mL mGH In 1-ROH; excitation wavelength, 295 nm; 0.1% TFA (pH 2.1). (A) 0.5 M phosphate buffer (pH 6.5); (6)
also that the endpoint of the transition at each temperature was close to the chromatographic elution composition, as indicated by arrows. The transition region and elution position were seen to shift to lower 1-PrOH values with an increase in temperature. When the mobile phase pH was changed to 2.1 (Figure 3B), the same red shift trends were observed as at pH 6.5, but the transitions were now less "cooperative"; i.e. at any temperature the transition proceeded more graduallythan at pH 6.5. Moreover, the transition appeared to begin earlier at pH 2.1, i.e. the value of 1320/1380started to decrease at a lower concentration of 1-PrOH than for pH 6.5. This result may be a consequence of the destabilizing effect of the acidic media on the protein structure43and the denaturing properties of TFA.17 The results suggest that the Trp environment of rhGH is somewhat more accessible at acidic pH, but it is also known that the helical "core" is only affected to a minor extent.31 The final conformational state is likely to be similar at the two pHs. The transitions in Figure 3A,B, in conjunction with other spectroscopic are indicative of a pronounced environmental change of the Trp residue which, as noted above, can be related to a conformational change of the protein, with the apparent formation of a similar structure at the (43)H a d , H. A.;Elzinga, P. A.; Kauffman, E. W. Biochim. Biophys. Acta 1988,955,154.
ANALYTICAL CHEMISTRY, VOL. 64, NO. 14, JULY 15, 1992 0.85
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Fbura 4. Effect of 1-RoH concentration on fluorescence emission Z,JZvalues of moH adsorbed on the C, RP support at various temperatwee. A 10-& aliquot of a 2.5 mglmL sduHon of rhGH In 0.5 M phosphate buffer (pH 6.5) was Injected Into the C, RP microcolumn. Excitalionwavelength = 295 nm. See Figure 2 for elution condltlons. (A) pH 6.5: (0)5 "C, (0)25 "C, (A)45 "C, (V)65 "C. (B) T = 45 O C : (0)pH 6.5, (0)pH 2.1.
endpoint of the transition at each temperature and pH investigated. We have already noted that the concentration of 1-PrOH necessary for elution corresponded to the completion of the conformational change. If the surface conformationalchangeswith 1-PrOHcomposition followed those in Solution, then this would suggest that desorption and elution were related to the observed structural change of rhGH (i.e. that elution was solvent driven). This picture has already been presented previously for the reversed-phase elution of standard proteins using 1-PrOH as organic modifier.44 In order to explore this model further, we therefore next examined the fluorescence change of adsorbed rhGH as a function of 1-PrOH concentration. B. Surface Fluorescence. Parts A and B of Figure 4 illustrate the effect of 1-PrOH, pH, and temperature on the emission characteristics of rhGH adsorbed on the Cd reversedphase surface. At pH 6.5 (Figure4A) and low organic modifier concentration (5 % ) at all temperatures, it can be seen that the 1320/Im values do not differ significantly from those observed in solution at the same composition and temperature (Figure 3A), e.g. 0.8 and 0.9 on the surface and in solution, respectively, for pH 6.5 and 5 % 1-PrOH, T = 25 OC. This correspondence could be attributed to the relatively stable (44)Sader, A. J.; Micanovic, R.; Katzenetain, G. E.;Lewis, R. V.; Middaugh, C. R.J. Chromutogr. 1984,317,93.
1627
structure of rhGH and suggest that the face of a-helix I1 (see Figure 1)which contains the tryptophan residue is pointed toward the solution phase. This conclusion is consistent with a-helix I11being the region of the hydrophobicsolute-surface interaction, as was the case in the association of the intermediate of bGH.= In the 15-25 % 1-PrOHconcentration region, a rather sharp change of the I320/1360 ratio of the protein was found (Figure4A),in agreementwith the change observed in solution. In addition, with higher temperatures the transition occurred at lower 1-PrOH concentration. Over 25% 1-PrOH, no measurement could be made due to .the elution of the protein from the microcolumn. The behavior of the adsorbed species at pH 2.1 was also similar to the solution results. Figure 4B shows the effect of changing mobile-phase pH from 6.5 to 2.1 on the I ~ ~ o / I w ratio at 45 "C. As expected, the transition was less sharp at the lower pH due to the destabilizing effect of the acidic media on the protein structure; however, at the endpoint of the transition I320/1360shifts close to 0.5 for both pHs, suggesting similar changes in the Trp environment. We also examined the structure of the desorbed species by measuring the fluorescence spectrum of the protein in flow cell I1 coupled in series to the packed flow cell. The fluorescence characteristics of the eluted species were found to be identical to the solution values at the same organic modifier concentration and temperature; i.e. the emission first moment was red shifted to 355 nm. This result provided further circumstantial evidence that a similar or related conformational change had occurred on the stationary phase as in solution. The correspondence of the solution and adsorption fluorescence behavior for rhGH with 1-PrOH as organic modifier suggests that desorption was solvent induced via a conformational change of the protein. It is likely that, in the c o m e of this change, solvation of exposed hydrophobic groups on the protein took place, and this led to elution. Interestingly, rhGH P0.3 mg/mL) formed a precipitate in the 1-PrOH concentration range 10-40 % v/v. However, no precipitate was observed if the rhGH was dissolved in 1-PrOH concentration above 40% (v/v) (see Figure 9). Moreover, less precipitate was observed during the structural change at room temperature than at 45 "C. The above behavior is similar to that observed for bGH with guanidine hydrochloride, where excess Gdn HCl led to redissolution of the pre~ipitate.~~ Interestingly, reduced and alkylated hGH was also found to yield precipitate in the unfoldingprocess.34 While the final state may well be different for hGH in 40% 1-PrOH relative to 5 M Gdn HC1, nevertheless, it is suggestive that a relatively stable intermediate can form in the pathway of conformational change. This intermediate, as in the case of bGH, may be able to associate hydrophobicallyin solution. It may be reasonable to assume that the addition of 1-PrOH at elevated temperature is sufficient to reduce the stability of the native state such that the intermediate may occur to a significant extent. This behavior may be an important point in the separation of rhGH from Met-hGH (see later). Finally, the solution-driven conformational change and subsequent elution were also likely a consequence of the significant amount of 1-PrOHadsorbed to the n-alkyl bonded phase.'s Such an adsorbed layer would in part present a less hydrophobic surface to the protein than the unsolvated C, alkyl chains themselves; in effect rhGH would appear to be partially protected from the strong hydrophobicforces of the surface. This conclusion is reinforced by the excellent recovery of the protein from the surface upon elution (data not shown). ~
(45) McCormick, R. M.; Karger, B. L. A d . Chem. 1980,52,2249.
ANALYTICAL CHEMISTRY, VOL. 64, NO. 14, JULY 15, 1992
1628
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Flgure 5. Effect of CH3CN concentration on fluorescence emlssion 1320/1360 values of rhGH In solution at various temperatures: ( 0 )5 "C; (0)25 "C; (A)45 "C; (V)65 "C. Arrows Indicate mobile-phase elution composltlonsof the protein from the C4RP column. Condltlons: sample, 0.5 mg/mL rhGH in CH3CN; excitation wavelength, 295 nm; (A) 0.5 M phosphate buffer (pH 6.5); (B) 0.1% TFA (pH 2.1).
Acetonitrile Mobile Phase. A. Solution Fluorescence. Parts A and B of Figure 5 show plots of 1320/1360as a function of CH3CN concentration at pH 6.5 and 2.1 and temperatures 5,25,45, and 65 "C. Again, other temperatures were explored in this range and were fully consistent with the trends shown in this figure. It is immediately evident that there is somewhat different solution behavior for rhGH with CH3CN than previously observed with 1-PrOH (Figure 3A,B). It is first to be noted that the change in the emission intensity ratio with organic modifier concentrationappears to be much more gradual than for 1-PrOH, particularly at pH 6.5. Even if surface tension of the solution is used as the abscissa rather than concentration of organic the change in the emission intensity ratio still remains more gradual for CH3CN. Moreover, the elution composition at low temperature, e.g. at 5 and 25 "C and at pH 6.5, as indicated by the arrows, occurs well before the point where the Trp is fully exposed to the solvent. Only at 45 "C and above does the elution appear to correspond to the complete redshift, as in the case of 1-PrOH. Interestingly, the change is not accompanied by a precipitate as in the case of 1-PrOH. In solution studies no opalescence was observed up to a protein concentration of 5.0 mg/mL at any CH3CN solution concentration tested. This fact may suggest that the effects observed may not be the same in the case of CH3CN as 1-PrOH. Indeed, separate studies based on fluorescence lifetime measurements point to the red shift being a
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of CH3CN concentration on fluorescence emission 1320/1380 values of rhGH adsorbed on the C, RP support at varkw temperatures. A 1 0 - ~ Laliquot of a 2.5 mg/mL solution of mGH In 0.5 M phosphate buffer (pH 6.5) was Injected In the C4 RP microcolumn. Excitation wavelength = 295 nm. See Figure 2 for elution conditions. (A) pH 6.5: ( 0 )5 "C, (0) 25 "C, (A)45 "C, (V)65 "C. (e) T I 45 "C: (0)pH 6.5, ( 0 )pH 2.1. Flguro 6. Effect
consequence of solvation rather than conformational change up to 45 0C.42Evidently, penetration of the protein by CH3CN is less than that by 1-PrOH also CH3CN may not be able to disrupt hydrogen intrapeptide bonds as well as 1-PrOH.17 In contrast to 1-PrOH, a change in solution pH from 6.5 to 2.1 with CH&N appears to result in greater structural and chromatographic change. For example, at 25 "C and 40% CH3CN the 1 3 ~ / 1ratio 3 ~ at pH 6.5 is 0.74 while at pH 2.1 it is 0.63. This result suggests that the lower pH may open the molecule to a greater extent than that at pH 6.5. B. Surface Fluorescence. Figure 6A shows the I320/Im values as a function of CHsCN concentration for rhGH adsorbed to the hydrophobic surface. It can be seen that for 5 and 25 "C the red shift is not very significant, and only at 45 and 65 "C does asubstantial red shift begin to appear. The results suggest that incomplete conformational change on the surface at these temperatures may occur when CH3CN is used. The 45 and 65 "C results appear more similar to that of 1-PrOH in that the red shift suggests the possibility of a conformationalchange in the elution region (>45% v/v CH3CN). Indeed, at 45 "C the solution fluorescence of the desorbed species presented in flow cell I1 agrees with the value measured in solution (see Figure 5A); Le. the red shift to = 355 nm has taken place. In addition, changes from pH 6.5 to 2.1 result in a further red shift at any composition (see
x
ANALYTICAL CHEMISTRY, VOL. 64, NO. 14, JULY 15, 1992
Figure 6B). This result is consistent with that found in solution; i.e. the protein is less stable in the acid medium in the case of CH3CN. The correspondencebetween the surface results of Figure 6A,B and the solution fluorescence results (Figure 5A,B) is again striking. It is suggested that the Trp residue is again pointed toward the mobile phase and can thus be potentially used as a probe of protein structural changes on the surface of the stationary phase. The results of Figures5 and 6 can help in the interpretation of the retention pattern observed for CH3CN in Figure 2. In contrast to 1-PrOH, the conformational changes in solution appear minor at least until approximately 45 OC at pH 6.5. The increase in retention with column temperature in Figure 2 may thus be due to the increased opening of the rhGH structure adsorbed to the reversed-phase surface with the rise in temperature. As the structure becomes more open, hydrophobic groups become exposed, leading to potentially stronger hydrophobic binding to the C4 surface or to other adsorbed rhGH molecules, as previously cited for other Thus, a stronger solvent (i.e. higher CH3CN concentration) is needed for elution to occur as the column temperature is raised. Above approximately 45 "C the migrationtime appears to level off with temperature increase. One interpretation of this result is that the surface conformational change is complete and the extent of binding to the stationary-phase surface becomes relatively constant with temperature. Finally, roughly parallel behavior in retention vs temperature is observed at pH 2.1 in Figure 2, except at any temperature, migration time is longer (i.e. more CH3CN is needed for elution) at the acidic pH. This result can also be explained on the basis of the greater extent of conformational change at any given CH3CN concentrationfor acidic pH leading to stronger protein binding. We have already noted the greater emission red shift corresponds to a more open structure of rhGH in acidic CH3CN media, e.g. Figure 5B. A comparisonof Figures 3 and 5 (solution) or Figures 4 and 6 (adsorption) reveals 1-PrOH to be a stronger modifier than CH3CN for inducing conformationalchange. This conclusion is not surprising given the greater molal surface tension lowering for 1-PrOH than for CH3CN. Moreover, for the same reason, at any given concentration of organic modifier, the extent of solvation of the C4 chains will be greater for 1-PrOH than for CH3CN.45 The surface-inducedconformational change in the case of CH3CN can lead to stronger binding of the protein to the stationary phase. At the same time, the possibility of protein-protein interaction while adsorbed to the surface will be greater as well. Such effects suggest that protein recoveries upon desorption using CH3CN as organic modifier may not necessarily be complete, as observed in other protein-RPLC ~tudies.'~J7 We therefore next examined recovery for rhGH using the CH3CN gradient. Protein recovery was determined by measuring the amount of desorbed rhGH in an HPLC fluorescence detector attached on-line to the flow cell. Minor corrections in the calibration plots for different CH3CN concentrations were taken into account. In the case of 1PrOH, recovery appeared to be complete, as evidenced by the absence of residual surface fluorescence at the conclusion of the gradient. However, as seen in Figure 7, recovery in the case of CH3CN at pH 6.5 was far from complete, being dependent on temperature and protein concentration. At 4 "C, recovery was complete and was thus set at 100% for the three protein concentrations of 1.0, 2.5, and 5.0 mg/ mL. Based on the low amount of CH3CN necessary for elution (see Figure 2) and the solution fluorescence of Figure 5A, it is reasonable to conclude that little or no conformational (46) Pearson, J. D.;Regnier, F.E.J . Liq. Chromatogr. 1983, 6, 511.
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1629
['C]
Flgwe 7. Effect of temperature and sample size on RPLC recovery of rhoH uslng CH&N as an organlc modlfler. Recovery Is estlmated by the amount of desorbed M.Condltlons: same as In Flgure 2. See text for details.
alteration of the structure had occurred under these conditions. As the temperature was raised to 25 and then 45 "C, recovery decreased. This trend can be correlated with the increased change of the structure of rhGH on the reversedphase surface. Not surprisingly, recovery was lower the greater the concentration of rhGH utilized. This result could be due to protein-protein interaction on the adsorbent surface creating a less soluble species. Recovery substantially increased as the temperature was raised. In summary, the RPLC behavior of rhGH is believed to be a consequence of conformationalchanges when both 1-PrOH and CH3CN are used as organic modifiers. However, the greater ease of conformational alteration in the case of 1PrOH appears in part to cause the elution behavior to be predominantly solvent induced, whereas in the case of CHsCN an important surface-induced conformational change is suggested. Central to the interpretation of this behavior has been the use of intrinsic fluorescence measurements in conjunction with the chromatographic results. As already noted, the intrinsic fluorescence measurements in this paper are further strengthened by other fluorescence and spectroscopic meas~rements.~~ On the basis of the results for rhGH, we next turn to the separation of rhGH from Met-hGH by RPLC. Separation of rhGH from Met-hGH. It has previously been shown that RPLC separation of the two variants, rhGH and Met-hGH, was possible at 45 OC, pH 6.5, and 28% 1PrOH.26,27Further results on this separation are shown in Figure 8A,B. In Figure 8A, the isocratic separation of the two variants is shown as a function of pH using 1-PrOHas organic modifier at T = 45 "C. Very poor separation was observed at low pH, with good resolution above pH 5.5, the optimum being near pH 6.5. Similar behavior was observed when CH3CN was used as organic modifier. An optimum column temperature was also found, as shown in Figure 8B. (In this case a shallow gradient was employed to obtain reasonableretention times.) Here, separation was best at 45 "C for both 1-PrOH and CH3CN; however, in agreement with Figure 2, retention was longest at 45 "C in the case of 1-PrOH and shortest for CH3CN. It is important to note that under all examined conditions that allowed separation, Met-hGH eluted later than rhGH. Given the results of Figure 8A,B and the studies on the retention behavior of rhGH, it was logical to presume that separation was somehow a consequence of conformational changes. We first explored the solution fluorescence of rhGH and Met-hGH to see if there were any differences in the stability of the two variants. In order to search for small differences, we measured the first moment (see Experimental
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ANALYTICAL
CHEMISTRY, VOL. 64, NO. 14, JULY 15, 1992
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Section) for the two variants under a variety of conditions. Figure 9 shows the change in first moment as a function of 1-PrOH concentration at 45 “C (the optimum temperature for separation). Because of the variability of the data in the transition region, no statistically significant difference in the 1-PrOHred shift curves of the two variants could be deduced. The similar stability behavior of the two growth hormones may be consistent with earlier studies which examined the proteins by a variety of physicochemical techniques and concluded that the proteins had similar if not identical 3D structure.47 Most interestingly in the present work, a small but measurable difference was observed for the two variants in their tendency to precipitate during the conformational change in 1-PrOH. As we have already noted, at the start of the 1-PrOH solution conformational change at 45 O C (-15% v/v 1-PrOH), we observed precipitation for protein concentration levels of 0.3 mg/mL or higher. With further addition of 1-PrOH the amount of precipitate increased and passed through a maximum at approximately 26 % v/v 1-PrOH, and at 40% v/v 1-PrOH a clear solution was again observed. Given the fact that precipitation may reflect on important bulk properties responsible for separation, we decided to (47) Jones, A. J. S.;O’Connor,J. V. Horomone Drugs, Proceedings of FDA-USP Workshop; U S . Pharmacopeial Convention,Inc.: Rockville, MD, 1982; p 335.
r
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Figure 0. Effect of 1-PrOH concentration on fluorescence r m b b n curve first moment. Conditions: protein concentratkns, 0.5 m~/mL, phosphate buffer (pH 6.5); T = 45 OC; excitation wavelength, 295 nm. Amount of precipitated protein as a functlon of 1-RoH concentration at 45 OC and at pH 6.5: (0) M,( 0 )Met-hQH.
explore this tendency in a quantitative fashion. The precipitated samples of protein from an originally 0.6 mg/mL, concentration and with different 1-PrOH concentrations were first centrifuged. The supernatant was removed, and the
ANALYTICAL CHEMISTRY, VOL. 84. NO. 14, JULY 15, 1992
1-PrOH concentration of this solution was increased to 40% v/v by the addition of 1-PrOH. The fluorescence emission intensities of these samples were measured and compared to a calibration curve of the protein at 40% v/v 1-PrOH (where no precipitation was observed). After appropriate volume corrections, the concentration of protein in the supernatant could be determined, and from this value the amount of protein in the precipitate could be assessed. Figure 9 shows the precipitation curves as a function of 1-PrOH concentration for Met-hGH and rhGH at 45 "C. For both proteins, there was a maximum in the extent of precipitation at roughly 50% of the amount of protein in solution. Beyond this maximum, 1-PrOH solvation predominated and the amount of precipitate was reduced until there was no precipitate observed at 40 % v/v 1-PrOH. As already noted, in the case of bGH, guanidine hydrochloride concentrations lower than 2 M were necessary to observe the precipitate.& Above this concentration the precipitate resolubilized. Significantly, despite very similar aggregation properties in aqueoussolution, Met-hGH began to precipitate at 1-PrOH concentrations lower than rhGH. In addition, Met-hGH appeared to reach the maximum in precipitation at a concentration of 1-PrOH lower than rhGH. As in the case of bGH,& it is reasonable to assume that precipitation was a consequence of hydrophobic interactions between protein molecules. The results in Figure 9 suggest that, in the process of the conformational change, Met-hGH appears to be somewhat more hydrophobic than rhGH. This increased hydrophobicity in the course of the conformational change may be a consequence of a greater accessibility of helix I11 (the known face for hydrophobic attachment for bGH33)or simply the hydrophobic nature of the Met residue itself.49As we have noted, in analogy to bGH, there is likely to be an intermediate in the pathway of structural change, and it would appear this intermediate is more hydrophobic for Met-hGH. It is necessary to emphasize that differencesin the two variants were observed in the process of conformational change, not in the native state itself (at low 1-PrOH concentration) or after the change had occurred, e.g. 40% v/v 1-PrOH. The behavior of Figure 9 can be used to interpret the separation, as well as the elution order, in the case of 1-PrOH. As we have already discussed, with increasing 1-PrOH concentration a solvent-induced conformational change occurred, leading to enhanced solvation of hydrophobicgroups and subsequent desorption and elution. Quite possibly, in the process of the conformational change, Met-hGH may become sufficiently more hydrophobic than rhGH that it would bind more strongly to the surface, requiring a greater amount of 1-PrOH for subsequent elution. This picture can be further used to interpret the temperature trends in Figure 8B where temperatures below 45 "C led to poorer separation. In agreement with earlier studies on bGH,& it was found that the extent of precipitation and hence conformational change decreased as the temperature was lowered. The hydrophobic difference between the two variants was therefore less pronounced. One possible reason for the lower amount of precipitation could be due to lower stability of the native state at the elevated temperature, leading to smaller thermodynamic differences between the native and the intermediate states. It should also be noted that the entropy-driven hydrophobic interactions would be expected to be stronger at the higher temperature. Finally, as noted, the solution behaviors of Met-hGH and rhGH in Figure 9 differ from that found in the guanidine hydrochloride unfolding of hGH.34 Whereas bGH can precipitate in this unfolding process, hGH was observed to be a two-state (48)Brems, D.N.Biochemistry 1988,27,4541. (49) Gellman, S. M. Biochemistry 1991, 30, 6633.
1631
unfolding proce~s.~3*W Besides the relative stability of the native and intermediate states, it needs to be recognized that the structural change in the case of 1-PrOH differs from that for guanidine hydrochloride. It is known that alcohols can enhance a-helix structure?l and this property may also increase the likelihood of formation of the molten globule state. This point will be discussed ~eparately.~~ Turning next to CHsCN, we have already noted that similar optimum conditions for separation of the two varianta were observed as for 1-PrOH, e.g. 45 "C and pH 6.5, and the elution order was similar as well. It is possible that separation was again related to a conformational change. If we w u m e hydrophobic protein-surface and adsorbed protein-protein interaction, then the hydrophobic difference of the two varianta exposed during the conformational change with 1PrOH may be observed for this organic modifier a~ well. In this case, the process may be surface driven. It may therefore be possible that the separation factors were somewhat related in the two systems.
CONCLUSIONS
This paper has explored the causes of RPLC retention behavior of rhGH as a function of organic modifier, temperature, and mobile-phase pH, as well as the separation of two varianta that differ by the addition of only one amino acid residue in approximately 200 residues. Central to developing this understanding has been the utilization of extrachromatographic data in exploring protein structural changes. The work demonstrates the value of solution and surface intrinsic fluorescence spectroscopy in elucidating potential separation mechanisms. The potential pivotal role of conformation change on the retention behavior has been emphasized. In agreement with earlier studies,u a conformational change, followed by enhanced solvation of the structurally altered state, is a possible mechanism of elution of proteins by 1-PrOH. Moreover, conformational change was a necessary ingredient to create a property difference between the two variants. This principle of the use of controlled conformational manipulationto induce property differences in closely related species would appear to be, in specific cases, a useful approach for variant separations. A knowledge of the relative stability of the molecule under various conditions ought to offer a logical starting point for seeking separation conditions for variant resolution. Of course, the principle of conformational manipulation is a frequently utilized approach by nature in the mechanism of biological function (e.g. see ref 17). It is worth noting that another variant separation mechanism involves specific protein surface contact with the adsorbent in the region of primary structure protein differences.3 Work is continuing on the elucidation of the structural details of the changes involved with the addition of organic solventa to solutions of growth hormone and related ACKNOWLEDGMENT B.L.K. gratefully acknowledges support of this work by NIH under GM-15847. We acknowledge the work of Mr. J. Caccia who carried out the separations shown in Figure 8B. This is contribution No. 498 from the Barnett Institute. RECEIVED for review January 28, 1992. Accepted April 6, 1992. Registry No. GH, 9002-72-6;CHsCN, 75-05-8; 1-PrOH, 7123-8. (50) Fridman, M.;Aguilar, M.I.; Hearn, M. T.W.J. Chromatogr. 1990,512, 57. (61)Acharya, S. hoc. Natl. Acad. Sci. U.S.A. 1987,84,7014.
