Peptide chemistry: Development of high performance liquid chromatography and capillary zone...

Charleen Miller

Peptide Chemistry: Jean Rivier The Salk Institute

for Biological Studies Development of High The Clayton Foundation

Laboratories for Performance Liauid Chromatography and Capillary Peptide Biology

100 10 North Torrev Pines Road La Joia. CA 92037 Zone Electrophoresis *

The development qfrelatively non-compressible supporting media qf small particle size as well as piimps that u‘eliver constant flow rates at high pressures has enabled investigators to perform rapid, high resolzit ion liquid chromatography jbr more than two decades. Studies initiated in this laboratory in 1975, evaluating the compatibility of unprotected peptides with commercially available chromatographic supports and development ofsolvent systems ultimately led to separations not previoiisiy observed with both synthetic peptides and native peptides from tissue extracts. It MUS rapid1.v realized however, that recovery of certain molecules could be problem- atic. To meet the challenges presented by the isolation ofnatural hormones (such as corticotropin releasing,fuctor and growth hormone releasing hormone) and proteins (such as inhibin and activin) and thrl need,for large quantities of highly purified peptides,for clinical invesstigations, our group invested heavily in identifving new supports (high carbon loading and 300 A” pore sizes) and solvent systems (triethylammonium phosphate and trijluoroacetic acid) compatible with reverse phase, size exclusion and ion exchange chromatographies .from a practical and economical perspective. More recently, we have contributed to the identification of unusual buffer systems (inclusive of organic mod$ers) compatible with capillary zone electrophoresis that will both tnodzilate the capillaries’ selectivity, increase resolution and serve as an orthogonal approach to determining peptide purity. From a pragmatic point of’ view, in this paper we highlight the original and timely contributions (technical and strategical) ofthis laboratory in the Jield qf analytical and preparative high performance liquid chromatography and capillary zone electrophoresis of synthetic and native biologically active peptides and proteins over the past twenty years. 0 1996 John Wiley & Sons, Inc.

INTRODUCTION AND RECOLLECTIONS

Peptide synthesis has been an ever expanding challenge as the length and complexity of the molecules being duplicated was increased. There is no doubt that in the 1950s and 1960s, the pillars of this dis- cipline met with innumerable and often insur- mountable difficulties that they addressed with all possible tools available to them. Their stepwise suc- cesses through the use of different strategies and

protecting groups in solution came with difficulty. This may explain why it was so difficult for most of these pioneers to abandon their rigor at identifying intermediates (for the purpose of ascertaining the purity of their final products), in order to grasp the elegance of the solid phase approach to peptide synthesis and embrace it. What these pioneers were unable to share were, in fact, their good intentions for a field that they represented and cherished. Ex- haustive characterization and identification of

* Dedicated to the late Dr. Roger Burgus. Biopolymers (Peptide Science), Vol. 40,265-3 17 ( 1996) 0 1996 John Wiley & Sons, Inc. CCC 0006-3525/96/030265-53

265

266 Miller and Rivier

their intermediates and final products was paramount. In terms of peptide isolation and characterization, they had mastered the art of crystallization, the beauty of counter current distribution (CCD), and the subtleties of thin layer chromatography. They had developed an intimate relationship with the family of peptides to which they had dedicated their lives. It was therefore not only blasphemy but anarchy to report on any peptide that would not have answered to all criteria of purity defined at that time by a powerful elite. Sharing these conser- vative convictions with a new generation of chemists dreaming of competing with and besting nature in her ability to design and make proteins led to the rift seen in the early 1970s between the advocates of classical solution synthesis of peptides and proteins and the solid phase approach.

Entering the peptide field from that of photo- chemistry where photon-induced isomerizations of nonconjugated double bonds led to novel classes (quadricyclanes) of small multicyclic crystalline molecules easily identifiable by nmr, it was particularly disturbing to find out that there were no quantitative methods to measure peptide purity independently from its origin. The vast experience of Dr. R. Burgus, who isolated and characterized ovine thyrotropin releasing hormone (TRH), lu- teinizing hormone/ follicle stimulating hormone releasing hormone [LH/FSH RH or GnRH (gonadotropin releasing hormone)], and somatostatin from huge quantities (i million) of sheep hypothalami in the laboratory of Dr. R. Guillemin, served us well during the purification and characterization of synthetic TRH and GnRH analogues. At the time, preparative thin layer chromatography, ion exchange chromatography, and partition chromatography on Sephadex supports were quite adequate, in most cases, to purify solid phase-generated mixtures to a high degree of purity [ >95% pure as ascertained many years after they had been synthesized using reverse phase high performance liquid chromatography (RP-HPLC, to be referred to as HPLC in this review unless otherwise specified) and capillary zone electrophoresis (CZE)]. Yet it is only with the discovery in late 1975 that reverse phase chromatography could separate very closely related synthetic unprotected peptides that we were finally freed from the hang- ing Damocles sword of our elders and were able to set new criteria of purity for peptides independently ofthe method used for their synthesis. These results were reported one year later at the European Peptide Symposium in Wepion, Belgium.' It was

very rewarding to see how quickly this technique was adopted by the peptide community at large. We hope to illustrate the fact that synthetic peptides will rarely, if ever, be homogeneous, but can readily be brought to a very high degree of purity using techniques that are now available. Although most uses for HPLC in the peptide chemistry laboratory were illustrated in our original paper, the number of instruments available, as well as the diversity of chromatographic supports and mobile phases, has increased dramatically. There are now numerous schemes using HPLC as one of the automated steps for submicrogram characterization as well as instruments for multigram purification of peptides and proteins. We will illustrate here, chro- nologically and by issues, some of the advantages and limitations of HPLC that we identified over the last 20 years through the use of complementary techniques such as analytical ion exchange chromatography and CZE.

REVERSE PHASE HIGH PERFORMANCE LIQUID CHROMATOGRAPHY: STEP I

By 1975, relatively noncompressible supporting media of small particle size as well as pumps that delivered high, steady flow rates had been developed, which enabled investigators to perform high resolution liquid chromatography at rapid flow rates with high column inlet pressures of up to 6000 psi. This new application of liquid chromatography, termed high pressure/ performance liquid chromatography, had been widely applied in the separation and analysis of a number of biologically important corn pound^,^.^ but there were, at the writing of our first report, I comparatively few reports on its application in the peptide field. HPLC of peptide derivatives such as phenylthio-, carbamyl-, or dimethylami- noaphthylsulphonyl-peptides4~5 or naturally occurring alkylcyclodepsipeptides, had been described. However, reports on separations of undenvatized peptides were still very limited. Schecter7 had described the separation of proteins on porous silica de- activated by Carbowax and Pickart, and Thaler' had reported the use of HPLC on silica gel to partition growth promoting peptides and proteins as well as histones. Finally, Tsuji and Robertson' had used reverse phase partition HPLC to separate peptide anti- biotics that were quite hydrophobic. In our first report, we described the use of HPLC in the separation of small hydrophilic peptides and peptide analogues ( (30 residues) that were closely related in structure. In doing so, we identified most analytical and prepar-

Peptidr Chemistry 267

ative applications of this powerful quantitative technique. From then on, we felt freed of the constant fear resulting from not knowing whether our synthetic peptides made incrementally on solid supports were as good as we thought they were or as bad as they were predicted by others to be. Interestingly and as illustrated below, there was still much to learn.

Most HPLC separations of peptides were, and still are, carried out at low pH. The eluting phase often referred to as “buffer” used originally contained a relatively low concentration of volatile ammonium acetate. ‘ , l o This buffer was first applied to HPLC because it was most commonly used for ion exchange chromatography of peptides and proteins at the time in our laboratories, had minimal absorption in the low uv range (2 10-220 nm), and was volatile. The pH and ionic strength of this buffer could be adjusted and it could be eliminated by repeated lyophilization form dilute acetic acid. This buffer, in combination with acetonitrile and the reverse phase columns available, yielded previously unprecedented separations as illustrated in Figures 1 and 2. Not only could these peptides (see Table I for structures) be baseline separated, but with some reasonable assumptions (e.g., all peptides absorb equivalently at 210 nm) could also be quantitated and recovered for further testing. Quantitation is obviously much more accurate if one compared members of the same family of peptides such as closely related impurities in synthetic crude or partially purified mixtures. As can be seen, isocratic conditions were used and, although the small hydrophilic peptides such as [ Arg’] vasopressin ( AVP), angio- tensin I1 (AII), gonadotropin releasing hormone (GnRH), neurotensin (NT) and a-melanocite stimulating hormone ( a-MSH ) yielded symmetrical elution profiles, the more hydrophobic peptides such as the human fragment (residues, 18-39) of adrenocor- ticotropin hormone ( hACTHI8-39), substance P (SP), and porcine glucagon (pGluc) showed a slightly tailing distribution. The possibility of such molecules and a number of their impurities to be sep arated in a matter of minutes, recovered from the eluting phase and, as we will see, quantitated, could not be matched by any other technique available at the time (partition chromatography on gels or in a CCD apparatus, thin layer chromatography, and electrophoresis).

It was amply illustrated that closely related analogues of the decapeptide GnRH, whereby one of the residues was deleted or substituted by the corresponding D-isomer, could be separated (see Ta- ble 11). Even more striking at the time was the observation that the introduction of a single methyl

group in a decapeptide (compare retention times of GnRH with that of [Ala6]GnRH) would generate a pair of analogues that could be easily separated in minutes. Table I1 includes examples of peptides that might occur as failure sequences in solid phase syntheses such as des- Gly6-GnRH and des-Ser4-GnRH, which can be separated from GnRH in this system. The diaste- reoisomers [ o-Ala6]GnRH and [ L - A ~ ~ ~ I G ~ R H were also separated. The sequence of the elution of [ o-Lys6]GnRH and [ D-Tyr6]GnRH followed the order that would be predicted on the basis of hydrophobic interactions with the chromatographic support.

Similarly, most analogues of the more hydrophobic tetradecapeptide somatostatin ( SRIF), could be separated from its diastereomers (except for [D-Phe”]SRIF) just as each of the diastereomers reported could be isolated from each other (see Table 111). The large difference in retention times (RT) of [ D - P ~ ~ ~ I S R I F and [D-PhelllSRIF might be explained by conformational differences between these two peptides that result in the exposure of more hydrophobic groups in the case of [ ~-Phe‘] SRIF. Even more striking is the difference in RT of [ Asp’] SRIF and SRIF (substitution of a side-chain amide by a carboxylic acid) compared with the much smaller one observed between [ Cys 14-NH2] SRIF and SRIF ( not baseline separated), which also results from the presence of an amide vs a carboxylic acid. It would seem unlikely that this large difference in RT could be entirely attributed to the difference in polarities of a protonated carboxyl and a carboxamide group; it was speculated at the time that the exposure of hydrophobic groups was affected by conformational differences between the two compounds. It is interesting to note that this observation, made in 1976, served as the basis of a rationale for quantitative structure activity re- lationships in the groups of Drs. M. Hearn’’,I2 and R. HodgesI3 fifteen years later.

These results showed that many peptides and their closely related structural analogues could be resolved well enough to aid in their identification and characterization. For determination of homogeneity of synthetic and natural peptides, however, it was recognized that it was not enough to merely separate components; it was also necessary to resolve them well enough to be able to quantitate the presence of a small amount or even a trace of some impurities in the presence of a large amount of the major product, a requirement usually much more difficult to meet using chromatographic methods. Figures 2 and 3 illustrate that trace amounts could


A 0

A11

0.1 100

E z 2

m B N - 0

v)

3 U s a

25 20

0 0 0 4 8 12 0 4 8 12

Time (min)

C

SRlF

D

SRlF

SP

I

A I L L < 2 4 6 8 1 0 1 2 2 4 6 8 1 0 1 2

Time (min)

00

z x 0 s

3 0

I

FIGURE 1 Isocratic separation of bioactive peptides using HPLC (panels A-D): Peptides (ca. 10 pg ea.) on pBondapak/C18 (10 pm particle size) column (0.4 X 60 cm). Buffer was CH3CN/0.01 MNH40Ac, pH 4; flow rate, 2.5 mL/min.

not only be detected by HPLC, but that they could also be quantitated to some extent. In Figure 2 it can be seen that, within 6 min, as little as 0.7% of [ D - H ~ s ~ I T R H can be determined in the presence of TRH (purified by HPLC). In Figure 3 we illustrate an even more difficult separation to quantitate; that is one in which the trace component fol- lows the main product, because the slight tailing of a large mass of a given molecule often interferes with the integration of a subsequently emerging minor component. Nevertheless, by reducing the organic solvent concentration and thus lengthen- ing the retention times, such a separation was achieved, as shown in Figure 3. A chromatogram

of [o-His2]GnRH (purified by HPLC in 0.5 mg batches of synthetic compound in this same system) showed only a trace of GnRH (the L-His2 containing diastereoisomer) if any. Chromatogra- phy of a mixture of this preparation with 0.5% of added GnRH showed a definite absorbance that was integrated as 0.1%. As can be seen, 1% added GnRH could easily be detected and properly integrated.

Should it be noted here that the introduction of histidine in peptides at the time was a challenge due to the tendency of histidine to racemize during ac- tivation (not only in solution but also on solid phase)? This challenge was essentially impossible

Peptide Chemistry 269

-

I-His21- T 1

0.01

z 0

N

m v) LL 3

7

c

a

0

C

TRH 1001-1

3H

A

TRH 50 pg

0.36 1-19

0.7%

0 2 4 6 8

Time (min)

6

z $ 0 ,\“

0.5 0

FIGURE 2 Separation of [DHis’ITRH and TRH by HPLC on pBondapak C I 8 (10 pm particle size) column (0.4 X 30 cm). Buffer was0.01MNH40Ac, pH 4; flow rate, 3 mL/min.

to address in the absence of a proper analytical system.I4 It is obvious that when testing the hypothesis that the introduction of a D-amino acid in a biologically active peptide such as the introduction of D-histidine in TRH or GnRH may yield a competitive antagonist, it is paramount that the analogue be devoid of any of the extremely potent L-isomer containing parent compound. Whereas [ D-H~s’ITRH was found erroneously to be as much as 50% as potent as TRH and [ D H ~ s * ] G ~ R H 10% as potent as GnRH by a number of investigators, we found that HPLC- purified [ D-His’] TRH and [ D-His’] GnRH were essentially inactive as agonists and antagonists. l 5 This demonstrated to us that from 100 to 20% racemka- tion could occur during the synthesis of TRH or GnRH and their analogues, respectively. The data presented in our original paper I therefore struck a very sensitive chord in the heart of most peptide chemists.

Whereas we have eluded to the (semi)preparative use of HPLC for the purification of synthetic samples for biological testing and analysis, it took further developments in terms of column technology and buffer systems to reach the present state of the art whereby kilogram quantities of peptides are purified using Good Manufacturing Pro- cedures. Yet when addressing the purification of minute amounts of small naturally occurring biologically active peptides, this technique was already most efficient. This was illustrated in the purification of a-endorphin from a partially purified pituitary extract (1.5 mg) after size exclusion, ion exchange, and partition chromatographies. a-En- dorphin is one of a series of peptides related to P- lipotropin (P-LPH) that compete with morphine at opiate receptors. l 6 a-Endorphin has the structure

shown in Table I, which corresponds to that of P- LPH (61-76).17 The major absorbance (at 7-8 min) contained the desired peptide among at least 20 other components (Figure 4). It should be noted that as the amount of acetonitrile increased, there was a dramatic shift in the baseline as the concentration of ammonium acetate decreased. This rendered the use of gradients somewhat impractical despite the introduction of some ammonium acetate in the organic (generally referred to as B) buffer. In this case, as in the “preparative” chromatography of TRH and GnRH isomers, we stated that “the preparative applications of this technique fall short of obtaining gram or kilogram quantities necessary for such purposes as extensive clinical tri- als, but enough peptide can be purified for most chemical and biological characterizations in the laboratory. . . . The studies just presented were by no means exhaustive in determining the ideal systems for individual peptides and were limited to peptides of 30 amino acid residues or less, but we felt that these data may begin to indicate the power and scope of the application of HPLC using non- polar bonded phases for synthetic and natural peptides, not only on an analytical scale, but also on a semi-preparative scale” (Ref. 1, page 93).

Isolation of peptides of biological interest from synthetic mixtures, as well as from natural sources, could now be accomplished with relative ease. For example, we applied such systems (ammonium acetate buffer/ CH,CN) to characterize neurotensin and bombesin analogues r e ~ p e c t i v e l y . ~ ~ ~ ~ ~ Ling et a1.20 isolated a- and y-endorphins from natural sources. Rivier et a1.21 demonstrated the homogeneity of synthetic somatostatin and glucagon-selec-


Table I Described in this Compendium

Structures and Molecular Weight (MW) of Peptides, the Chromatographic Properties of Which Are

-

hACTHI8-39

AVP A11 Azaline BN oCRF

r/hCRF

a-endorphin P-endorphin

G V l A

pClucagon

m G n R H h C R F

(porcine)

GRP

H2Al-53 amide

Inhibin-3 I-OH

NT SP SRIF SRIF-28

TRH

Arg-Pro-Val-Lys-Val-Tyr-Pro-Asn-Gly-Ala-Glu-Asp-Glu-Ser-Ala-Glu-Ala-Phe-Pro-Leu-

Cys-Tyr-Phe-Gln-Asn-Cys-Arg-Pro-Gly-NH2 M W 1083 Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-OH MW 1045 Ac-~-NaI-~-Cpa-D-Pal-Ser-Lys(Atz)-D-Lys( Atz)-Leu-ILys-Pro-D-Ala-NH2 MW 1 546 pGlu-Gln-Arg-Leu-Gly-A~n-Gln-TrpAla-Val-GIy-His-Leu-Met-NH~ M W 16 1 9 Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-

Glu-Phe-OH MW 2467

Met-Thr-Lys-Ala-Asp-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Leu-Asp-Ile- Ala-NH, MW 4665

Met-Ala-Arg-Ala-Glu-Glu-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Met-Glu-Ile- Ile-NH, MW 4753

Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Clu-Val-Leu-Glu-

Tyr-G1y-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr-OH M W 1 774 Tyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-G1n-Thr-Pro-Leu-Val-Thr-Leu-Phe-Lys-Asn-

Cys-Lys-Ser-Hyp-Cly-Ser-Ser-Cys-Ser-Hyp-Thr-Ser-Tyr-Asn-Cys-Cys-Arg-Ser-Cys-Asn-

His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr- Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-

pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 MW 1 182 Tyr-Ala-Asp-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-GIn-Leu-Ser-Ala-Arg-Lys-Leu- Leu-Gln-Asp-Ile-Met-Ser-Arg-GIn-Gln-Gly-Glu-Ser-Asn-Gln-Glu-Arg-Gly-Ala-Arg-Ala- Arg-Leu-NH, MW 5035

Trp-Ala-Val-Gly-His-Leu-Mt-NH, MW 2785

Ala-Gly-Leu-Gln-Phe-Pro-Val-Gly-Arg-Val-His-Arg-Leu-Leu-Arg-Lys-Gly-Asn-Tyr-Ala- Glu-Arg-Val-Gly-Ala-Gly-Ala-Pro-Val-Tyr-Leu-Ala-Ala-NH, MW 5605

His-Asn-Lys-Gln-Glu-Gly-Arg-Asp-His-Asp-Lys-Ser-Lys-Gly-His-Phe-His-Arg-Val-Val-Ile- His-His-Lys-Gly-Cly-Lys-Ala-His-Arg-Gly-OH M W 359 1

pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu-OH MW 1672 Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gl y-Leu-Met-NH, M W 1 346 Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH M W 1 638 Ser-Ala-Asn-Ser-Asn-Pro-Ala-Met-Ala-Pro-Arg-Glu-Arg-Lys-Ala-Gly-Cys-Lys-Asn-Phe-

pGlu-His-Pro-NH2 MW 362

Ala-Ile-Ile-Lys-Asn-Ala-Tyr-Lys-Lys-Gly-Glu-OH M W 346 I

Hyp-Tyr-Thr-Lys-Arg-Cys-Tyr-NH, MW 3035

Phe-Val-Gln-Trp-Leu-Met-Asp-Thr-OH MW 3480

Ala-Pro-Val-Ser-Val-Gly-Gly-Gly-Thr-Val-Leu-Ala-Lys-Met-Tyr-Pro-Arg-Gly-Asn-His-

Ac-Ser-Gly-Arg-Gly-Lys-Gln-Gly-Gly-Lys-Ala-Arg-Ala-Lys-Ala-Lys-Ser-Arg-Ser-Ser-Arg-

Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH MW 3 137

tive somatostatin analogues made in gram quantities by solid phase procedures.

During the course of these studies we became aware of poor recoveries, especially when dealing with polypeptides (P-endorphins) and small proteins (insulin in particular). This could be best demonstrated when we started using HPLC for the purification of radioactive tracers (data never reported until adequate conditions were found, see below) or when trying to recover corticotropin releasing factor (CRF) activity from tissue extracts (see below). This observation was particularly troublesome as it was obvious that the validity of any analytical technique depended to a great degree on the ability to observe all components of the given mixture.

Conditions in Figure 5 , for example, are those that were used tentatively to separate human &endorphin from ovine &endorphin (identical to [ Hiss7, Gln9']-human ,8-lipotr0pin~,-~~ and from porcine /3-endorphin (identical to [ Vals3, Hiss7, Gln 9' ] human P-lipotropin, ) .22 It should be noted that the only difference between &-endorphin and &-endorphin is the presence of an extra methyl group at position 83 of the original /3-LPH sequence (the substitution of one Ile residue for Val). The basic conditions used for that separation (pH > 8), however, were found to be detrimental to the column, which finally collapsed due to the postulated slow dissolution of the support.22

We were thus faced with having to either retire columns after about ten experiments or with find-


Table I1 HPLC of GnRH and GnRH Analogues (see Table I for Sequences)

Compound Conditions" R T ~ (min)

GnRH Des-Gly6-GnRH Des-Ser4-GnRH [Sar' 'IGnRH [ D - A I ~ ~ I G ~ R H [ L - A I ~ ~ I G ~ R H [ D - L ~ s ~ I G ~ R H GnRH [ L - A I ~ ~ I G ~ R H [ D - T ~ ~ ~ I G ~ R H

22% EtOH/0.01MNH40Ac, pH 4, 1.5 mL/min 22% EtOH/O.O 1 M NH40Ac, pH 4, 1.5 mL/min 22% EtOH/O.O 1 MNH40Ac, pH 4, 1.5 mL/min 22% EtOH/0.01MNH40Ac, pH 4, 1.5 mL/min 22% EtOH/0.01MNH40Ac, pH 4 ,4 mL/min 22% EtOH/0.01MNH40Ac, pH 4 , 4 mL/min 22% CH3CN/0.01MNH40Ac, pH 4 ,2 mL/min 22% CH,CN/O.O 1M NH40Ac, pH 4 , 2 mL/min 22% CH3CN/0.01MNH40Ac, pH 4 , 2 mL/min 22% CH3CN/0.01MNH40Ac, pH 4 ,2 mL/min

11.8 12.7 12.5 13.0 5.9 6.5 3.5 6.2 7.7

13.0

a pBondapak CI8, 0.4 X 30 cm. RT = retention times from injection.

ing new systems that would be more compatible with the column supports. The loss of the peptides seemed to be due essentially to irreversible binding to the column through high nonspecific hydrophobic or ionic interactions. The observation that the use of basic conditions (showing unique separations such as that shown in Figure 5 ) leading to the slow dissolution of the column pointed the way to the use of detergents (or ion pairs), as originally suggested by G. Hawk (from Waters Associates) for full recovery. We had shown that the addition of sodium dodecyl sulfate (SDS) in our buffer dramatically increased recoveries, but were reluctant to systematically use it even for analytical purposes since recovery of samples of biological interest would then require removal of the SDS, thus making it impractical for use on a regular basis.

By using a sodium phosphate buffer,22 which was nonabsorbing in the uv range where the absorption ofa peptide bond is maximal (i.e., 190 nm and above),23,24 we found that when running gra-

Table I11 Analogues (see Table I for Structures)

HPLC of Somatostatin and Somatostatin

Compound RT (min)"

Somatostatin (SRIF) [Asp'ISRIF [D-C~S'~]SRIF [D-Phe' 'ISRIF [~-ser ' , ]sRIF [ D - T ~ ~ ' ~ ] S R I F [ D - P ~ ~ ~ I S R I F

12.5 24.2 15.1 12.7 15.2 15.6 19.4

a pBondapak CI8, 0.4 X 30 cm, 35% EtOH/O.OIMNH,OAc, pH 4.0, 1.5 mL/min.

dients we could keep level baselines by avoiding the absorption of the carbonyl group of the acetate or formate counterions originally used. We were determined to use 200-2 10 nm as the wavelength of detection so that we could keep high sensitivity and reliable response for any peptide bond containing components of our test mixtures. This was clearly in contradistinction with the approach of Uden- friend and collaborators, who used postcolumn derivatization (orthophthalaldehyde) and the volatile pyridine formate based buffer for increased recovery and ~ens i t iv i ty .~~

An ideal chromatographic solvent system, then, should give high resolution and high recovery for closely related peptides or proteins; the ideal system would also be sensitive (nonabsorbent in the uv); would give reproducible results from day to day; would be versatile, so that it could possibly be used for the separation of other classes of compounds; would be compatible with the support and the pumps (i.e., be noncorrosive or not too viscous), and last but not least, would be compatible after the elimination of the organic solvent with in vitro and in vivo biological systems, so that additional procedures such as lyophylization, size exclusion chromatography, or dialysis could be avoided in the screening of columns for biological activities. Triethylammonium phosphate (TEAP) in particular and trialkylammonium salts in general were found to fulfill these criteria and allow for a variety of pH ranges.22

It is evident that any chromatographic system that does not allow for complete recovery of the different components of a given mixture cannot be used as a quantitative analytical tool. We were especially concerned by that particular aspect of


0.02

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I ~ [D-His*]-GnRH, 94 pg

......_ [D-Hisz]-GnRH, 94 pg + 0.5 pg (0.5%) GnRH found 0.1 O/b

_ _ _ _ _ _ [D-Hisz]-GnRH, 94 pg + I pg ( 1 . I 96) GnRH found 1.1 ?o

Z

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8

I

I I I I I J O - 0 5 10 15 20 25

Time (rnin)

FIGURE 3 Separation of [D-H~s'IG~RH and GnRH by HPLC on pBondapak CI8 (10 pm particle size) column (0.4 X 60 cm). Buffer was 0.0 1 M NH,OAc, pH 4; flow rate, 2.5 mL/min.

HPLC when analyzing small synthetic peptides for homogeneity. ' 8,1 932 ' Indeed, one could imagine that a nonrepresentative percentage of a polypep- tide mixture applied onto the column might irre- versibly adsorb to the column, or simply disappear in the system (more specifically, in the injector), thus invalidating quantitation.22

Since recovery of weighable material was difficult to achieve accurately, and biological activities were not providing us sufficiently accurate

1 .o

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measures of recovery for this purpose, we arrived at the conclusion that the only way to assess recovery was to follow counts per minute (cpm) emitted by a radioactive peptide. The proviso was that loaded cpm and eluted cpm would be measured under the same strict conditions to eliminate such problems as quenching of the solvent in the case of a /3 emitter. Technically (and it is not trivial), we also had to make sure that no material was lost in the injector (a possibility that was real at the time).

Too

- 40

0

0 5 10 15 20

2

Y 0

Time (min)

FIGURE 4 HPLC of a native a-endorphin fraction ( 1.5 mg), on pBondapak CI8 ( 10 pm particle size) column (0.4 X 30 cm). Program 0-80% B 20 min, where buffer A = 20% CH3CN/ 0.01 MNH,OAc, pH 4.0 and B = CH3CN; flow rate, 2.5 mL/min.

0.1

E 0 cu 7 0.05 cn U 3 a

0

2100

1800

1500

1 1 2 0 0 - I $ 900

600

300

0 -

P-Human

-

- -

2

(0

- 4 a -

-

I I I I I 0 5 10 15 20 25

Time (min)


100

Z

Y 0 8

32

0

FIGURE 5 Separation of @-endorphins (ca. 45 pg each) on pBondapak Cyanopropyl ( 10 pm particle size) column (0.4 X 30 cm). Buffer was 0.05M H3P04, pH was adjusted to 7.50 with NaOH; flow rate, 2 mL/min.

The only way to reliably do so was to push the solution of the peptide contained in a syringe in front of a plug of pure buffer that had been introduced in the syringe prior to the peptide solution.22

Figure 6 shows the elution profile (cpm X elution time) obtained when a preparation of [ 3H] -GnRH (10,000 k 1000 cpm) was run under the conditions described in the caption. A total of 11,000 * 1000 cpm were collected in the different fractions, indicat-

Time (min)

Too

FIGURE 6 Separation of [3H]-GnRH [lO,OOO cpm: .-.- with GnRH (4 p g ) : -) on pBondapak Cya- nopropyl ( 10 pm particle size) column (0.4 X 30 cm). Buffer was TEAP. Flow rate, 1.5 mL/min.

ing that all the counts had been recovered. This experiment could be repeated and was reproducible, not only in terms of high recovery, but also in terms of resolution. A second experiment, not shown here, showed 50,140 cpm recovered and a similar profile, when 50,000 cpm were injected.

Also not shown here was the separation of GnRH from somatostatin, insulin, and cytochrome c in comparatively good yields with a linear load-response curve for the larger two molecules suggesting good recovery in that system.

The discovery of TEAP as a buffer compatible with the chromatography of unprotected peptides was important because the first silica supports could almost never be completely end capped, and whereas the phosphate ion formed a strong counterion to any basic moiety in the peptide, it was suspected that the triethylamine would serve (similarly) as a hydrophobic counterion to any residual free silanol on the silica and hence displace any peptide that may tend to interact through ionic forces with the silica support. This phenomenon was later dissected by Sokolowski et al., who studied peak tailing and retention behavior of tricyclic antidepressant amines and related hydrophobic ammonium compounds in reverse phase ion-pair liquid chromatography on alkyl-bonded phases.26 Interestingly, Sokolowski et al. identified triethylamine as among the very best, if not the best amine to use in their system.


an

8-Ovine

8-Porcine

0

L I

25 31

30

z % 0 8

4

7

Time (min)

FIGURE 7 Separation of p-endorphins (ca. 15 pg each of porcine P-endorphin, ovine p- endorphin and human P-endorphin) on a pBondapak Cyanopropyl ( 10 pm particle size) column (0.4 X 30 cm). Buffer was CH,CN/TEAP pH 3.0; flow rate, 1.5 mL/min.

We then returned to see if our troublesome separation of ,&endorphins would behave more pre- dictably using this buffer system. Figure 7 shows the elution profile generated after injection of the three 0-endorphins shown in Figure 5 under acidic conditions (TEAP buffer). In terms of resolution, the acidic system, when run at room temperature, seems slightly more effective. It is interesting to note that &-endorphin, which is more acidic than the other two @-endorphins (see above for sequences), had now become more hydrophobic. This was expected since the different available side chain and C-terminal carboxyl groups are now in the nonionized form. It should be pointed out that p,- and Po-endorphins are still eluted in the same order, indicating that whatever the pH, the ratio of the partition coefficients of those two peptides remained the same. This is also to say that if any conformational change took place as a result of lowering the pH, residue 23 of Po- and P,-endorphin must still remain exposed for interaction with the support.

Most significant for us, however, was the observation that, for the first time, CRF activity in crude tissue extracts could be recovered from reverse phase supports. We will expand later on this observation as we discuss the role of reverse phase supports and their respective selectivity.

By then we had found that, for neutral or basic peptides, better resolution was obtained at low pH (2.25-3.5). This is the range in which most acidic

functional groups in peptides are not dissociated and all basic groups protonated. TEAP, in this pH range, has the advantage of being compatible with the column packing materials as exemplified by the excellent performance of columns used for more than a thousand different runs. As shown above, the uniqueness of this buffer was demonstrated by using porcine &endorphin as a model peptide and, except for a sodium phosphate buffer at pH 7.5 (Figure 5 ), no other buffer (ammonium acetate, carbonate and formate; sodium acetate or phosphate) at any pH from 3 to 7 or concentration (0.01-0.5 N ) were found to give good recoveries based on absorbance coupled with good resolution.**

Whereas TEAP buffers at various pH (TEAP at pH 6.5 gave remarkable chromatographic profiles for gastrin for example) satisfied most of our needs (high resolution and recovery, uv transparency and compatibility with biological systems), these buffers were not volatile. We suggested and used triethylammonium formate (0.25 N, pH 3.0) which was shown to give good recoveries and resolution for somatostatin and @-human endorphin. Yet it could only be used when large enough quantities of material were available so that the problems due to absorption of the buffer, such as shifting of baseline when running gradients, and lower sensitivity in the 200-230 nm region, could be avoided. The report by Bennet et al. at about that time, that 0.1 % aqueous trifluoroacetic acid (0.1 % TFA)/


-100

z Y 0 s

CH3CN2' could be used as a volatile buffer compatible with CIS silicas, revolutionized peptide isolation and characterization. The use of acidic TEAP 2.25/acetonitrile ( CH3CN) and 0.1% TFA/ CH3CN27 buffers on C18 silicas became routine for the preparative purification and desalting of synthetic peptides in our laboratory. We have found that approximately 90% of the hormonal peptides could be purified in that manner. However, there were instances where the use of the relatively acidic TEAP 2.25/0.1% TFA/CH3CN buffers failed to produce acceptable levels of purification or were not appropriate at all. Examples of very hydrophilic (basic), relatively large peptides and acidic peptides for which other systems had to be found will be described later.

With a rather satisfactory set of buffers, it was now time to concentrate our efforts on the role played by different supports in the chromatographic process.

We illustrate in Figure 8 the effect of different derivatizations of a similar silica by presenting the traces of the same preparation of a partially purified synthetic GnRH eluted on these different supports. All parameters, such as column size, flow rate, temperature, amount loaded, and detection mode, were kept constant. Different concentrations of the organic buffer had to be used, however, so that identical retention times would be obtained for the major component of the mixture. These data demonstrated the critical role of the packing material for resolution of different components of a standard peptide mixture composed of a major component and closely related impurities. It is noteworthy that the best resolution was obtained on the most hydrophobic columns as judged by the higher concentration of organic solvent needed to elute the peptide mixture at a comparable RT. Even though we had not studied recoveries in that system (i.e., alkylphenyl or pCI8 columns), it did not seem to be a problem for small peptides. As will be shown next, the most significant parameters that influence a separation of peptides are those associated with the composition of the buffer and the characteristics of the supports. Other parameters, which included the role of flow rate and of temperature, played a comparatively minor role.

0.1

$ z N m 0 LL 3 4

c

HPLC AND NATURAL PRODUCTS

-

We will now illustrate the influence of buffer composition, column supports, and temperature, among others, on separations, by describing the

I: I A-----J -17.2

OL, I I I J o 0 10 20 30

Time (min)

0.1

? N

m - (I) U 3 4

0

Time (min)

Time (rnin)

FIGURE 8 Effect of column packings on the separation of GnRH from unidentified impurities: Top Trace: Load, GnRH (50 p g ) ; pBondapak Cyanopropyl ( 10 pm particle size) column (0.4 X 30 cm). Buffer was TEAP flow rate, 1.5 mL/min. Middle trace: Load, GnRH (50 p g ) ; pBondapak Alkylphenyl ( 10 pm particle size) column (0.4 X 30 cm). Buffer was TEAP; flow rate, 1.5 mL/ min. Bottom trace: Load, GnRH (50 pg); pBondapak CI8 ( 10 pm particle size) column (0.4 X 30 cm). Buffer was TEAP flow rate, 1.5 mL/min.

different HPLC steps used in the purification of CRF from ovine hypothalamic extracts and rat brain extracts.

Isolation of Ovine Hypothalamic CRF2* (See Table I for Sequence)

Guillemin and RosenbergZ9 and Saffran and Schally3' independently postulated and demonstrated, in 1955, that corticotropin (ACTH) release


from the pituitary was stimulated by a hypothalamic substance, which they named corticotropin releasing factor (CRF). By 198 1, CRF had gained the label “putative” or “elusive” because it had as of yet not been characterized.

Using the HPLC techniques reported above, we isolated enough pure native ovine CRF to allow its characterization 28 and ultimate total synthesis by the solid phase strategy. The synthetic replicate exhibited high potency and intrinsic activity to release immuno-like ACTH and @-endorphin from both pituitary cells maintained in monolayer cultures2’ and in vivo in several rat preparation^.^' CRF is an amidated 4 1 -peptide (see Table I for sequence). The main difficulties encountered by us and others (Ref. 28 and references therein) in car- rying out this project were associated with ( a ) the bioassay, because a number of substances, including vasopressin and ACTH,32-36 can lead to false positive or negative; (b ) the complexity of the peptide/protein mixtures generated during extraction; (c) the occurrence of losses due to degradation (chemical as well as enzymatic) or adsorption to column supports or test tubes in which fractions were collected; (d) the small amount of purified peptide that was available for sequence analysis; and (e) the total synthesis for comparative chemical and biological studies. This isolation project, the purification of ovine CRF using HPLC techniques developed in our laboratory, was the driving force to understanding and optimizing HPLC- based purification techniques both analytically and semipreparat ively .

As part of a project directed toward the isolation of GnRH in the Laboratories for Neuroendocrinol- ogy at The Salk Institute, 490,000 sheep hypothalami were extracted in a mixture of ethanol : acetic acid : chloroform, defatted with a mixture of ether : petroleum ether, and partitioned in an n-butanol : pyridine: 0.1 % acetic acid (5 : 3 : 1 1 ) system as reported by Burgus et al.37 The lower phase (2 kg) of this last step contained a fraction of the total CRF- like activity exhibited by the defatted starting material. A succession of classical purification steps including ultrafiltration or dialysis against 2 N acetic acid and batch size exclusion chromatography on Sephadex G-50 eluted at 4°C with 2 N acetic acid yielded two zones exhibiting CRF-like activity. The low molecular weight fraction eluting at 2 Ve/ V, was later found to have the composition of [ Arg’] vasopressin. The large molecular weight fraction eluting at 1.3 Ve/ V, was suspected to be CRF because it elicited a much higher secretory rate at a maximum concentration of added sub-

stance than did [ Arg’] vasopressin and showed en- couraging properties in a series of in vitro ~tudies.~’ Treatment of the larger molecular weight active fraction (guanidine hydrochloride ( Gn-HCI )/ AcOH, pH 2.5, as eluate) gave one single peak of activity that was only partially retarded, thus ex- cluding the possibility that the activity had been associated noncovalently with larger molecular weight carriers. The pool of this zone from several columns was the starting material for the HPLC purification of ovine CRF. The protein content of this highly viscous pool was estimated to be 1.0- 1.5 g/300,000 hypothalami on the basis of absorbance at 210 nm (0.75 mL of this stock solution corresponded to 1 550 hypothalami abbreviated 1550 H.).

Early attempts to purify CRF by HPLC using pBondapak C18 or even pBondapak CN and 0.02 N NH40Ac, pH 4.0/CH3CN buffer as described in Burgus and Rivier’ resulted in complete loss of CRF biological activity. This disappointing observation was the main incentive to search for a buffer system that would have minimal absorbance at 200-210 nm and be nontoxic or volatile so that bioassays could be carried out without any further manipulation of the samples except for lyophilization. The trialkylammonium buffers and the triethylammonium buffer (TEAP), 22 in particular, were found to indeed increase recovery as well as overall column performance. The role of the added alkyl- amine to the mobile phase was to inhibit competitively the participation of the solute in the ion-exchange or adsorption reactions with the nonbonded silanols on the stationary phase as suggested earlier.

The first step in the HPLC purification scheme of ovine CRF was a rough separation with emphasis on recovery of biological activity rather than resolution and had as its main purpose an increase in the concentration of proteinatious material. Be- cause more than 100 runs were necessary to process the active Gn-HCI containing fraction from the P- 10 gel permeation column, emphasis was put on reproducibility of the absorbance pattern so that each column would not have to be tested individu- ally for biological activity. This goal was met, and a typical profile where biological activity was found in the hatched fraction (i.e., fraction 5 in the assay reported in Table 4 ) is shown in Figure 9. The pool of the active zone of a hundred such runs generated a fraction that we labeled 28- 125-00 ( 150,000 hypothalami in 8.75 mL). In this step, Gn-HCI was removed and approximately 1 00-fold purification was achieved.

That CRF eluted at a higher concentration of


Table IV Representative Assay of Column Fractions Generated in Figure 9

Dose ACTH Secreted Substance Tested (H/mL) (pg/mL) SEb

Control Starting material Column fractionsa

4 4 5 5 6 6

- 1 .0

0.3 3.0 0.3 3.0 0.3 3.0

155 2175

130 337 883

1850 217 527

39 212

8 64

173 54 31 11 -

a Fraction 3 tested in another assay was found to be inactive. Standard error.

acetonitrile than did other releasing factors at acidic pH was a possible indication of its overall hydrophobicity under those conditions and/or of its size, which we did not suspect at the time. Why would nature use a peptide greater than a few residues to trigger the release of another hormone (ACTH) only 39 residues in length? At the time, hydrophobicity was considered as the main culprit to explain CRF’s propensity to adsorb to glassware, polypropylene tubes, and other surfaces including HPLC packing materials. Addition of an organic modifier or, more practically, keeping the CRF- containing HPLC fractions in polypropylene tubes at -20°C in the original mobile phase that eluted

_17 I

them, allowed us to retain biological activity throughout our purification schemes.

Three independent schemes of purification using three different aqueous buffers/acetonitrile gradients and four different columns (some of which became available as the project progressed) could lead to a CRF preparation that would be pure enough for microsequence determination (i.e., > 85% purity).40 These are summarized in Figures 10-12.

At a time when column characteristics were poorly understood and desired features for high resolution and recovery of peptides were neither recognized nor available, our attempt to isolate CRF from the 28- 125-00 pool was mostly empirical. As mentioned earlier, however, we developed the TEAP buffer to solve the problem of free silanols partially. Many columns other than those reported in the figure captions, including Supelco Sil CI8 (5 pm), DuPont ODs, and CN (5 pm) columns, and several solvent systems made of different organic modifiers (CH,CN, 1 -propanol, 1- butanol, 2-propanol, or mixtures thereof) and NH,OAc or TEAP at different concentrations (0.1-0.5 N ) or pH (2.25 to 7.2), were tested without success. In most cases either biological activity was lost or no separation was achieved. It is only when we used a pBondapak ClX (but not Supelco Sil or DuPont CI8) immersed in ice water, thereby following initial size exclusion conditions, that the TEAP/ CH3CN buffer system would yield acceptable resolution and recovery of biological activity.

I I I I I I I

0 5 10 15 20 25 3(

Time (min)

100

2

51 y 0 8

39

15

D

FIGURE 9 First HPLC step to generate pool 28-125-00. Load: 1550 H. in 750 pL of the pool after Gn-HCI P-10 column. Conditions: pBondapak Cyanopropyl ( 10 p m particle size) column (0.46 X 30 cm), buffer was TEAP, pH 3.0; flow rate, 1.5 mL/min at room temperature.


0.4 r

1 I I j I ( I I , I h l j l l l l , l I , O d 5 10 15 20 25 30

Time (min)

0.4

E 0 z 0.2

a

N

In LL 3

0

oC,RF I I I I , I I 1 _I, 0 5 10 15

Time (min)

I00

z I 0

u, 8

38

31

0

/ /

1' I

I

10 15 20 25 30 Time (min)


Optimized gradient conditions on the CI8 column are shown in Figure IOA. Similarly, it took lowering of the temperature to find isocratic conditions on the pBondapak CN columns (Figure 1 OB) that would achieve some further purification. At that stage we became aware of the fact that the nonvol- atile TEAP buffer was interfering with the Edman degradation used in microsequencing. Desalting using the “volatile” triethylammonium formate (TEAF) at room temperature and a sharp gradient of CH3CN is illustrated in Figure 1OC and yielded approximately 10 nmol of highly purified ovine CRF.

The main drawbacks of this original isolation scheme were a relatively low overall yield due to adsorption on the columns and the need for making sharp cuts that emphasized purity vs recovery. Because of our inability to monitor column effluent at 2 10 nm at the desalting step (CRF did not seem to absorb at 255 or 280 nm, and TEAF would not be transparent in the uv at the concentration used), we had to follow CRF by biological testing or amino acid analysis while arbitrarily frac- tionating the column effluent.

Most of these difficulties were later resolved by using large pore size silica (300 A) that had been properly derivatized (C,,) and end capped, i.e., Perkin-Elmer ODS-HC SIL-X-1 ( 10 pm particle size; Figure 1 1A and B) or Vydac (5 pm particle size; Figure 12 ).

One should note that a fairly sharp cut of the active zone shown in Figure 1 1A could be further resolved by using 0.1% TFA buffer (Figure 1 1B). Because the same column was being used in both cases, we do not think that this selectivity was due to differences in column load or gradient shape, but was rather attributable to the different eluotropic properties of TEAP vs 0.1% TFA buffer. This seems to hold true in many instances and has been taken advantage of in many purifications of synthetic peptides as well as naturally occurring substances as further illustrated in this review.

Whereas acceptable recovery of CRF-like activities could be obtained in the 100 A (pBondapak) support by cooling the column (thus driving the equilibrium of mass transfer from the solid support toward the eluate), one could do the reverse and increase resolution (as illustrated in Figures 1 1 and 12) by using end capped CI8, 300 A silica at room temperature or higher. Higher resolution was obtained on 5 pm particles (Vydac; Figure 12) than on 10 pm particles ( Perkin-Elmer column, Figure 1 1 ) as one would theoretically expect. The use of solvent systems having a relatively high concentration of alkyl ammonium salt also became less critical due to the absence of residual silanols. This allowed for the purification and desalting of 28- 125- 00 in one step using 0.1 % TFA/CH3CN as eluate (Figure 12). Rechromatography of purified ovine CRF showed a single absorbance in the TFA system as expected. Amino acid and sequence analyses of this material were consistent with a structure having 4 1 r e~ idues .~~ ,~ ’ A synthetic replicate [Met(0)21]CRF could be made2* and exhibited the physicochemical and biological properties of the natural product as it was isolated.

From this complex isolation project that was carried out for more than ten years, it was concluded that the ability to follow and purify CRF activity from ovine hypothalami was the direct result of both a reliable CRF assay and of a better understanding and constant improvement of HPLC technology. Indeed, we demonstrated that what may have seemed to be the most intractable mixture could be resolved, provided that an opti- mum use of column and eluate characteristics be taken advantage of. By 198 1 (initial publication of these results), the field had already evolved so much that there was a significant choice of column types from different manufacturers and of buffer systems. In fact, we used this next generation of columns and compatible solvent systems that exhibit different selectivities to achieve the isolation of rat CRF4’ (see below) which was later demonstrated

FIGURE 10 First scheme and three-step purification of CRF from pool 28-125-00. (A) Load: 2150 H in 125 pL of pool 28-125-00. Conditions: pBondapak CI8 ( 10 pm particle size) column (0.46 X 25 cm); buffer was TEAP, pH 3.0; flow rate, 1.2 mL/min at 0°C. (B) Load 7500 H. in 1 mL of pool of the active fraction shown in (A). Conditions: pBondapak Cyano- propyl ( 10 p m particle size) column (0.46 X 30 cm); buffer was TEAP, pH 2.25; flow rate, 1.2 mL/min at 0°C. (C) Load: 12,000 H. in 1 mL of pool of the active fraction shown in (B). Conditions: pBondapak Cyanopropyl ( 10 pm particle size) column (0.46 X 30 cm) . Buffer was TEAF, pH 3.0; flow rate, 1.2 mL/min at room temperature.


I , I 1 I $ l I ,I

1 .o

s 0 N

cn LL 3 Q

7

0.5

1 1 1 1 1 1 1 1 1 I ~ ; C I , I I I I I I I I J ~ 0 5 10 15 20 25 30 35 40 45

Time (min)

B 100

z I 0

58 0,

8 37 27

0

FIGURE 11 Second scheme and two-step purification of CRF from pool 28-125-00. ( A ) Load: 1400 H in 80 pL of pool 28-125-00. Conditions: Perkin-Elmer ODs-HC SIL-X-1 ( 10 pm particle size) column (0.26 X 25 cm). Buffer was TEAP, pH 2.25; flow rate, 0.7 mL/min at room temperature. (B) Load: 15,000 H. in 13 mL of a pool of the active fraction shown in ( A ) . It was injected 1 mL at a time every 90 s in a Rheodyne injector having a 2-mL loop. Conditions: Perkin-Elmer ODS-HC SIL-X-1 ( 10 ym particle size) column (0.26 X 25 cm). Buffer was 0. I % TFA; flow rate, 0.7 mL/min at room temperature.

to have an identical sequence with that of cloned human CRF.42

Since oCRF was purified from a side fraction of an isolation program directed originally at the isolation and characterization of GnRH, and that this side fraction contained only a small percentage of the total ACTH-releasing activity in ovine hypothalamic extracts, and that in that side fraction the methionine residue in CRF had been oxidized, we felt it necessary at the time to establish the structure of the predominant CRF in fresh tissue extracts. The rat was chosen as a source for a more thorough isolation program because its CRF differed from ovine CRF with respect to immunologic and chromatographic behavior. Furthermore, it was suspected that it would be more rigorous to do all future physiologic studies (mostly carried out in rats) using an endogenous form of the hormone.

Isolation of Rat Hypothalamic CRF41i42 (See Table I for Sequence)

The initial steps of extraction of peptides from hypothalamic tissues that ultimately led to the isolation of rat growth hormone releasing hormone ( rGRF)43 and rat corticotropin releasing factor ( rCRF)4' included acetone defatting, acid extraction, and size exclusion chromatography on Seph- adex G-50. Indeed, impurities that contaminate the bioactive components of tissues include not only peptides and proteins, but lipids, carbohydrates, enzymes, salts, and cellular debris. The most common extraction method, using acid, acetone, or petroleum ether, generally extracts most of the lipids, while insoluble materials are isolated by centrifugation. Carbohydrates or glycosylated proteins can be isolated by affinity chromatography on


0.2

5 z 5 0.1

a

cu

rn L L 3

0

-100

5 10 15 20 25 30 35 Time (min)

FIGURE 12 Third scheme and one-step purification of CRF from pool 28-125-00. Load: 1400 H. in 80 pL of pool 28-125-00. Conditions: Vydac CI8 (5 pm particle size) column (0.45 X 25 cm) No. 14228-3. Buffer was 0.1% TFA/CH3CN; flow rate, 1.2 mL/min at room temperature.

concanavalin or boronate gels. The most difficult problem, in fact, is often one resulting from the presence of enzymes with proteolytic activity that may destroy the active entity or generate fragments, resulting in new entities that may have similar physicochemical properties to those of the active component or interfere with radioimmunoas- says ( RIAs) by destroying or competitively binding the radioactive tracer, for example. Enzymatic activity can either be retarded by working at low temperature or partially destroyed at high temperature in the presence of denaturing agents. A third method of protection against enzymatic degradation is to use protease inhibitors such as phenyl- methylsulfonylfluoride ( PMSF) , pepstatin, etc.

In the case of rat CRF, lyophilized hypothalamic fragments provided by Dr. A. Parlow ( Harbor-UCLA Medical Center) under the aegis of the NIADDK were defatted with acetone and the resulting powder was extracted at 290°C with 10 volumes of a mixture of 1 N acetic acid (HOAc), 0.1 N HC1, 0.5% 0-mercaptoethanol, 10 m M EDTA, and 5 pg/mL pepstatin A. The hot slurry was immediately ground in a blender, cooled in an ice bath, and centrifuged. The supernatant was saved while the precipitate was re-extracted with the above mixture with the addition of 20 m M NaCl. The combined supernatants were defatted by multiple extraction with 2 volumes ether-petro-

leum ether ( 1 : 2) . This protocol was adapted from the one used for the isolation of ovine CRF (oCRF) in the sense that P-mercaptoethanol and pepstatin were added to keep any methionines present in their reduced form and inhibit degrada- tive enzymes, respectively. The aqueous phase was subjected to size exclusion chromatography at 4°C on a Pharmacia K 2 15 / 100 column packed with 85 cm Sephadex G-50 fine, topped with 5 cm Seph- adex G- 10, V, = 3 1 L. The eluate was 3 N HOAc with 0.2% P-mercaptoethanol. In this system, GRF could be purified away from most of the somatostatin-like peptides since the GH-releasing region eluted immediately before the somatostatin-28- rich fractions and was well separated from the zone containing somatostatin- 1 4.44

Several zones exhibiting CRF-like immunoactivity were found. A high molecular weight zone appearing in the void volume of the size exclusion step (representing < 10% CRF-like immunoactivity) and a zone (representing = 90% CRF-like immunoactivity ) also containing GRF-like activity with a partition coefficient K,, of 0.20-0.31 were both biologically active as well as immunoac- tive. Multiple low molecular weight areas (KaV = 0.5- 1.1 ) exhibited lower intrinsic activities (maximal secretory rate at maximum concentration of added product) than did oCRF in the in vitro bioassays and were not active in our CRF assay. We


suspect that the smaller molecular weight bioactive substances with lower intrinsic activity than CRF may be related to neurohypophysial peptides or catecholamines. The nature of the high molecular weight fraction (Kav = 0.0-0.15) was not further investigated. The zone representing 90% of the total CRF-like immunoactivity detected after size exclusion was further purified batchwise by preparative HPLC with the Waters Associates preparative LC-500 system. Under these conditions rCRF and rGRF activities could be clearly separated. Reten- tion volume for rat CRF was 1900-2200 mL while that for rat GRF was 1600- 1900 mL.

A total of 100,000 rat hypothalami was processed in three batches of 60,000, 20,000, and 20,000 hypothalami, which had a total dry weight of 500 g. Final yields of purified rCRF were approximately 3.0, 0.8, and 1.0 mg, which had respective purities of 50-55%, 80-90%, and > 90% as shown by sequence and amino acid analyses. Over- all recovery was > 50% as assessed by RIA of both starting materials and final product. This corresponds to an approximate lo '-fold purification. Figures 13- 18 illustrate the importance of column support and eluate in the chromatographic separation of peptide/proteins from natural sources. It should be noted that each separation was first carried out analytically using the equivalent of a few hypothalami to assess recovery and overall quality (high resolution and selectivity). After optimization, a first semipreparative step (Figure 13 ) on Vy- dac C4 was selected. As can be seen, the technique of multiple injections was implemented that allowed for the concentration of the material at the top of the column prior to elution with a gradient of acetonitrile. Good recovery of activity was found in fractions 12 and 13. This step achieved some concentration of the active fractions as well as led to the elimination of a significant amount of uv absorbing material that was both more hydrophilic and hydrophobic than the desired fractions. Sev- eral similar runs were carried out and active fractions were pooled on the basis of uv profiles. Mate- rial pooled from fractions 12 and 13 was then applied after dilution ( 1 : 3) with water to an analytical Vydac diphenyl column and eluted with the TEAP buffer at pH 2.25 (Figure 14). This system again achieved a remarkable separation, with most of the impurities being more hydrophilic than CRF found in fraction 10. It should be noted that the diphenyl column that was used was identified by the manufacturer as V- 1590-3 [as we will see later, this same column (see Figure 17) was used with 0.1 % TFA with dramatic results, whereas a

different column used with the same buffer (see Figure 16) also gave major differences in selectivity]. The next step in the isolation of rCRF was carried out on a CI8 analytical column using the 0.1 % TFA buffer (Figure 15 ) . The desired fraction (23) was found in the dead center of the overall absorbances. At the time, column manufacturers had major problems with column reproducibility; yet for us this turned out to be a blessing. This is best illustrated by the separation shown in Figure 16. While using a different Cj8 column with the same buffer and a very similar gradient we were again able to increase purity significantly. The activity was found in fraction 10, a pool of which was rerun on the same diphenyl column that was used in Figure 14. While we kept the protein load about the same on all purifications by increasing the number of hypothalami equivalent that were loaded, it should be noted that identification of the bioactive fraction by following uv profiles only would have been impossible. In fact, it is only at this last step (Figure 17) that we felt confident that the absorbance associated with the active fraction represented in fact that of the purified rCRF. Indeed, rechromatography of this material yielded the symmetrically shaped peak shown in Figure 18. This last step was used to concentrate the fractions generated by repeated chromatographies of the earlier step and free them from the P-mercaptoethanol that had been added at each step to minimize oxidation of the methionine residue(s). This turned out to be a necessary step prior to sequence analysis.

Several laboratory procedures were followed rig- orously in order to eliminate potential systematic as well as random errors during this purification scheme. First, P-mercaptoethanol was aliquoted into all fractions within minutes of their collection (0.5% by volume). This served two purposes that we could identify: ( a ) it kept a reducing atmo- sphere above the fractions (thus protecting methionine residues from oxidation) and (b) P-mercaptoethanol seemed to have a beneficial effect in pre- venting the protein fractions from binding to the polypropylene tubes in which they were collected. Second, we realized that peptides in nanogram quantities in volatile buffers such as the TFA solutions would disappear upon lyophilization, even when carried out in a SpeedVac. It was therefore paramount to add sterile bovine serum albumin (at least 1 mg/tube) prior to lyophilization of the aliquoted fractions for bioassay in order to recover biological activity.

In contradistinction with oCRF, which was iso-


- 2.0 -

E t

- 100

FIGURE 13 Second chromatographic step for the purification of rCRF from fractions 37- 42 of a preparative run on a Vydac CIS (20 pm particle size) cartridge ( 5 X 30 cm) using TEAP (pH 2.25) at a flow rate of 75 mL/min and a gradient ofCH3CN going from 4.2% to 36% in 40 min. Load: 5000 H. in 14 mL of fraction eluting between 1900 and 2200 mL. Conditions: Vydac C4 ( 5 pm particle size) No. 1830B-10A column ( 1.0 X 25 cm). Buffer was 0.1% TFA; flow rate, 3.0 mL/min at room temperature.

lated in its oxidized methionine form from a minor side fraction of extracts originally used for the characterization of leuteinizing hormone releasing factor (see above), rCRF was isolated from fresh rat hypothalamic extracts in its native form and found to be the major ACTH releasing substance with high intrinsic activity in vitro. Within a short time

of the report of the structure of rat CRF, that of human CRF, obtained by cloning, was found to be identical to that of rat CRF.42 It should be noted that it took another fourteen years and the application of quite different strategy to identify and characterize a second rCRF homologous to the fish ur- otensin. This peptide was named u r ~ c o r t i n . ~ ~

Time (min)

FIGURE 14 Third chromatographic step for the purification of rCRF from fractions 12 and I 3 from Figure 13 (5,000 H. in 6 mL). Conditions: Vydac diphenyl ( 5 pm particle size) No. V-1590-3 column (0.45 X 25 cm). Buffer was TEAP, pH 2.25; flow rate, 1.2 mL/min at room temperature.


Time (min)

FIGURE 15 Fourth chromatographic step for the purification of rCRF from fraction 10 from Figure 14 (4,000 H. in 4 mL). Conditions: Vydac diphenyl ( 5 pm particle size) No. V-1830-1 I column (0.45 X 25 cm); buffer was 0. I % TFA; flow rate, 1.2 mL/min at room temperature.

Conclusions Derived from the Use of HPLC in the Isolation of Natural Products

Recovery of biologically active protein generated via recombinant DNA technology presents the same types of problems as those encountered in the isolation from tissues or biological fluids, with the possible advantage, however, that the concentration of the desired material, when the process has been optimized, can be significantly higher than

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that found in tissue extracts. Other bioproducts that may complicate and confuse the decision- making process on how to perform fractionation of the active zones are those generated by chemical alteration (either oxidation of methionine or re- duction of disulfide bridges), which may consider- ably alter the in vivo or in vitro biological potencies of a peptide without altering its ability to be fully recognized and quantitated in an RIA. Similarly,

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FIGURE 16 Fifth chromatographic step for the purification of rCRF from fraction 23 from Figure 15 ( 10,000 H. in 3 mL). Conditions: Vydac diphenyl(5 pm particle size) No. V-163 I - 3 column (0.45 X 25 cm). Buffer was TEAP, pH 2.25; flow rate, 1.2 mL/min at room temperature.

Peptidr Chemistry 285

-

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FIGURE 17 Sixth chromatographic step for the purification of rCRF from fraction 10 from Figure 16 (5,000 H. in 3 mL). Conditions: Vydac diphenyl(5 pm particle size) No. V-1590-3 column (0.45 X 25 cm). Buffer was 0.1 % TFA; flow rate, 1.2 mL/min at room temperature.

there are cases in which RIA activity can be destroyed while biological activity remains unaltered. These observations have led us to opt for redun- dancy, when possible, in the number and type of biological tests that can be used to identify an activity in a column effluent. It is also obvious that steps can be taken to prevent oxidation of methionine. One possibility is to keep chemicals such as dimethylsulfide present throughout purification and until isolation. P-Mercaptoethanol can also be used, but may reduce disulfide bridges or generate mixed disulphides under certain conditions (high

0.4

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pH) . This can be desired if one of the contaminat- ing activities has cystine in its structure, as is the case for somatostatin [see isolation of GRF (which triggers the release of growth hormone) in the presence of somatostatin (which, in the same system, inhibits the release of growth hormone in a non- competitive manner)46]. Such treatment with 0- mercaptoethanol, however, can be considered to be an irreversible procedure that would exclude finding another disulfide-bridged bioactive molecule in these extracts. It is to be remembered that any chemical alteration of an extract may greatly

I r

FIGURE 18 Seventh chromatographic step for the purification of rCRF from fraction 9 from Figure 17 (20,000 H. in 3 mL). Conditions: same as in legend Figure 17.


facilitate the isolation of one identity to the detri- ment of any other. When starting materials are unique and difficult to obtain, however, such chemical alterations may not be tolerable. Another problem often encountered is that associated with the storage of column fractions while assays are being carried out. It is now accepted that when dealing with small quantities, the natural substances are best kept in solution at low temperature in the absence of air or in the presence of oxygen scaven- gers. Freezing and thawing may not be advisable. We have also suggested that the presence of an organic solvent and such molecules as P-mercaptoethanol or dimethylsulfide may successfully compete with peptides for binding sites on glass and polypropylene tubes4’ Large quantities of bioactive mixtures from extraction steps or synthetic peptides are best kept as a lyophilized powder at low temperatures and in the dark.

Other conclusions from this work include the observation that end-capped, large-pore CIS derivatized silica (Vydac, 5 pm particle size, 300 A pore size) gave significantly better resolution of CRF and, in general, of peptides larger than 25 residues than similarly derivatized silicas with pore sizes ranging from 80 to 120 A.47 Usage of similar silicas with different selectivities obtained by derivatization and end capping (C4, diphenyl, and C18) and of two solvents (TEAP and 0.1% TFA) with different ion-pairing capabilities had resulted in good resolution and excellent recovery of rCRF biological and immunological activities.

It should be noted that since this early work on the isolation of selected natural products using HPLC, a large number of other successful isolation projects have appeared in the literature. We have summarized it in a review that also addressed some aspects of purification of native peptides only partially presented here.48

ANALYTICAL/SEMIPREPARATlVE HPLC AND SYNTHETIC PEPTIDES: STEP It

Whereas one can see major differences in the chromatographic behavior of the two native CRF extracts (oCRF, Figures 9-12, and rCRF, Figures 13- 18 ) relative to the impurities associated with the desired hormones as the result of using quite different chromatographic supports, the most tell- ing examples illustrating progress made in the late 1970s and early 1980s in terms of column development and solvent use are shown in the next several examples.

HPLC of L-Phe-L-X Dipeptides

By 1979, HPLC was already recognized as the tool of choice for the analysis of amino acid derivatives and synthetic peptides as well as for their isolation in pure f ~ r m . ’ , ’ ~ . ’ ~ , ~ ~ While previous reports might not have addressed themselves to the recovery of peptides, it became obvious that certain families of peptides would elute from the commercially available columns almost quantitatively, whereas others would be unpredictably and partially displaced from the top of the column where they had been adsorbed. With the introduction of the TAAP buffers (trialkyl ammonium phosphates) and more specifically the TEAP2* buffer, these effects could be minimized. However, recoveries of larger proteins such as insulin or cytochrome c were not quantitative even when using TEAP with the more polar pBondapak CN columns. Independently, O’Hare and Nice, 50 studying hormonal polypeptides, had shown good resolution and recoveries while using an acidic phosphate buffer and a CIS derivatized Hypersil column. New techniques of derivatization of the silica (spherical 5 micron particles), high carbon loads, “capping” of the residual silanol groups, and new packing technolo- gies were pioneered by Karger et al., who developed column supports ( Cf8 coverage > 3 phf/m2: original surface area 200 m2/g end capped using tri- methylchlorosilane, original pore size ca. 100 A), the performance of which was dramatically improved over that of the commercially available columns. Among other applications (separation of somatostatin analogues, see below), we used such columns for the separation of dipeptides. Two years earlier, l5 we had shown that diastereoiso- meric dipeptides of the type L-Phe-L-X and L-Phe- D-X, where X was one of the twenty common amino acids, could be separated thus allowing for a rapid quantitation of racemization. We also suggested that such a system could be used for rapid amino acid analysis. Figure 19 demonstrates the feasibility of such analysis (see conditions in caption). Similarly, conditions were found using this new type of column and the TEAP buffer for the separation of phenylthiohydantoin (PTH) amino acids.5’

Recapitulating, it was becoming obvious that an in-depth knowledge and understanding of the properties of the crude peptide mixtures and that of the desired moiety were key to the development of a successful strategy that will lead to its isolation. If the desired product is to be isolated from a synthetic mixture, one must recognize that the impu-


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FIGURE 19 HPLC of L-Phe-L-X dipeptides. Column Cls-2 #1 ( 5 pm particle size, 0.46 X 25 cm) courtesy of B. Karger. Load ca. 1 nmole each L-Phe-L-Z dipeptide; X = amino acid assigned to each peak. Buffers were A = H,O/TEAP, pH 2.25 ( 1 : 1 ); B = H,O/TEAP pH 2.25/CH3CN ( 5 : 3 : 2) . Gradient conditions: isocratic 10% B (40 sec) 6 min linear gradient to 45% B, isocratic 45% B (8 min) 2 min linear gradient to 100% B; flow rate, 1.5 mL/min. Temperature 25 f 1°C.

rities could be closely related, both structurally and functionally. In the case of peptides synthesized by the solid phase approach, the most efficient chromatographic systems have to be used in order to separate entities that may differ because of the different chirality of one amino acid, its deletion, or its chemical alteration (alkylation, oxidation, etc.) . This is not to exclude chain termination (for extensive review of chemical peptide synthesis, see Ref. 52 ).

HPLC of Three Analogues of Somatostatin with Biological Selectivity

It is given that solid phase-generated synthetic mixtures grow more complex as the size of the peptides increase (independently of the strategy and protocols used). As recovery of the large peptides (>25 residues) from the original reverse phase packings became poorer with increasing size, we realized some of the limitations of the analytical, semipreparative, and preparative HPLC approach. This led us to investigate the role of support in the separation of peptides of sizes ranging from 10 to 5 1 residues.

Figure 20A illustrates what could be expected early on in terms of resolution from semipreparative columns using 10 pm particles. It took a column 1.2 m in length to achieve baseline separation of [ D-Trp'] -somatostatin, somatostatin, and [ D- Cys 14] -somatostatin. By comparison, the same peptides were separated on the newly developed 25 cm analytical column (see caption to Figure 20B) packed with spherical, 5 pm CIS silica with an aver- age pore size of 100 A. An important benefit of the use of high efficiency columns was improved sensitivity since > 25 ng of peptides could be monitored with uv detection.

HPLC of Peptides Including Insulin and Glucagon

During the evaluation of columns (analytical or semipreparative) packed with 5 pm CI8 (100 A) particles prepared from different batches of silica gel, it was found that glucagon would elute either as a sharp symmetrical peak (Figure 2 1 ) or as a tailing peak poorly resolved from insulin (not shown), while all other peaks would elute with reproducible retention time and with good peak sym-

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FIGURE 20 HPLC of an equimolar mixture of somatostatin, [ ~-Cys'~]somatostatin and [DTrp8]somatostatin (15 pg each). Panel A, pBondapak CIS (10 p m particle size) column (0.7 X 120 cm) column composed of four units used in series. Buffer was 35% CH3CN in 0.01 MNH40Ac, pH 4.15; flow rate was 6.0 mL/min. Panel B, one analytical CI8 ( 5 p m particle size) column (0.46 X 25 cm) packed with 5 pm silica with 100 A pore size. Buffer was TEAP, pH 2.25; flow rate, 1.5 mL/min.

metry. The mass and biological recovery of glucagon was also found to be variable. Fresh solutions of all peptides were used in all experiments to avoid possible artifacts due to peptide degradation. In all cases, acetonitrile was used as the organic solvent and TEAP at pH 2.25 as the buffer.

Using glucagon as our standard for good resolution and recovery, we investigated the influence of surface area (and pore diameter) on peak shape and recovery under elution conditions shown in Figure 2 1. Lewis et al.53 had reported the beneficial effects of working with silicas possessing wider than 100 A pore diameter for the chromatographic separation of proteins (in this case, it was presumed that the wider pore material is better able to allow free access of the large size proteins into the porous matrix). We (in the isolation of ovine and rat CRF),

had found the beneficial effect of large pores for peptides as small as CRF and as shown here also for glucagon and the 27 amino acid gastrin releasing peptide (GRP) related to bombesin (see Table I ) .39

We therefore selected a 300 A, 5 pm porous silica material for which the surface area was estimated to be 100 m2/g in contrast to the 100 A materials for which the surface area was 170 m2/g. Figure 22 illustrates a separation of our standard peptide mixture on a 300 A CIS semipreparative column. Most interestingly, the irreproducible chromatographic results for glucagon were not observed with the 300 pore material. We suggested that the surface area (and pore diameter) of the silica support played an important role in compen- sating for the physicochemical properties of linear peptides of 25 or more residues and that differences

Pepptide Chemistry 289

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GnRH

SRIF 1’”

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FIGURE 21 HPLC of an equal load ( 7 pg) of GnRH, neurotensin (NT) , bombesin (BN), substance P (SP), somatostatin ( SRIF), insulin and glucagon. Conditions were: Semi-prep CI8 column 100 A pore size ( I X 25 cm). Buffer was TEAP, pH 2.25; flow rate, 2.5 mL/min; back pressure was 900 psi.

in silica surface and/or bonding chemistry may influence the results. As mentioned earlier, we also studied the isolation of partially purified synthetic GRP. GRP also seemed to have chromatographic properties similar to that of glucagon; only when using a 300 A pore size silica base could we find isocratic conditions that would elute it as a sharp symmetrical peak with a k‘ of 3-7. The same chromatographic conditions using 100 A pore size columns would give rather broad (and often tailing) peaks, the shape of which could be improved by use of gradients.39

From this point on, new criteria for the purity of synthetic peptides were evolving; yet it is only with the application of these new methods of derivatization on silica with large pores that unique selectivity could be achieved and that the separation of large molecules such as insulin could be achieved reliably. This is illustrated in the following two sub- sections.

HPLC of GnRHs from Different Species (Use of TEAP at Neutral pH)

The GnRH family is neither related to other peptide families nor a member of a peptide superfam- ily. However, GnRHs belong to an ever growing family with nine members that are distinct decapeptides isolated from various vertebrate^.^^ One or more of the nine GnRH forms is found in indi-

vidual species from all classes of vertebrates. Stud- ies of species with multiple forms of GnRH in the brain have shown that each endogenous form of GnRH can release gonadotropins in vitro, provided the pituitaries are from the same s p e ~ i e s . ~ ~ , ’ ~ However, the multiple GnRH forms in the brain of a single species are assumed to have functions in addition to that of releasing pituitary hormones based on variation in their structures (see Sher- wood et al.”) and brain locations (see Davis et aL5‘). Little is known about these functions.”

Interestingly, all these peptides have conserved residues at positions 1, 2, 4, 9, and 10. Because more than one GnRH is found in most species (with the exception of mammals) the role of such molecules is the source of much interest for the understanding of the evolution of the reproductive system.

Whereas all separations presented here were carried out on reasonably sized columns (0.46 to 1 cm in diameter and 25-30 cm in length), it was mostly because packing technology at the time made it particularly difficult to obtain high resolution from narrower columns, which are now classified as microbore ( l mm internal diameter), narrow bore (2.1 mm internal diameter), and analytical (4.6 mm internal diameter). Yet all the advantages associated with such microbore and narrow bore columns (mainly high sensitivity and economy of solvents) were well acknowledged. The parallel development of precision HPLC instrumentation has led to the successful final steps of numerous isolation projects. We illustrate in Figure 23 a separation of nine native GnRH decapeptides (see Table V for sequences) carried out on a narrow bore column using TEAP pH 6.5 and a CIX support at 40°C. The peptides differed by one or several residues and were mostly baseline resolved. Two of the peptide peaks were fused but different retention times were identifiable (Figure 23 ) .

Occasionally a peptide is encountered that cannot be purified in the acidic milieu of the normal TEAP 2.25 10.1 % TFA buffer systems due to a lack of solubility. Such peptides are usually moderately to highly acidic and/or hydrophobic; at an acidic pH (pH < 4), these compounds may be temporar- ily soluble yet will aggregate upon standing or upon loading onto the preparative cartridge. One such group of peptides are those related to the GnRH precursor protein, pHGnRH, a 69-peptide. The preparation and lack of biological activity in the systems in which [ pGlu ’ ] -pHGnRH ( 1-26 ) was tested have been reported elsewhere.59 This peptide was found to be extremely insoluble in TEAP 2.251


0 L, 0 5 10 15 20 25

Time (min)

FIGURE 22 HPLC of an equal load ( - 70 Fg) of GnRH, NT, BN, SP, SRIF, insulin, and glucagon. Conditions were: Semi-prep CIx ( 5 pm particle size, 300 A pore size) column, ( 1 X 25 cm). Buffer was TEAP, pH 2.25; flow rate, 2.5 mL/min.

CH3CN and a strategy using TEAP at pH 7.0/ CH3CN was developed. At that pH, the compound could be easily dissolved and chromatographed." With the knowledge that these purified peptides would probably not be soluble under acidic conditions, we were faced with the problem ofdesalting the

purified fractions. Since plain waterlacetonitrile or dilute acetic acid/acetonitrile would result in unac- ceptable tailing of the product and major losses under the usual desalting conditions, we investigated the use of ammonium acetate (NH,OAc) as an elu- tropic, volatile salt. In this case, after repeated lyo-

1 1 ' 1 ' I I I I ' I I I I " " I 20 25 30 35 40

Time (rnin.)

FIGURE 23 Separation of synthetic GnRHs from different species. Load: 0.2 pg each GnRH; Vydac CIx ( 5 pm particle size) column (0.2 1 X 15 cm). Buffer was TEAP pH 6.5; flow rate, 0.2 mL/min.


Table V Primary Structures of Characterized GnRHs

hypothalamic Porcine/Ovine pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH,

pClu-His-Trp-Ser-Tyr-Gly-Leu-Gln-Pro-Gly-NH2 brain Sea Bream

pGlu-His-Trp-Ser-Tyr-Gly-Leu-Ser-Pro-Gly-NH2 brain Chum Salmon

pGlu-His-Trp-Ser-Tyr-Gl y-Trp-Leu-Pro-Gl y-NH2 brain Dogfish 111

pGlu-His-Trp-Ser-His-Gly-Trp-Leu-Pro-Gly-NH2 brain Catfish I

pClu-His-Trp-Ser-His-Gly-Leu-Asn-Pro-Gly-NH2

brain Ratfish brain Dogfish I1 brain Catfish I1

pGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH2 brain Lamprey I

pGlu-His-Tyr-Ser-Leu-Glu-Trp-Lys-Pro-Gly-NH2 brain Lamprey Il l

pGlu-His-Trp-Ser-His- Asp-Trp-Lys-Pro-Gly-NH,

brain Chicken I brain Alligator I

brain Chicken I1 brain Alligator 11

philizations, we found this particular buffer to give good recovery of the highly purified desired product.

HPLC of lnsulins from Different Species6'

In order to compare column supports, eight different insulins (Table VI) were separated using ( 300 A pore size) columns that were derivatized three ways (CI8, phenyl, and C,) and using two buffer systems (0.1% TFA/CH3CN and TEAP 2.25/ CH3CN). The use of C,, phenyl, and diphenyl (not illustrated here) columns was not extensive at that time and was relatively new to the separation of peptides and protein^.^'.^* Their use, however, played a major role in the isolation of rat hypothalamic growth hormone releasing factor and corticotropin releasing factor (as shown a b ~ v e ) , ~ ' , ~ ~ . ~ ~ as well as salmon GnRH64 and human pancreatic growth hormone releasing factor.62

During these experiments we found that the CIS column was the most hydrophobic (as defined by peptide elution volumes) and that the phenyl column was the least hydrophobic, there being a small but noticeable difference between the phenyl and C4 column. Pearson and Regnier6' found that in a TFA / 2-propanol system, the retention times were similar for chain lengths from C2 to CZ2 and proposed that the proteins used in their studies, of which insulin was one, ". . . only interact with the extreme top portion of alkyl chains. . . ."65 Our findings of different retention times for the C, and

C18 columns using a TFA-acetonitrile buffer war- ranted further investigation into the interaction between the organic modifier (acetonitrile vs propanol) and the reverse phase support. In the TEAP system (Table VII ) , the elution order was the same and the phenyl column was still unable to resolve ovine and rabbit insulin. There was, however, a striking change in the comparative hydro- phobicities of the columns. The retention times on the phenyl and C4 columns were nearly identical, while the retention times on the CI8 columns were much greater.

The separation of eight insulins on a C4 column using 0.1 % TFA is illustrated in Figure 24 and a 0.1 % TFA separation is compared to a separation carried out using TEAP 2.25 (see Table IX). These separations were remarkable, but not unexpected (see separation of P-endorphins, 66 when one con- siders that rabbit and human insulins differ by a methyl group only, i.e., the substitution of threo- nine for serine at position B30, Table VI). In general, under identical gradient conditions, elution of insulins with TEAP resulted in lower capacity factors and better separation though it should be noted that the separation of human insulin from rabbit insulin is greater in 0.1 % TFA than in TEAP. The peak shapes of rat I and rat I1 insulin are already broad in 0. l % TFA. This problem is even greater in the TEAP buffer and was thought to be due to the slower exchange kinetics of the solute interacting through the "bilayer" of triethylamine phosphate compared to that in 0.1% TFA.67

All three columns and both buffers can be uti- lized for effective separation of insulins. TEAP 2.25 generally allowed for greater separation and quicker analysis but some compounds chromatographed more broadly. The 0.1% TFA buffer was not as selective but gave better peak shape on the more hydrophobic insulins. The CI8 column showed greater resolution of the more hydrophilic insulins. The phenyl column did not show as much resolution overall, yet porcine and human insulins separated best on that column in TFA.

From these studies we could conclude that as a function of their ion-pairing capacity, mobile phases may have significantly more pronounced effects on a given separation than the particular bonded phases. We have found that the supports and mobile phases reported here are compatible and their usage results in most remarkable separations.

HPLC of Template Assembled Synthetic Proteins68 Template-assembled synthetic proteins (TASP) are tools to investigate the protein folding problem.


Table VI Structure of Insulins

5 10 15 20 A-chain (human): Gly-Ile-Val-Glu-G1n-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys-Asn-OH

B-chain (human): Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-GIu-Arg- 5 10 15 20

25 30 GI y-Phe-Phe-Tyr-Thr-Pro-Lys-Thr-OH

~~~

Modifications of the Insulin Structure

A-Chain Position B-Chain Position

Source 4 8 9 10 1 2 3 9 27 29 30

1. Chicken 2. Bovine 3. Ovine 4. Rabbit 5 . Human 6. Porcine 7. Rat I 8. Rat I1

Glu His Glu Ala Glu Ala Glu Thr Glu Thr Glu Thr Asp Thr Asp Thr

Asn Ser Glu Ser Ser Ser Ser Ser

Thr Val Val Ile Ile Ile Ile Ile

Ala Phe Phe Phe Phe Phe Phe Phe

They consist of a template (cyclic in some cases) onto which are built linear chains. The structure of these assemblies is then studied spectrometrically under different sets of conditions (CD, nmr, and x- ray) in order to reveal nonlinear chain connectivi- ties. In order to elucidate the potential of solid phase peptide synthesis for building up this new family of macromolecules, we (in collaboration with the group of Dr. Mutter)68 designed TASP with the following structural features:

1. A cyclic ( 1 - 10) template: Ac-Cys-Lys-Ala- Lys-Pro-Gly-Lys-Ala-Lys-Cys-NH2

Table VII Comparison of Retention Times on C4 vs Phenyl vs CI8 in 0.1% TFA”

Retention Time (min)

c4 Phenyl CIS

Chicken insulin 14.31 13.20 15.46 Bovine insulin 17.81 17.38 18.81 Ovine insulin 18.23 17.70 19.26 Rabbit insulin 18.60 17.70 19.76 Human insulin 18.85 17.86 20.0 1 Porcine insulin 19.16 18.36 20.35

Ala Asn Ser Val Asn Ser Val Asn Ser Val Asn Ser Val Asn Ser Val Asn Ser VA1 Lys Pro Val Lys Ser

Ser Thr Thr Thr Thr Thr Thr Thr

Lys Ala Lys Ala Lys Ala Lys Ser Lys Thr Lys Ala Lys Ser Met Ser

2. four identical chains covalently bound to the c-amino function of the lysines. These chains were eleven, fifteen, and eighteen amino acids long as shown: Glu-Leu-Leu-/Glu- Ala- Leu-Glu-/ Lys- Ala- Leu- Lys-Glu-Ala- Leu- Ala-Lys-Leu-Gly-.

The peptides were assembled using a combination of Boc and Fmoc strategies on a methylbenzhydrylamine solid support. Cou- pling reagents were diisopropylcarbodiimide and benzotriazolyloxy tns- ( dimethylamino)- phosphonium hexafluoro-phosphate ( mostly to introduce dipeptides) ; disulfide formation was achieved using potassium ferricya-

Table VIII Comparison of Retention Times on C, vs Phenyl vs CIS in TEAP 2.25”

~ ~~~


C4 Phenyl CIS

Bovine insulin 9.63 9.82 20.88 Ovine insulin 10.30 10.35 22.00 Rabbit insulin 10.50 10.35 22.36 Human pancreatic insulin 10.90 10.82 22.80 Porcine insulin 11.26 11.25 23.22

a Gradient: from 40 to 60% B in 25 min; flow rate: 2 mL/ min. Buffer A: 0. I % TFA in water; buffer B: 0.1% TFA in water- acetonitrile (40 : 60).

a Gradient: from 40 to 48% B in 25 min; flow rate: 2 mL/ min. Buffer A: TEAP 2.25; buffer B: TEAP 2.25-acetonitrile (40 : 60).


? F

Time (min)

FIGURE 24 Separation of (5 pg each) A, chicken insulin, 9.23 min; B, bovine insulin, 15.70 min; C, ovine insulin, 16.66 min; D, rabbit insulin, 17.43 min; E, human insulin. 18.00 min; F, porcine insulin, 18.75 min; G, rat I insulin, 19.96 min; H, rat I1 insulin 22.36 min. Vydac C4 ( 5 pm particle size, 300 A pore size) column, V-1830-4 (0.46 X 25 cm). Buffer was 0.1% TFA; flow rate, 2 mL/min.

nide. Although the crude synthetic mixtures showed a major component by HPLC in the case of the 54- and 70-peptides, isolation of the 82-peptide in a relatively pure form was quite challenging and followed purifications schemes presented below for a cholecystokinin-58 analogue and a rat histone fragment. Here we want to emphasize the role of particle size in a separation on a semipreparative scale. Columns were custom made and provided by The Separations Group. There is no doubt that the smaller the particle size, the better the separation of the different components of the mixture and the purer the product ultimately isolated; this could not be emphasized enough. We all recognize that the main component of the mixture shown in Figure 25 sits on a mound of closely related impurities shown as a significant baseline shift. When one collects fractions, not only does one collect the main peak but also the area under this peak, all the way down to baseline. We went through the exercise of drawing a flat baseline under each chromatogram, vertical lines at the beginning and the end of each peak and a line separating the top of the peak (the desired product) from its

base (which must account for a large number of closely related impurities in small amounts). Cutting the areas under each of the peaks and their base and weighing them separately allowed a rough quantitation of the purity of each of the fractions. Our esti- mation is that the material isolated with the 15-20 pm silica would be ca. 55% pure, the material isolated with the 10-1 5 pm silica would be 60% pure, and the material isolated with the 10 pm silica would be ca. 65% pure-that is if there is no other single component (aside from the impurities responsible for the “mound”) in significant amount coeluting with the desired product. After this first step of purification it is clear that another one with different selectivity should be used in order to separate the desired product from the underlying complex mixture of impurities. This was achieved using further purification systems taking advantage of column and buffer selectivity as shown below. Based on HPLC, CZE profiles, and sequence analysis, it was concluded that this 70-TASP was at least 90% pure. Conformational analysis, using CD of this TASP and individual chains, suggest a definite participation of the template in inducing and maintaining the predicted a-helical secondary structure of the 4-helix bundle.

Temperature Effects

Early studies on the influence of temperature concluded that the higher the temperature, the lower

Table IX Comparison of Retention Times Using 0.1% TFA vs TEAP 2.25 on a Cq Columna


O.I%TFA TEAP2.25

Chicken insulin 15.86 14.68 Bovine insulin 18.53 17.72 Ovine insulin 18.93 18.25 Rabbit insulin 19.1 1 18.55 Human insulin 19.58 18.80 Porcine insulin 19.70 19.06 Rat I insulin 19.8 1 19.6 1 (Broad) Rat I1 insulin 20.90 2 1.03 (Broad)

“Gradient: from 30 to 55% B in 25 min; flow rate: 2 mL/ min. Buffer asdefined in Table VII (TFA) orTable VIII (TEAP).


I 0.4

5 0 N N -

0.2 :: 3 a

0

-

0 2 0 4 0 60 Time (min)

FIGURE 25 Semi-preparative separation of a TASPaEffect of particle size. Crude T4- ( 4 c ~ , ~ - A c ) (5.3 mg, MW ca. 7500 Da) on Vydac C4 (300 A pore size) column (2.2 X 25 cm). Buffer A was 0.1% TFA ( 5 % CH3CN/H20). Buffer B was 0.1% TFA (80% CH3CN/H20). Gradient: 45-10'-45-45'-65% B; flow rate, 8.0 mL/min.

the resolution. Even if retention volume was kept constant at two different temperatures, resolution at the higher temperature was poorer than at the lower one.22 Yet peak shape was improved by increased temperature. This was against common sense and it is only with the development of silica- based supports with high carbon loading and/or capping that the expected improvements in resolution with high temperature were observed. We now explain the early results by the less than optimal characteristics of the derivatized silicas and the unique properties of such peptides as GnRH and CRF. Most analyses nowadays are run above am- bient temperature.

Temperature effects on the chromatographic profile of peptides with constrained conformations can be quite drastic as exemplified by the chromatographic behavior at different temperatures of conotoxin M 1, a 14-amino acid ~ e p t i d e . ~ ~ Both the synthetic and natural peptide gave badly skewed peaks during chromatographic purification at 25"C, which contrasted with the sharp peaks obtained after trypsin or chymotrypsin treatment and with those obtained for conotoxin GI, GIA, and GII. A number of additional experiments strongly suggested that the broad peaks might be due to nonequilibrium behavior of the peptide during elution from the HPLC column.

Material was isolated from the first third of a typical peak and rerun under the same condi-

tions. The complete profile was regenerated (data not shown), eliminating explanations such as chemical breakdown or modification. In addition, a similar broad peak was seen on testing a different reverse phase column that had given sharp peaks with other conotoxins. Finally, the basic features of peak shape were independent of loading in an 80-fold concentration range (0.5- 40 nmol) of peptide.

The strong forward skew of conotoxin MI peaks suggested to us the occurrence of a slow intercon- version between two or more forms of the peptide in solution, rather than nonequilibrium partitioning between mobile and stationary phases. A 5-fold increase in concentration of the ion-pairing agent in the chromatographic elution buffer, to 0.5% TFA, led to the expected increase in retention time, but did not cause any obvious change in peak shape. Regardless of the cause of disequilibrium, we expected an increased temperature to improve peak shape by speeding up the attainment of equilibrium. A marked improvement was obtained at 50 and 60°C.69

The two modes of nonequilibrium behavior should give different results on lowering the temperature during chromatography. If disequilibrium is between sorption / desorption, one expects the problem to become worse at low temperature. By contrast, disequilibrium between two peptide conformations, when carried to an extreme,

Peptide Chemistry

should lead to separation of two sharp peaks with an elevated baseline between them. This is precisely the result that was obtained by immersing the column in an ice bath during chromatography of conotoxin MI. This result strongly indicated that the anomalous behavior was due to retarded equilibrium between conformational states of a single ~ e p t i d e . ~ ~

HPLC of Inhibin-31-OH, Use of Heptafluorobutyric Acid (HFBA)

We have mentioned a number of challenges resulting from the unique physicochemical properties of peptides related to solubility, hydrophobicity, size, and secondary structure. At the other end of the spectrum of solubility, we have found very basic peptides to be so hydrophilic that they could not be adsorbed to the usual column supports ( CI8) using the standard buffers. In these cases, we have found the use of the strong ion-pairing heptafluorobutyric acid (HFBA) to be extremely helpful at a concentration of 0.05-0.1 % (data not shown). We have also found HFBA to be a particularly powerful ion- pairing agent for the chromatography of arginine-, histidine-, and lysine-rich peptides.

Inhibin-3 1 -OH is a 3 1-peptide that was isolated from human seminal plasma o n the basis of its inhibin-like activity.” Because of our interest in the characterization of inhibin of gonadal origin the function of which is to inhibit follicle-stimulating hormone secretion and our observation that it was a rather large and complex protein, it was particularly important for us to test such a small peptide and, above all, be sure of its identity and purity. We assembled this peptide on a Merrifield resin using standard protocols, cleaved and depro- tected the peptide with hydrofluoric acid, and after extraction and lyophilization, obtained a very complex crude mixture eluting as a major hill from which three identifiable peptides emerged. The fraction containing the desired peptide was identified and isolated using the usual TEAP and TFA buffers. Figure 26A shows the elution profile of the purified material as a single symmetrical absorbance. To our surprise, this material gave an unac- ceptable amino acid analysis ( a poor ratio for arginines, lysines, and histidines could not be explained). Because (HFBA) is a common sequencing reagent and is readily available, and since it had been described as a counterion for HPLC purposes, we used it for analysis of this preparation of inhibin-3 1 -OH, rationalizing that if the peptide was impure because of the deletion

0

E

2 K

0.1

3 a

A

5 10 15 Time (min)

B

I

I

I

5 10 Time (rnin)

20

00

z I 0,

12 0 8

30

1

295

100

z : 0 -0 0-

24

2

1

FIGURE 26 HPLC analysis of inhibin-3 1-OH (load: ca. 20 pg) . Vydac CIS, ( 5 pm particle size, 300 A pore size) column (0.46 X 25 cm). Panel A: buffer was 0.1 % TFA. Panel B: buffer was 0.1% HFBA; flow rate, 2.0 mL/min.

of one or several arginines, histidines, or lysines, a strong cation pairing reagent would most likely help discriminate between these different entities. To our surprise (see Figure 26B), the presence of several major impurities became apparent. These could be eliminated subsequently in a preparative run using HFBA to yield highly purified inhibin-3 1 -OH, which was ultimately shown to be inactive in our assay systems.


PREPARATIVE HPLC AND SYNTHETIC PEPTIDES, STEP 111

Introduction

By the late 1970s, extrapolation from analytical to semipreparative, and finally, to preparative (gram amount) separations of small biologically active peptides from nine to fourteen amino acids (gonadotropin releasing hormone and analogues, and somatostatin) had been shown possible and was performed routinely in our laboratory using commercially available stainless-steel 1 X 30 cm CIS columns from Waters Associates.’.’’ Similar semipreparative results dealing with small protected or unprotected peptides had also been reported by other^.^^-^^ Unique was our use of the radially compressed Prep Pak CI8 cartridges ( 5 X 30 cm) from Waters Associates with the Prep LC-500 apparat~s.~’ Its limitations were ( a ) lack of flexibility due to the unavailability of gradient capabilities of the preparative instrumentation (>20 mL/min); (b) poor mechanical as well as inadequate chromatographic characteristics of the C18 silica for peptides (even relatively small ones) in the Prep Pak CIS cartridges ( 5 X 30 cm) from Waters Associates. This led to poor recovery of peptides and is without mentioning that large quantities (gram amounts) of small peptides are costly, thus limiting the number of trial runs that could be performed in order to optimize the chromatographic conditions. The first convincing evidence, however, that preparative HPLC had great potential was that even with these drawbacks, it performed better for the large-scale purification of somatostatin and its analogues than countercurrent distribution, which was the only available preparative technique that could practically be scaled up.78

Extrapolating what had been learned from analytical work presented above, it became obvious that preparative separations should also best be carried out on end-capped CIS silicas with large pores.47.6 1.79 While such material ( 15-20 pm) was made available to us by Vydac, we were able to convince Waters Associates to provide us with frits and empty cartridges (5 X 30 cm), which we learned to dry pack efficiently. The use of large particle size for preparative work is dictated by two considerations: the high cost of small particle size and back pressure. We will summarize here the material presented in our first paper describing the preparative purification of synthetic peptides of all sizes.”

First, we demonstrated that these particular car-

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::

0

cu T-

Z 0.16

3 a

0 I

00

z

r 0

0,

8 35

15

0 ~

0 10 20 30 40 Time (min)

FIGURE 27 Analytical run on preparative cartridge. Load: synthetic peptides (structures shown in Table 1 ). Column: PrepPak 500,30 X 5 cm I.D., packed with Vy- dac CI8 ( 15-20 p m particle size) material (46 atm radial compression). Buffer was TEAP, pH 2.25; flow rate, 100 mL/min.

tridges could be used analytically. Indeed, we hy- pothesized that if we could demonstrate that a fraction of a milligram of peptides-such as GnRH, neurotensin, bombesin, substance P, somatostatin, insulin, and growth hormone releasing factor (a member of the glucagon family of peptides)- could be recovered from such large cartridges (5 X 30 cm) and that the elution profile would be symmetrical, we would be convinced that recovery was high if not quantitative. Figure 27 illustrates such an analytical run. As can be seen, the elution profile of the two most critical compounds in our past experience (insulin and GRF) was almost per- fectly symmetrical, considering the fact that the pumping system was, at times, capricious and the gradient maker somewhat primitive. The symmetrical elution profile suggested that true partitioning took place between stationary phase and mobile phase and as a corollary would exclude any “memory effect” of the column. The memory effect, in simple terms, is the ability of the column to adsorb part of the material that is being chromatographed in one run and release part of it in subsequent runs. Such memory effects were often blamed (and right- fully so) for contamination of natural as well as synthetic peptides. It was not unusual for one of us to review papers relating to the isolation of a known natural product whereby yield of the native material would increase dramatically as the purification proceeded-results that generally left the


investigators astonished and most of the time won- dering which material had interfered in their RIA that had led to underestimation of the amount originally in the extracts. One early solution was to dedicate columns and, at times, injectors to particular projects as we did for the isolation of the CRFs from different species. At the time, to validate our chromatographic systems and reject the possibility of contamination, we would screen our column eluate using sensitive RIA developed for the particular compounds. To the dismay of the peptide chemist, RIA would easily detect contamination on the order of less than 1 in 100,000, definitely three orders of magnitude more sensitive than any spectrometric technique. It was therefore rewarding to find out that cartridges filled with the Vydac derivatized silica (300 A pore size) would give very good recovery of peptides and not show any detect- able tendency to retain material from one run to the next as long as either a TEAP or a TFA containing buffer was used and that adequate cleaning protocols were followed.

We illustrate three different representative strategies for the purification of crude synthetic peptides using hGRF( 1 -40)-amide, synthetic conotoxin GVIA, and azaline as examples. Many more such examples have been described by us.60,80-85

Purification of hGRF ( 1-40) -Amide

The neuroregulation of growth hormone secretion is mediated in part by a stimulatory GRF and an inhibitory peptide, somatostatin, both of which reach the hypophysis by the hypothalamic-hypo- physial portal system. GRF was first isolated from a human pancreatic tumor and characterized independently by two g r o ~ p s . ~ ~ . ~ ~ Whereas Guillemin and collaborators isolated three different entities with biological activity including the mature form, which is an amidated 44-peptide, in the tumor made available to us, only the fragment 1-40 could be identified. At the time it was not clear to us whether the native form had a C-terminus free carboxyl or would be amidated. Both peptides were synthesized and only the free C-terminus peptide co- eluted with the native material. We describe here the preparative purification of hGRF ( 1 -40)-amide.

First, an analytical profile developed in TEAP pH 2.25 on a CI8 column was obtained in order to ascertain that there is a major component (Figure 28). When it is not the case, fractions could be collected manually from this preliminary run for analysis by mass spectrometry using, for example, a sensitive time of flight instrument. After examina-

tion of the elution profile of Figure 28, and the determination of the relative position ( 16 min) and abundance (major component) of the desired product, isocratic conditions are found whereby the desired peptide is eluted within 4-6 min. Al- though we often screen our columns using the same buffer system as the first one used in our purification, we have found it advantageous to screen our columns with the desalting buffer. This gives us an opportunity to identify impurities that are not separated under the original conditions while it also reveals that further purification (resulting from different solvent-induced selectivity) may be obtained with the later buffer system. In this particular example, isocratic conditions whereby the desired material is eluted with a retention time of 3.5 min were selected (Figure 29). These isocratic conditions will be used for the monitoring of the eluate from the preparative cartridge as illustrated below in Figure 3 1.

Parenthetically, we should mention that the selection of isocratic conditions may present some interesting surprises. In the case ofthe isocratic separation of neuropeptide (NPY) from its fragment NPY (20-36) using the 0.1% TFA/acetonitrile buffer system, the absorbances attributed to NPY and NPY(20-36) change their order of elution with baseline separation (after overlapping each other) within a range of 2.4% acetonitrile and the corresponding retention times of 8.5 and 3 min (see Hoeger et a1.60 for illustration). This phenomenon is likely due to the different sizes of the two peptides being separated whereby at high concentrations of acetonitrile a large peptide such as NPY may not fully enter the chromatographic support, with the mode of separation becoming mostly one of size exclusion. This phenomenon, in fact, has been exploited in the case of preparative separations (unpublished results).

Crude hGRF( 1-4O)-amide (6.3 g) was purified in three runs. The 40-peptide was loaded on a Vy- dac cartridge, 17-pm particle size, in TEAP 2.25-acetonitrile buffer (Figure 30). A gradient of acetonitrile was applied (ca. 1% acetonitrile/3 min). As a general rule of thumb we used gradients that consist of a graded increase in the concentration of CH3CN at the rate of 1% every 3 rnin at a flow rate of 100 mL/min. We have found that gradients of acetonitrile shallower than 0.2% per min at 100 mL/min resulted in peak broadening with little improvement on overall separation or recovery.

Fractions were collected approximately every 50-75 mL and analyzed isocratically in TEAP-ace-


0.

I I I I I

10 20 30 Time (min)

FIGURE 28 Analytical profile of crude hGRF( 1-40)-NH2 from HF cleavage (20 pL, ca. 20 pg) under gradient conditions. Vydac CIS ( 5 pm particle size) column (0.46 X 25 cm). Buffer was 0.1 % TFA; flow rate, 2.0 mL/min.

tonitrile buffer (Figure 3 1 ). It should be remembered that in these cases, resolution achieved by the column was a direct function of the size of the collected fractions. The fractions from each of the three preparative runs that most resembled fraction 7 in Figure 30 were then pooled and rerun on

I- \

I I

0 ’ 5 10

Time (min)

FIGURE 29 Analytical profile of crude hGRF( 1-40)- NH2 from HF cleavage (20 pL, ca. 20 p g ) under “isocratic” conditions. Vydac CI8 ( 5 pm particle size) column (0.46 X 25 cm). Buffer was 0.1% TFA in H20; flow rate, 2.0 mL/min.

a Vydac C4 cartridge, 17-pm particle size in TEAP 2.25-acetonitrile, as were the fractions from each of the three runs that most resembled fraction 8 in Figure 29, as seen in Figure 32. The isocratic analyses of the fractions generated in the different preparative runs allowed the selection ofthe highly purified fractions selected for further chromatography. The fractions from each of the three runs that most resembled fraction 9 in Figure 31 were then pooled and rerun on the preparative HPLC, as were the three fractions that most resembled fraction 10 from Figure 29. In this manner, all the impure fractions were processed to a uniform purity with the aid of two different cartridges, C18 and C4. These fractions, which had a total volume of 3.0 L after being diluted with water to ensure that the peptide adsorbed to the cartridges, were pumped onto the C4 column and desalted with 0.1 % TFA- acetonitrile ( Figure 33 ). The three fractions were lyophilized and yielded 60, 345, and 137 mg, respectively, with HPLC purities of 98.0, 99.1, and 94.2%, respectively.

Preparative Purification of Conotoxin GVIA

Voltage-sensitive calcium channels are critical components of neurons. Their role in neurotransmitter release is well documented. In addition, electrophysiological work has indicated that calcium channels are essential for the generation of complex firing patterns that are found in certain neurons. Conotoxin GVIA (See Table I ) , first iso-


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m N N

3 4

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-

-

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0 10 20 30 40 50 60 70

100

2 54 0 8

33

21

0

Time (min)

FIGURE 30 Preparative profile of crude hGRF( 1-40)-NH2 from HF cleavage (400 mL, 2.1 8). Column: Vydac ClX (17 pm particle size) in PrepPak 500 cartridge ( 5 X 30 cm; 40 atm radial compression). Buffer was TEAP, pH 2.25; flow rate, 100 mL/min.

lated and characterized from the fish-hunting cone snail Conzis geogruphus, 8 ' 3 7 inhibits neuronal voltage-activated calcium channels. Because of a limited supply of native material (which had to be isolated from a complex mixture of other toxins found in the venom of the C. geogruphus) and the need of relatively large quantities for the understanding of the molecular biology of Ca2+ channels, we developed a synthetic approach that led to the total synthesis of this toxin. Conotoxin GVIA contains three disulfide bridges (which could yield theoretically I5 different secondary structures) and two hydroxy-prolines resulting from posttransla- tional modification of a proline. Synthesis was carried out on a solid support and cyclization was car-

ried out by air oxidation yielding a complex mixture of compounds. The analytical HPLC-uv trace ofthe crude toxin run in 0.1% TFA/CH3CN is presented in Figure 34. A major component (starred) could be identified as the desired product (determined by coelution with a sample of the native peptide). As shown earlier, we have found that, in order to gain highly purified synthetic peptides, at least two sets of purification conditions should be used, since purification in two systems (TEAP followed by TFA) and analysis in the second (TFA) minimizes the probability of missing any impurities. We have observed, with disulfide- bridged polycyclic peptides, the importance of this practice, as well as that of carefully determining the

I ' I , , 5 0 5 0

I , , 5 0 5

I00

2 P 0 s

32 31.5

3

Time (min)

FIGURE 31 Isocratic screen of fraction 5- 12 generated in preparative purification shown in Figure 30 (20 pL, ca. 20 pg each). Vydac CIx ( 5 pm particle size) column (0.46 X 25 cm). Buffer was 0.1% TFA. 3 1.5% and 32% CH3CN isocratic, after fraction 6; flow rate, 2.0 mL/min.


1 .o-

5 0 0 N

B 0.5 ul LL 3 Q

-

100

2

H 0 a?

34

22

0

10 20 30 40 50 60 Time (min)

FIGURE 32 Preparative profile of partially purified hGRF( 1-40)-NH2: 3 X fraction 8 (300 mL, ca. 0.2 g) from preparative purification shown in Figure 30 and screened in Figure 3 1. Vydac C4 ( 17 pm particle size) in PrepPak 500 cartridge ( 5 X 30 cm; 40 atm radial compression). Buffer was TEAP, pH 2.25; flow rate, 100 mL/min.

analytical isocratic conditions. Normally we have found that relative peak location does not dramatically change in going from TFA to TEAP for linear 10- to 50-peptides; however, for some polycyclic peptides we have observed the early elution of the desired product out of the peptide by-product en-

2.0 -

E 2 1.0-

:: 0 m

3 a

-100

2

Y 0 8

-30 " 0 10 20 30 Time (min)

FIGURE 33 Preparative desalting of hGRF( 1-40)- NH2 fractions from five preparative C4 purifications shown in Figure 32 (3000 mL, ca. 550 mg). Vydac C4 ( 17 pm particle size) in PrepPak 500 cartridge ( 5 X 30 cm; 40 atm radial compression ). Buffer was 0.1% TFA; flow rate, 100 mL/min. A.B. = air bubble spike.

velope. One such occurrence was observed during the purification of synthetic w-conotoxin GVIA. The analytical isocratic conditions were determined for this peptide and the crude material was then applied to a CI8 cartridge. The compound was then eluted using TEAP 2.25/CH3CN buffers; the profile for this purification is given in Figure 35. Based on the previously obtained analytical HPLC (Figure 34), it was expected that fractions 9, 10, and I 1 would contain the desired peptide as we have found that relative elution order does not change dramatically from one system to the other; however, analysis of these fractions by isocratic HPLC indicated that the peptide was not present in these fractions but rather was located in fractions 4 and 5 (Fig. 35 ). These two fractions were then pooled and desalted using the volatile 0.1 % TFA/ CH3CN buffer system (Figure 36). The analytical HPLC of the final product obtained is illustrated in Figure 37; the synthetic material was demonstrated to coelute with the natural product and had the anticipated biological activity."

The reason for the unusual behavior of these peptides in TEAP 2.25 (i.e., the early elution of the desired product) is unclear; however, it does point out the importance of isocratic screening of fractions and the advantages of using a two-buffer system purification scheme. In the case of these polycyclic peptides, the differences between the effects observed upon usage of the TEAP or TFA buffers


I I I 1 I I I

0 5 10 1 5 2 0 2 5 30 Time (min)

FIGURE 34 Analytical HPLC profile of crude Conotoxin GVIA ( 15 pL, ca. 2 pg) on Vydac CI8 ( 5 pm particle size), column (0.46 X 25 cm). Buffer was 0.1% TFA; flow rate, 2 mL/min.

was advantageous in the sense that the desired product eluted away from undesired impurities. It is as if TEAP promotes a more stable secondary structure, whereby intramolecular hydrogen bonding is favored for the native structure only; this would then limit the interaction of the desired peptide with the support, thus resulting in early elution. A rationale for this observation would be that the cyclic entity may appear as having a smaller radius than the other expected species, which sup- posedly include ( a ) correct peptide with incorrect disulfide bridging and ( b ) polymeric unprotected or partially protected peptide and their fragments. The TFA buffer system, on the other hand, may disrupt the internal hydrogen bonding through the

2.0

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fii 1.0

a

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formation of ion pairs and consequently increase the indiscriminate interaction of the peptide with the support. It must be realized that such effects are not commonly seen for linear peptides.

Purification of Azaline (See Table I for Structure)

Mammalian GnRH plays a major role in the mod- ulation of reproductive functions. A concerted effort directed toward the development of potent and long-acting agonists and antagonists has been sustained by many l a b ~ r a t o r i e s . ~ ~ . ~ ~ Whereas some of the superagonists are now available for therapeutic use, the relatively low potency of the competi-

0 10 20 30 40 50 60 Time (rnin)

FIGURE 35 Preparative HPLC profile of Conotoxin GVIA (930 mg in 100 mL buffer). Vydac CI8 ( 15-20 pm particle size) in PrepPak cartridge (5 X 30 cm). Buffer was TEAP, pH 2.25; flow rate, 100 mL/min; back pressure, 25 atm.


2

E 0 m (v

v) LL 3

c

m l

a

0

FIGURE 36

Time (min)

PreDarative desalting of Conotoxin GVIA fractions 4, 5 from Figure 35 (ca. - 100 mL diluted 1 : 1 with HzO). Vydac CIS ( 15-20 Km particle size) in PrepPak cartridge (5 X 30 cm). Buffer was 0.1 % TFA; flow rate, 100 mL/min; back pressure, 25 atm.

tive antagonists coupled with the finding that some of them release histamine in a variety of tests’” including in h u m a n ~ , ~ ~ have been the main road-

I I I I J 0 5 10 15 20

Time (rnin)

FIGURE 37 Analytical profile of purified Conotoxin GVIA (from fractions 5-7 from Figure 36 (ca. 15 F g in 15 KL); Vydac Ci8 (5 Fm particle size) column (0.46 X 25 cm). Buffer was TEAP pH 2.25; flow rate 2 mL/min.

block to their acceptance and use. Because the histamine releasing property of these analogues was correlated to both their overall hydrophobicity and the presence of strongly basic side chains, analogues such as the Nal-Glu antagonist ( [ AC-D- Nal i,D-Cpa2,D-Pa13,Arg5 ,4-(p-methoxybenzoyl)- ~-2-Abu‘,~-Ala ‘“1 GnRH)92 were developed and extensively studied in humans.” It was recognized, however, that for contraceptive as well as other purposes that require sustained administration, the therapeutic index (relative potency at inhibiting gonadotropin secretion over relative potency at stimulating histamine release) of Nal-Glu is still too low. To address this challenge, we have described an approach to generate trifunctional, moderately basic, novel amino acids by the selective modification of the w-amino function of orni- thine, lysine, 94 and paraaminophenylalanine (Aph), some of which (mostly those containing Aph) have high potency, low histamine-releasing activity, and long duration of action.” Azaline is a short acting member of this safe family of GnRH antagonists. Some unexpected results obtained during the purification and characterization of this peptide are described here. We first carried out a “standard” preparative purification of this peptide, which appeared to be quite pure as a crude prepa-


0.0

5 P

:: N -

0.0

3 a

I l l l ~ l i l l l l l l

3 5 10

Time (min)

FIGURE 38 CZE trace of partially purified Azaline. Voltage: 20 kV (constant); current: 1 13 pA; 50 cm X 75 pm fused silica capillary at 30°C. Running buffer was 100 mM sodium phosphate, pH 2.50.

ration using both TEAP pH 2.25 and desalting with dilute AcOH in order to obtain the acetate salt (data not shown). Although being routinely characterized using HPLC in different acidic systems, the CZE profile shown in Figure 38 was obtained, which indicated the presence of 20% of an impurity when all other analytical systems tested indicated the presence of a single component. This suggested that an impurity with a different overall charge than that of the desired peptide was present. Our past experience suggested that we run this preparation at a different pH. It was found that this impurity could be separated from azaline by using TEAP at a pH of 7.0 (Figure 39). Repurification of this peptide at pH 7.0 (coupled with isocratic analytic HPLC screening of the fractions at pH 7.3) , followed by conversion to the acetate salt and lyophilization of the “acceptable” fractions, provided the highly purified azaline containing less than 1% of this closely associated impurity.

The use of high pH mobile phases (>7) on silica-based columns is usually avoided as column performance and life are reduced compared with low pH mobile phase use. However, a recent study has shown that certain silica-based packings can be used for long periods at pH 9 without significant changes in chromatographic proper tie^.^^ Bonded- phase degradation at low pH is mainly due to hy-

drolysis of the covalent Si-0-Si group whereas column failure at high pH results from silica support dissolution. Kirkland et al. found that densely bonded monomeric dimethyl-Cls ligand better protect the silica support from dissolution than bulky diisopropyl- and diisobutyl-substituted bonded ~ i l a n e s . ~ ~ Also, whether the bonded CIS stationary phase is monomeric or polymeric does not significantly influence column degradation at high pH. The rate of silica dissolution is a strong function of the nature of the porous silica support (differences in preparation methods) and the purity of the support ( more highly purified, less acidic silicas dissolve more rapidly while less pure silicas seem to be more stable at high pH). Acetonitrile was compared to methanol as an organic modifier at high pH and found to prolong column life in this study.

It should be noted that polymeric organic materials exist for reverse phase separations of peptides and proteins. We have only limited experience with these supports, yet none of these tested so far compared favorably with the best silica base supports even with the limitations of pH at which they can be used. It should also be noted that many peptides are not very stable at basic pH.

0.2

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0 z 0.1 N

rn U 3 Q

0

/-

L

I I 1 I 0 5 10 15

Time (min)

00

z I 0

0,

s

13

!4

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FIGURE 39 HPLC trace of partially purified Azaline (20 pL, 10 pg) at neutral pH. Vydac CI8 ( 5 pm particle size) column (0.46 X 25 cm). Buffer was TEAP pH 7.3; flow rate, 2.0 mL/min.


Remarks

Whereas certain manufacturers of chromatographic supports advertise the predictability of separations from an analytical scale to a preparative scale, we have often found and taken advantage of differences in selectivities between our analytical and preparative columns. It is not unusual to observe that an analytically hydrophobic impurity would behave hydrophilically to the main product on the preparative column. The exact reason for this common difference in selectivity has not been fully explored, but may be due to such variances in the analytical and preparative columns as caused by differences in end capping, particle size, or the use of rigid (metal) vs. malleable (plastic) column walls. With respect to the impurities, we have found that, in general, hydrophilic impurities (on preparative supports) were easier to eliminate than hydrophobic ones; whether this may be due to a certain amount of peak tailing encountered in the purification of peptides on reverse phase packing or whether other factors may be involved was not investigated. It results, however, in poorer recovery of the main product when removing a given amount of hydrophobic impurities riding the tail of the main peak than when eliminating an equivalent amount of hydrophilic impurity from its sharp front.

CHALLENGING PREPARATIVE SEPARATIONS THAT DEMAND THE USE OF COMPLEMENTARY CHROMATOGRAPHIES: STEP IV

Ion Exchange Chromatography

Purification of Rat Histone H2AIFs3 Amide. Ge- netic material is organized in a compact structure by the association of DNA with proteins. Approxi- mately half of these proteins are histones, which are highly basic owing to the presence of a large pro- portion of lysine and arginine residues. The protein H2A is one of the core proteins around which the DNA is coiled.

It has been reported that histone H2A purified from bovine ovaries inhibited the binding of GnRH to rat ovarian membranes and showed anti- gonadotropic activity in cultures of rat ovarian cells. Independently, we purified a protein from rat testes that inhibited the binding of GnRH to bovine pituitary membranes. After extensive purification, a product was identified by SDS polyacryl- amide gel electrophoresis, and a partial sequence

0.2 -

: * ;; 0.1 - 2 c m

3 Q

- 12

0 I I I I I I 6 5 10 15 20 25 30 35

T O

Time (min)

FIGURE 40 Analytical HPLC profile of crude rat histone H2AI-53-NH2 ( 15 pg in 10 pL) on a Vydac CI8 ( 5 pm particle size) column (0.46 X 25 cm). Buffer was 0.1% TFA; flow rate, 2.0 mL/min.

obtained using pulsed liquid-phase Edman degradation. These data and an amino acid analysis indicated that the purified peptide responsible for the activity may be a fragment of rat histone H2A. In order to confirm the biological activity of the fragment, it was necessary to synthesize and purify rat histone H2AI-53 that, for ease of synthesis, was amidated at its C-terminus (see Table I for sequence). We identify here the problems encountered during the purification of the synthetic mixture and propose solutions of general applicability that encompass an alternative to all-RP-HPLC purifications of pep tide^.^'

The analytical profile obtained with HPLC is shown in Figure 40. The crude histone appeared to be remarkably pure. The conditions were those usually used in this laboratory to identify the major component (0.1% TFA) and give an estimate of its overall purity. Because peptides half the size of H2Al-53-NH2 generally give profiles that are con- siderably more complex, 6o the validity of this result appeared doubtful. Consequently, the crude material was analyzed by narrow-bore cation-exchange chromatography, which revealed that there were indeed other major components present (see Fig- ure 41A). Using mass spectrometry, it was determined that the desired product corresponded to the last-eluting component in that chromatogram ( m/ z 5604.8; retention time ca. 31 min). Other fractions eluting were shown by mass spectrometry to be mixtures with both higher and lower masses. Suspecting that the separation shown in Figure

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3

7 - 1 .o

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/-- Prptide Chemistry 305

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0 I I I I

0 10 20 30

Time (min)

FIGURE 41A Crude rat histone H2AI.53-NH2 ( 100 pg in 125 pL) . Column: Pharmacia Mono S Precision, 1.6/5 (5.0 X 0.16 cm id.) . Buffer were [A] = 50 mM boric acid (pH 9.6), [ B] = 1 .OMNaCI in A, with a gradient from 0 to 100% B in 20 min; flow rate, 0.1 mL/min.

41A had not been fully optimized, it was recalled that acetonitrile had been used in the separation of peptides and proteins by size-exclusion chromatography (see below) ." The data shown in Figure 4 1 B demonstrate the beneficial effect of the addition of acetonitrile to the buffers, and also suggest that the order of elution had probably not been altered. This was confirmed by isolation of the last-eluting component (uv detection at 2 14 nm), which corresponded to the desired product. Because the separation shown in Figure 41B was repeatable and showed very good resolution and recovery, scale- up conditions were identified that were also reproducible. We concluded that the use of such systems would benefit those peptide chemists attempting the synthesis and purification of large peptides and proteins. As can be seen, optimization of the chromatographic conditions will play a major role in most isolation projects. The use of acetonitrile in buffers for size exclusion chromatography therefore could be expanded with extremely beneficial consequences to ion exchange chromatography and CZE, as shown later in this report.

Further purification and desalting by HPLC of the desired fraction yielded H2AI-53-NH2 with a purity probably close to or greater than 90%. De- spite the fact that chromatographic conditions that took advantage of ionic and hydrophobic properties of the peptide mixture were used, evidence of residual amounts of impurities were found from a critical evaluation of the mass spectrometric data,

peptide mapping, sequencing data, and CZE, a technique that unfortunately cannot be used pre- paratively as yet (see below).

We clearly can say here that the desired peptide present in extremely small amounts in our synthetic mixture never could have been isolated in a highly purified form if it had not been for the dis-

1 .o

- 0.5 m f2 2

I 1 I I I '0 0 5 10 15 20 25 30 35

Time (min)

FIGURE 41B Crude rat histone H2AI.53-NH2 (60 pg in 50 pL). Column: Pharmacia Mono S Precision, 1.6/5 (5.0 X 0.16 cm id.) . Buffers were [ A ] = 50 mM boric acid ( CH,CN/H20: 35/65) ( p H 9.7), [ B] = OSMNaCl in A, with a gradient from 0 to 100% B in 20 min; flow rate, 0.1 mL/min.


criminating first ion exchange chromatography step. This is also apparent in the next example.

Purification of [Phe(p-CH2S03Na) 52, Nle32,53,56, NalS5] -CCK-58. Cholecystokinin (CCK) was first described as a 33 amino acid p~lypeptide.~' Since that time, several different molecular forms of CCK have been detected in vivo (CCK-58, CCK- 39, CCK-22, CCK-8, and CCK stim- ulates pancreatic exocrine secretion, gallbladder contraction, and intestinal motility, and also may act as a neurotransmitter / neuromodulator in the central nervous system. A relatively small number of these published biological investigations, however, have focused on the large forms of CCK (CCK-39 and CCK-58) for lack of synthetic rep- lica. While CCK-39 was first isolated from porcine intestine by Mutt and Jorpes in 197 I , ''I it was not until a decade later that CCK-58, the largest circu- lating form of CCK, was isolated and characterized from canine intestine and later from other species including human.lo3 Despite their relatively large abundance in the intestine and blood, neither CCK-58 or CCK-39 can be extracted to date in quantities large enough to sustain a major effort in the understanding of their biological function in vivo resulting in slow progress in the understanding of CCK physiology in mammals. The total synthesis of the title compound was reported earlierIn4 and we present here the highlights of the problems encountered during the purification of the synthetic mixture and the development of a mixed mode purifications of peptides as an alternative to an all-HPLC approach.

The analytical profiles obtained with HPLC and ion exchange chromatography are shown in Fig- ures 42 (panels A and B), respectively. It is striking to see that the HPLC profiles show no major product with most of the uv absorbing material eluting as a very broad peak. On the other hand, the profile of the material eluting from the cation exchange column indicates that most of the uv absorbing material eluted within the first two void volumes and that there was left several identifiable components and a major product eluting at about 16 min. Not shown is the profile of the same separation carried out on a semipreparative scale [ 8 5 mg on a Mono S column, 10 pm, HR 10/ 10 ( 10 X 1 cm), all other conditions being identical to those shown in Figure 42Bl. The desired fraction was further purified and desalted using a reverse phase column. The final product was isolated in poor yield in an unexpected pure form (>90%) in view of the com-

plexity of the crude mixture yet could be fully characterized both chemically and biol~gically. '~~

With these two examples and others to be found in the literature (protein purification), we hope to have demonstrated that HPLC, used in conjunction with other chromatographic techniques (such as ion exchange chromatography and size exclusion chromatography not documented here), can expand the range of highly purified peptides from 50 residues97 up to peptides 82 residues in length.68.''5 With the ready availability today of such orthogonal and highly resolutive chromatographic techniques (including CZE, see below), there remains no reason for the use of crude, poorly characterized materials for biological and immunological investigations.

Size Exclusion Chromatogrgphy

With the development of new hgid supports (in contradistinction with the soft g Is also used for partition chromatography the e.,b asic principle of this technique was applied underhigher pressures. One major obstacle to be overcome was to find noncompressible supports compatible with the biomaterials to be chromatographed, i.e. ( a ) nonspecific adsorption had to be minimized and (b ) controlled pore size had to be achieved. This early work was reviewed by Cooper and Van Der- veer.Io7 We, and others, had shown22,49,5' that peptides and even small proteins (molecular weight < 12,000) could be eluted under optimized conditions from reverse phase supports.

The reasons that larger proteins (molecular weight > 12,000) could not be eluted include insol- ubility of the proteins under the chromatographic conditions, inadequate pore size of the support, and inappropriate kinetics of exchange of the proteins between the different phases. With the availability of supports (Waters Associates PAC 1-125 in this case) that had been designed for the chromatography of large hydrophilic polymers/ proteins and using our past experience in peptide/ protein separation using HPLC technology, we un- dertook to test the compatibility of TEAP/TEAF buffers on such a column for peptide/protein separation and/or molecular-weight determination. Our ultimate goal was to develop a uv transparent, biologically compatible or volatile buffer that would give high resolution and good recoveries.66

Figure 43 illustrates the effect of acetonitrile concentration on the resolution of different peptides and proteins. We found that the separation of larger proteins was improved at lower concentra-


A

A

".LO - z

U J U 3 d

0 i J W I J O I 0 5 10 15 20

Time (min)

FIGURE 42 Analytical HPLC ( A ) and ion exchange (B) elution profiles of the crude [Phe( p-CHzS03Na)52 ,Nle32.53,56 ,Na155]-CCK-58. ( A ) Experimental conditions: Vydac-C,8 ( 5 pm particle size, 300 A pore size) column (0.21 X 15 cm). Buffer was TEAP, pH 2.25; flow rate, 0.2 mL/min. (B) Experimental conditions: Mono S 1.6/5 column prepacked with 10 pm Mono S resin (the charged group on the gel is CH2-SO;). Buffer A was 50 mM sodium borate in 35% CH3CN/65% H 2 0 , pH 9.0 buffer B was A + 0.5MNaC1; gradient: 0-10070 Bin 2 mL; flow rate, 0.1 mL/min.

tion of acetonitrile (better overall solubility) whereas for smaller peptides, higher concentration of acetonitrile seemed favorable. However, as is often the case for peptides and proteins exhibiting a large spectrum of solubility characteristics due to their inherent primary and tertiary structures (low to high isoelectric points, more or less hydrophobic and globular or random in conformation), no gen- eralization is possible. For the first time however, a combination of an aqueous buffer and a significant

amount of an organic modifier was successfully used for the size exclusion of a peptide/protein mixture using a high-pressure system.66

In the range of 15-30% acetonitrile, the different components of the mixture were being separated according to size in a size exclusion mode with very little nonspecific adsorption with good peak sym- metry. It was believed that the addition of triethylamine to the buffer contributed to these desirable effects.


CYT c

0 5 10 15 20 25 Time (rnin)

0 5 10 15 20 Time (rnin)

z

0 J Y

30

0 5

0.1

E K t? B 0.05

3 4

0

" I \

I 20 25 5 10 15

Time (rnin)

20 25 0 5 10 15

0 , 1 Time (inin)

FIGURE 43 Influence of CH3CN concentration on elution pattern. Conditions: load, 50 p1 protein-peptide mixture. Buffer was TEAP pH 2.25. Retention times (sec) for the different components are for 18% and 30% CH3CN respectively: BSA, 695 and 729; cyt c, 829 and 861; @-endorphin (human), 1027 and 101 I ; hACTH (18-39), 1 1 1 1 and 1075; GnRH, 1185 and 1167;TRH, 1227and 122l;aceticacid, 1311 and 1273.

Also noteworthy is the low pH (below most isoelectric points) of the aqueous buffer used for the separation shown in Figure 43. A low pH ( < 3 ) is recommended in HPLC for most pep tide^*^.^^,^' (exception: acidic peptides that are insoluble under those conditions: for example, gastrin I for which a dilute 1 : 1 TEAP buffer at pH - 6.5 is recommended lo*) . Obviously, proteins that would be insoluble under the conditions used could hardly be expected to elute from any column. It is remarkable, however, that among the proteins present in this mix, cytochrome c has a PI of 10.6 whereas bovine serum albumin has a PI of 4.4-4.8. Recov- ery studies using integrated areas under the peaks and different loads ( 5 , 10, 20, and 40 pg) have

shown good linearity for all components of the mixture when using 30% acetonitrile and the TEAP pH 2.25 buffer at room temperature.

The effect of the concentration of the TEAP buffer on retention times was studied. Whereas proteins eluted earlier at low concentrations of the buffer than at higher buffer concentration, peptides had the opposite tendency. Although no simple in- terpretation of those results could be given, two phenomena may be involved: ( a ) a dependence of Vi upon TEAP concentration would be compatible with some interaction of the eluate and the stationary phase26; (b ) the particular solvent system had an effect on the Stokes radius of the peptide and protein studied. At high concentration of TEAP,

Pepti& Chemistry 309

proteins appear smaller than they really should be (salting out effect or hydrophobic interaction) whereas peptides appear larger than they really are (ion pairing and solvation effect). That indole was being unexpectedly retarded in this system indicated that other compounds (most prevalent in the case of very hydrophobic and cyclic peptides) may also show anomalous behavior resembling that shown to operate in the hydrophobic interaction mode of chromatography.

The effect of temperature on the separation of the different peptides/proteins of our standard mix clearly showed the advantage of working at high temperatures ( 50°C). Using the retention times obtained from an extensive list of peptides and proteins, we could plot the log molecular weight vs. retention times (in seconds). We found a linear relationship for molecular weights ranging from 1000 to 44,000 Da. This is somewhat different from the suppliers specifications for that particular column used where the linear range was reported to be between 2000 and 80,000 Da using a quite different buffer system. The correlation coefficient derived from linear regression analysis was found to be -0.985. It is interesting to note that at this pH, peptides and proteins with a high PI: bradyki- nin, dynorphin, cytochrome c, and soybean trypsin inhibitor-appear to be larger in size than they are, probably due to some ion pairing effect or repulsive effect of the support, both already discussed. This system, in fact, turned out to be particularly useful to study the apparent size of cyclic GnRH analogues6' as well as TASP.68 As mentioned earlier, TASP molecules that were investigated con- sisted of a 4-helix bundle T4-( 4a,) containing four identical amphiphilic helices (our study included helices that were 1 1, 15, and 18 amino acids long) that are covalently attached via amide bond formation to the e-NH2 side chains of lysines in the template molecule. The fifteen amino acid long chain was also acetylated for comparison to the free amino chain. The molecular weights were calcu- lated to be 5567,7336,7505, and 8757. TASP were analyzed using HP size exclusion chromatography and TEAP buffer at pH 2.25. Acetonitrile was used ( 30%) to assist solubility and temperature was maintained at 40°C in the Bio-Rad Bio-Sil TSK- 125 column. The results of the GPC suggested that the eleven amino acid chain TASP gave an apparent MW of 6853. This shortest chained TASP eluted early suggesting a random secondary structure while the fifteen and longer TASP eluted later than expected, suggesting a more compact secondary structure.

Finally, we have used size exclusion chromatography on HPLC as one technique for characterization and evaluation of purity of the small protein human [Tyr5', Nle32.53,56, Na155]-CCK-58 Io9 described earlier. The peptide eluted with the retention time corresponding to the expected molecular weight as compared to that of other peptides chromatographed under these same conditions. A symmetrical profile was obtained and a 1 % impurity detected. The later elution of the impurity indicates that a smaller molecular weight component was present.

CAPILLARY ZONE ELECTROPHORESIS

We started this article by emphasizing the importance of HPLC in the characterization of peptides generated by the solid phase and how this methodology ultimately allowed objective comparison of the purity of peptides generated in solution, enzy- matically, or by the solid phase. We proposed that CZE would be to the 1990s what HPLC had been to the 1980s. We present here some unusual separations that could be achieved using CZE under a number of less conventional conditions than those offered by the manufacturers, such as using TEAP buffers or standard buffers in the presence of acetonitrile.

Analysis of D- or L-0-Methyl-Phenylalanine Enantiomers

Current academic and pharmaceutical research has focused on the characterization of natural extracts (plants, marine organisms, or bacterial broths) and on the development of methodologies for generating chemical diversity ( peptide or pepti- domimetic libraries) for the discovery of new bioactive leads. The latter approach depends on auto- mation of chemical methods for solid phase syntheses and the identification of novel scaffolds. Several monomeric building blocks that mimic the peptide backbone have been proposed recently and include peptoids, I I I azoles, 2-isoxazolines, I l 3

oligocarbamates, oligosulfones and oligosulfox- ides, pyrrolinones, 'Is vinylogous backbones, the more classical oligomers with pseudopeptide bonds described by Spatola et al.,Ii7 and the @- methyl amino acids."' The latter amino acids are of unique interest for the design of rigid peptide based analogues in that they have built-in rota- tional constraints of their side chains that will complement constraints brought about by the intro-

Miller and Rivier 310

0.01

E 2 0.00:

L

-J r

m

4

I

I I \ r B' \

\ \ \ \ -

A' , I \ I

\ \ -

0 110 I 20

Time (min)

FIGURE 44 CZE profile of D,L-PMe-Phenylalanine [trace A: threo ( 1 % ) : erythro (99%); trace B: threo (43%): erythro ( 5 7 % ) ] . Beckman P/ACE 2050, fused silica capillary ( 50 cm X 75 pm) at 30"C, Voltage: 20 kV, 0.1 M sodium phosphate, pH 2.5.

duction of cycles. Such rigid analogues are particularly important for the definition of the bioactive conformation of peptide hormones. We were inter- ested in these amino acids and the effect of their introduction in GnRH antagonists (manuscript in preparation). Unlike Kazmierski et al., 'I8 we opted for reason of scale and cost for a combination of crystallization steps for the separation of the diastereomers and enzymatic resolution or frac- tional crystallization of optically active salts for the separation of the enantiomers (Simon and Rivier, in preparation).

In Figure 44 we present a CZE-based method for the quantitative determination of diastereomeric ratios.

More recently, we have reported synthetic path- ways to orthogonally protected betidamino acid and methylbetidamino acid scaffolds ("-mono- acylated aminoglycine derivatives) for solid phase peptide synthesis and their use in the design of bioactive GnRH 19,120 and somatostatin 1 2 ' analogues. The structural preferences of betidamino acids and their corresponding mono- and bis-methylated derivatives (as compared to those of amino acids and P- methyl amino acids) were investigated using molecular mechanics in combination with a continuum solvation model '22 to calculate the total energy of the Ac-Xaa-methyl amides as a function of backbone di- hedral angle. Interestingly, on the basis of theoretical structural preferences as well as of limited biological evidence, we suggest that P-methyl-betidamino acids

may mimic quite precisely P-methyl-amino acids without the burden of a second asymetnc center.

Separation of Neuropeptide Y Diastereomers

As part of a structure-activity relationship investigation of NPY, we synthesized the entire series of D-isomer substitutions to explore the role of backbone modification at each position in the se- q ~ e n c e . ' ~ ~ Separation of these 35 analogues of NPY in which a single D-amino acid replaced the corresponding naturally occurring residue was performed by HPLC and CZE to ensure the quality of the synthetic peptides to be used for structural and biological studies recognizing that contamination of any of these peptides by the L-isomer of the substituted amino acid would yield NPY and significantly influence biological activity and potency.

The use of racemic mixtures of amino acids used in peptide synthesis or epimerization at any one of the many amino acid a-carbons during synthesis could lead to the undesired incorporation of D-isomer impurities. Such impurities represent one of the greatest challenges to chromatographic detection systems, since they differ by only a single inversion of a peptide backbone a-carbon. Analogues for our studies were assembled by standard solid phase peptide synthesis methodology, which virtu- ally eliminates the occurrence of epimerization, and were subsequently purified by preparative HPLC. Standard characterization of synthetic peptides included assessment of purity by HPLC, amino acid analysis, and determination of molecular mass by mass spectroscopy. Although these three methods allow (in one way or another) the detection of the most common side products in peptide chemistry (adducts and truncations), the latter two techniques will not distinguish diastereomers and thus could not detect racemic contamination that could lead to ambiguous biological results. In this study of a series of NPY analogues we confirmed both the uniqueness of each derivative, differing only by the inversion of a single chiral center and the resolutive power of HPLC and that of CZE, especially when combined with the use of TEAP buffer.124

Initially, separation between NPY and 26 of the 35 analogues was achieved by standard analytical HPLC methods using a Vydac CIS column and a gradient buffer system comprised of TEAP at pH 2.25 and acetonitrile at 25°C. Under the same gradient conditions at 40"C, efficiency increased, allowing the separation of three additional N- and C-terminal analogues. At both temperatures, the greatest

NPY


100

I I1 I I I I I 0 5 10 15 20 25

Time (min)

FIGURE 45 Coelution of diastereomers of NPY by HPLC using TEAP 2.25/CH3CN. Load:NPY(3pg);a, [ D - I ~ ~ * ~ ] N P Y ( 1 pg);b, [ ~ - I l e ~ ' ] N p y ( 1 pg);c, [D-L~u~ ' ]NPY ( 1 pg); d, [ ~ - T h r ~ ~ ] N p y ( 1 pg);e, [ D - A s ~ ~ ~ I N P Y ( 1 pg);f, [ D - A ~ ~ ~ ~ I N P Y ( 1 pg). VydacC18(5pm particle size) column (0.21 X 15 cm). Buffer was TEAP, pH 2.25; flow rate, 0.2 mL/min; temperature, 40°C.

selectivity was exhibited by analogues modified in the region proposed to exist as an a-helix, as expected since these residues are held in close proximity by hydrogen-bonding and hydrophobic interactions and critically depend on the orientation of the side chains to maintain conformational stability. Coinjection of six such centrally modified analogues with NPY is shown in Figure 45.

The unresolved diastereomers could be separated on the same solid support by using 0. l % TFA as the mobile phase modifier; however, separation factors were smaller and retention times were longer. Three of the remaining seven unresolved analogues were separated ( a = 1.02-1.96) by changing the solid phase support to Vydac diphenyl derivatized silica and a buffer system consisting of 0.1 % TFA and acetonitrile.

As a final approach to separating the remaining diastereomers, CZE was employed. It was expected that, although the global charge of each NPY dia- stereomer would be identical, the altered local orientation of a charged group might be exposed, and thus could be expressed by differing migration times. Under our standard CZE analysis conditions (sodium phosphate buffer, pH 2.5) [ ~ - S e r ~ ] - and [ D - T ~ ~ ~ ~ I N P Y were resolved from NPY (Figure 46A). The other isomers-[ D-Tyr I ] NPY, and [ D-LYs~] NPY-were not resolved under these

conditions. All four isomers were finally resolved by changing the electrophoretic buffer to 0. I A4 triethylammonium phosphate at pH 2.5 (see Figure 46B). Under these CZE conditions, the entire series of 35 isomers was reevaluated and resulted in the separation of 32 out of 35 isomers, emphasizing the need for multiple analytical techniques and systems for the characterization of peptides.

Analysis of an Hydrophobic CRF Antagonist Using CZE in the Presence of Acetonitrile

As discussed earlier in this article, the addition of acetonitrile in buffers used for ion exchange chromatography as well as size exclusion chromatography was found to be extremely beneficial. We hy- pothesized that acetonitrile increases the solubility of the peptides and facilitates partitioning among phases (stationary and mobile). Because most separations have to be carried out in the presence of a support or of capillary walls in the case of CZE, it is extremely important that such interactions be minimized. We have already mentioned that spe- cific coatings of the capillaries will facilitate some separations. We show in Figure 47A (broad elution pattern) that the addition of acetonitrile to the buffer used for the separation of a hydrophobic


CRF analogue (Figure 47B, three impurities pre- ceding the main adsorption) is very beneficial and allows better discrimination between the desired product and closely related impurities.

0.006

0.004

2 N

v) U. 3

- a

0.002

A

NPY /

-dt------ \

\ \ D

I 10 15 20 25

Time (min)

0.006 r t ' 0.004 2 cu v) u. 3 Q

0 ,002

A

\

\r-A---YrG D I I

20 25 30 35 Time (min)

FIGURE 46 CZE trace of NPY, A and diastereomers [D-T~~ ' ]NPY; B, [ D - S ~ ~ ~ I N P Y ; C, [o-Lys4]NPY; and D, [ ~ T y r ~ ~ l N P y . Beckman P/ACE 2050, Fused Silica Capillary (50 cm X 75 pm) at 30°C. Panel A, Voltage: 12 kV, 62 pA, 0. I Msodium phosphate, pH 2.5. Panel B, Volt- age: 20 kV, 100 PA. Buffer was 0.1 M TEAP, pH 2.50.

0.003 r A

P I

0 L,--

0.003

E, 2 N la (I) LL 3

c

a

0

10 15

B

20

L

15 20 25

FIGURE 47 CZE trace of a CRF antagonist. Panel A: (C-3 124), Beckman P/ACE 2050; Supelco P-175 Capil- lary; (50 cm X 75 pm) at 30°C; Voltage: 15 kV; Buffer was O.1M sodium phosphate, pH 2.5. Panel B: (C- 3 I36), same as above; Voltage: 12 kV. Buffer was 0.1 M sodium phosphate, pH 2.5 in (95 : 5 , H 2 0 : CH3CN).

General Observations

CZE is now routinely used in our laboratory for the analysis of native and synthetic peptides and proteins as a complementary method to HPLC analysis. The high sensitivity, high automated throughput, high resolution, and low solvent con- sumption of CZE make it an invaluable technique.

Capillaries used for CZE are fused silica exter- nally coated with polyimide. A small area of polyimide is removed to form a window for online detection. We have used bare fused silica and coated capillaries. A capillary coated with a neutral hydrophilic bonded phase (Supelco CElect P 1 75 ) has been extremely useful in our work with hormonal peptides that have isoelectric points > 7. Basic peptides analyzed using a bare fused silica capillary often separate as broad peaks. The hydrophilic coat- ing seems to minimize interactions with the capillary wall. One example of this was the separation of a mixture of [ D- and L-Agl( Me,A~)~]-acyline using a fused silica capillary or CElect P175. All


other conditions of the separation remaining the same, no separation of the D, L mixture was observed using the fused silica capillary while the same mixture was well separated (allowing quantitation 54 : 46) by using the CElect P175 capillary.

We have found that acidic phosphate buffers are useful for CZE separations of most peptides except some negatively charged ones. For those cases, a borate buffer at pH 8.5 is used. In either case, the peptide must be positively charged or there must be enough electroosmotic flow to cause net move- ment of the peptide toward the cathode (in the an- ode + cathode configuration ).

CONCLUSION

Whereas HPLC has played and will continue to play a major role in the life of the peptide/protein chemists, it should not be relied upon as the only chromatographic approach (even with the use of more than two-buffer systems) for the purification and analysis of peptides greater than 40-50 residues in length. Selection of the column supports and buffers for peptide and protein separation is often critical. Preparative cation-exchange chromatography proved to be an excellent orthogonal technique for the purification of peptides (small and large) and because it has been found that narrow-bore cation-exchange chromatography and CZE complement HPLC for the determination of the purity of synthetic peptides, such techniques should find their way in every laboratory dealing with bioorganic molecules. The addition of organic modifiers in such chromatographies as ion exchange and capillary zone electrophoresis should be encouraged. Although we have shared here and tried to exemplify some of our most striking observations while purifying natural products and synthetic peptides, what has been presented is by no means exhaustive, nor can we pretend that the techniques used have been optimized to their limits with reference to given peptides. We also recognize the limitations of this purely empirical approach when it comes to the development of a mechanistic understanding of the observed phenomena. When used critically and with discrimination, HPLC has been a magnificent tool both for the isolation of natural products and for the purification and characterization of peptides synthesized by Merrifield's solid-phase approach. What should be realized is that thanks to these chromatographic developments and constant improvements in the synthetic area (new strategies, activating agents, and amino

acid derivatives resulting in improved quality of crude peptide mixtures) we can observe a consistent extension of the limit in size and complexity of available synthetic peptides. As mentioned by one of our colleagues, the recent availability of other highly resolutive techniques ultimately broke the HPLC monopoly and allows examination of peptide preparations from more than one angle, very much as taught originally by the pioneers of peptide chemistry. Finally, we peptide chemists owe much to those who have dedicated their lives to the understanding of the different chromatographic processes and extended their knowledge to the development of unique chromatographic supports and instrumentation.

MATERIALS AND METHODS

Preparation of TEAP Buffer

We thought that it would be helpful to describe here again how we prepare the TEAP buffers at pH 2.25, 5.2, and 7.0 because of their recognized usefulness in a peptide laboratory for both analytical and preparative separations for reverse phase, ion exchange, size exclusion chromatographies, and CZE.

To several liters of distilled HPLC grade water, under stirring, is added concentrated phosphoric acid ( 1 % by volume) and within a few minutes analytical grade tn- ethylamine ( 1% by volume). It may take as much as half an hour for all the triethylamine to be dissolved, at which point the pH can be adjusted with either base or acid, although we have found little difference between the eluotropic behavior of a TEAP buffer at pH 2.25 or 2.5. Fol- lowing the same sequence of operation but using a 0.4% concentration of phosphoric acid and a 0.85% concentration of triethylamine, one obtains a TEAP at pH 5.2. Finally, using a 0.1% concentration of phosphoric acid and a 0.3% concentration of triethylamine, one obtains a TEAP at pH 7.0. We have found these three pHs to be the most useful. These different TEAP buffers are used as is for preparative purposes. For analytical purposes we recommend that the above buffers (up to 10 L) be fil- tered on a CI8 preparative cartridge discarding the first two void volumes. These buffers keep well in the cold room; however, kept at room temperature they are good growth media. It is therefore important to keep glassware and HPLC leads free from bacterial contamination.

Conversion of a Peptide Trifluoroacetate Salt to Its Acetate SaltGo

Technical problems, in the 1970s, associated with the conversion of peptides from their trifluoroacetate salts to their acetate salts using classical anion exchange supports (based on cellulose, Sephadex, or polystyrene) led us to


investigate the possibility of using HPLC for a quick and convenient alternative. We came up with the following protocol. Peptide trifluoroacetate salt is dissolved in a large excess of ammonium acetate ( I M ) and the pH adjusted to 7.0. This solution is applied to a preparative column/cartridge and additional ( 2 void volumes) ammonium acetate ( 1 M ) is eluted. Ammonium acetate is then displaced ( 3 void volumes) with dilute acetic acid (0.5%). A sharp gradient of acetonitrile is then applied to displace the peptide from the column. The same procedure can be applied to convert the peptide in a triethylammonium phosphate buffer into its acetate salt.

Work was supported over the years by numerous grants and contracts from NIH and in part by the Hearst Foun- dation. We thank those who contributed day after day to improving the quality and reliability ofthe analytical and preparative separations: Jerry Desmond, Richard McClintock, Dean Kirby, John Porter, Robert Galyean, John Dykert, Laura Cervini, Sabine Lahrichi; and Drs. Harry Anderson, Jerry Boublik, Anthony Grey Craig, William Gray, Jean-FranGois Hernandez, Carl Hoeger, Maria Teresa Machini de Miranda, Antonio de Miranda, and Lajos Simon. We thank Drs. Roy Eksteen and Barry Karger for their early collaborative contributions in the development of reverse phase columns. We thank our colleagues, biologists and biochemists, Drs. Manfred Mutter, Baldomero Olivera, Marilyn Perrin, Nancy Sherwood, Jochem Spiess, Wylie Vale, and Ms. Joan Vaughan who challenged us with unusual separation problems of natural products and synthetic constructs. We want to acknowledge the early involvement and en- thusiasm of Gerald Hawk (with Waters Associates at the time) and Lance Hellinger (with Perkin Elmer at the time) who brought to our attention the unusual properties of different column supports. We thank Kervin Har- rison of The Separations Group for making available to us experimental supports and Pharmacia and Perkin El- mer for contributing experimental instrumentation. We thank Debbie Johns for manuscript preparation and Dr. Carl Hoeger for a critical review of the manuscript.

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2. Grushka, E. ( 1974) Bonded Stationary Phases in Chromatography, Ann Arbor Sciences, Ann Ar- bor, MI.

3. Brown, P. R. ( 1973) Biochemical and Biomedical Applications, Academic Press, New York-Lon- don.

4. Gil-Av, E. (1974) in Peptides, Wolman, Y., Eds., John Wiley & Sons, Israel University Press., New York-Toronto, Jerusalem, pp. 247-256.

5. Yoshida, N., Zimmerman, C. L. & Pisano, J. J.

(1975) in Peptides, Walter, R., Meienhofer, J., Eds., Ann Arbor Sciences, Ann Arbor, MI, pp. 955.

6. Hardy, P. M., Prout, R. A. & Rydon, H. N. ( 1974) J. Chem. Soc. 802.

7. Shechter, I. ( 1974) Anal. Biochem. 58,30-38. 8. Pickart, L. R. & Thaler, M. M. (1975) Prep. Bio-

9. Tsuji, K. & Robertson, J. H. ( 1975) J. Chromatog.

10. Hancock, W. S., Bishop, C. A. & Hearn, M. T. W. (1976) FEBSLett. 72,139-142.

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